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BNL-52351 (Rev. 12/93)UC-900

(General, Miscellaneous,and Progress Reports -


Laboratory DirectedResearch & Development

ProgramAnnual Report to the Department of Energy

December 1993

Gregory J. Ogeka and Anthony J. RomanoSpecial Assistants to the

Associate Director for Administration

B R O O K H A V E N N A T I O N A L L A B O R A T O R YA S S O C I A T E D U N I V E R S I T I E S . I N C .

U P T O N , N E W Y O R K 1 1 9 7 3 - 5 0 0 0




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The use of additional color on the cover and divider pages of this documentwas paid for using non-Department of Energy Funds.


This report was prepared as an account of work sponsored bv an agency of the UnitedStates Government. Neither the United States Government nor any agency thereof,nor any of their employees, nor any of their contractors, subcontractors, or theiremployees, makes any warranty, express or implied, or assumes any legal liability orresponsibility for the accuracy, completeness, or useftilr.rss of any information,apparatus, product, or process disclosed, or represents that its use would not infringeprivately owned rights. Reference herein to any specific commercial product, process,or service by trade name, trademark, manufacturer, or otherwise, does not necessarilyconstitute or imply its endorsement, recommendation, or favoring by the United StatesGovernment or any agency, contractor or subcontractor thereof The views andopinions of authors expressed herein do not necessarily state or reflect those of theUnited States Government or any agency, contractor or subcontractor thereof

Printed in the United States of AmericaAvailable from

National Technical Information ServiceU.S. Department of Commerce

5285 Port Royal RoadSpringfield, VA 22161

NTIS price codes:Printed Copy: A06; Microfiche Copy: A01

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The Laboratory Directed Research and Development(LDRD) Program is directed by Martin Blume, DeputyDirector and is administered by Henry Grahn, Associ-ate Director for Administration (ADA). Preparationof the FY 1993 report was coordinated and edited byGregory Ogeka and Anthony Romano, Special Assis-tants to the ADA who wish to thank RoseMarie Buschfor her assistance in organizing and typing the docu-ments. A special thank you is also extended to BethBlevins and Virginia Morante for their help in proof-reading sections of the report and to the Photographyand Graphic Arts Division for their help in publishing.


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•7-able of Contents


Introduction 1

Management Process 2

Broad Technical and Scientific Categories 7

Project Summary Table 8

Project Description and Accomplishments - FY 1993


GLOBAL CHANGECalcification in Marine Alga: A Molecular Approach to a Major Pathway of

Global Carbon Cycling 19

RADIATION THERAPIES AND IMAGINGStudies on the Cellular Toxicity of Cocaine and Cocaethylene 23Uranium Neutron Capture Therapy (UNCT) 26Design of a Dedicated X-Ray Source for Coronary Angiography 31Cryogenic Targets for the Production of PET Isotopes 34Development of Microdialysis Technology 37Analysis of Structures and Interactions of Nucleic Acids and Proteins by Small

Angle X-Ray Diffraction 42Enhancement of Microplanar Radiation Therapy (MRT) of the Intracerebral

Rat 9L Gliocarcoma by Preinfusion with a Diiodofluorecein-HistoneConjugate 47

Relaxographic MRI and Functional MRI 49

GENETIC STUDIESA New Malaria Enzyme - A Potential Source for a New Diagnostic Test

for Malaria and a Target for a New Antimalarial Drug 53Effect of a Bacterial Spore Protein on Mutagenesis 54Structure and Function of Adenovirus Penton Base Protein 56Human Melanocyte Transformation 59Exploratory Applications of X-Ray Microscopy; Determination of the

Higher Ordered Structure of Eukaryotic Chromosomes 61Nuclear Techniques for Study of Biological Channels 63Induction and Repair of Double-Strand Breaks in the DNA of Human

Lymphocytes 66Genetics of Drug Addiction 68

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Project Description and Accomplishments - FY 1993 (continued)


High Gain Harmonic Generation Experiment 73Tunneling Microscopy Studies of Nanoscale Structures 75RF Sources for Accelerator Physics (CAP) 78An EBIS Source of High Charge State Ions up to Uranium 80The Structure and Reactivity of Electrode/Electrolyte Interfaces 82Investigation of the Utility of Maximum-Entropy Methods for the Analysis

of Powder Diffraction Data 84Accelerator Technology 85

OTHER - MISCELLANEOUS AREASStructural Investigations of PT-Based Catalysts 93Structure-Sensitive Properties of Advanced Permanent Magnet Materials:

Experiment and Theory 94A Very High Flux Research Reactor 96


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Brookhaven National Laboratory has three primarymissions. The first is to conceive, design, buildand operate in a safe and environmentally soundmanner, complex research facilities for the benefitof the entire scientific community. These facilities,such as particle accelerators and colliders, nuclearreactors and synchrotron storage rings are used forfundamental scientific studies and both basic andapplied research in energy-related, physical, lifeand environmental sciences.

The second mission is to carry out basic scienceresearch efforts in long-term high-risk programswhich have potential long-term payoffs. Many ofthese programs employ the unique facilities men-tioned above; others take advantage of the specialexpertise and ancillary support services and facili-ties at the Laboratory. The ease of engaging incollaborative efforts with outside users from univer-sities, industries and other government laboratoriesgreatly enhances the effectiveness of the programsand encourages wide based use of the special facili-ties present at Brookhaven.

The third mission is to contribute to the technologybase of the nation. The Laboratory is engaged inthe development of new technologies and facilities.The process of transferring this new knowledge tothe commercial sector helps advance and broadenthe nation's preseni basic and applied researchcapabilities. Brookhaven is involved in the educa-tion of scientists and engineers through a widevariety of cooperative research programs and hasother extensive educational programs covering abroad spectrum, reaching elementary schoolthrough university students and faculty.

As a national resource, Brookhaven makes avail-able, when feasible, its unique facilities and ex-pertise to state and federal agencies and to theprivate sector.

Today, new ideas and opportunities, fosteringthe advancement of technology, are occurring atan ever-increasing rate. It, therefore, seems ap-propriate that a vehicle be available which fos-ters the development of these new ideas andtechnologies, promotes the early exploration andexploitation of creative and innovative concepts,and which develops new "fundable" R&D pro-jects and programs. At Brookhaven NationalLaboratory (BNL), one such method is throughits Laboratory Directed Research and Develop-ment (LDRD) Program. This discretionaryresearch and development tool is critical inmaintaining the scientific excellence and vitalityof the Laboratory. Additionally, it is a means tostimulate the scientific community, fostering newscience and technology ideas, which is the majorfactor in achieving and maintaining staff excel-lence, and a means to address national needs,within the overall mission of the Department ofEnergy (DOE) and the Brookhaven NationalLaboratory.

The Project Summaries with their accomplish-ments described in this report reflect the above.Aside from leading to new fundable or promis-ing programs and producing especially notewor-thy research, they have resulted in numerouspublications in various professional and scientificjournals, and presentations at meetings andforums.

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•H anagement Process


INTRODUCTION: The Department of Energy's(DOE) Laboratory Directed Research & Develop-ment (LDRD) Program at Brookhaven NationalLaboratory (BNL) was formally established underthe guidelines set forth in DOE Order 5000.1 inMay 1984. From inception through September1994, a period spanning ten fiscal years, the Labo-ratory has authorized $19.4 million in ExploratoryR&D, consisting of 134 separate projects.




















HISTORICAL PERSPECTIVE: BrookhavenNational Laboratory was established in 1946.Throughout its history, certain projects of an ex-ploratory nature, sometimes referred to in the pastas "seed money projects," were supported withoverhead funding. In 1979, as a result of a Re-view Audit in that year, the seed money account-ing procedures were formalized, and oversight bythe DOE Brookhaven Area Office Manager wasfirst established. This seed money program oper-ated at a variable level of funding, which averagedabout 0.1 percent of the Laboratory's operatingbudget over the period 1979 to 1984.

In May 1984, the program was expanded. Theexpanded program embraced the new ExploratoryR&D guidelines of DOE Order 5000.1. The newprogram, called the Exploratory Research Pro-gram, was put into effect for FY 1985 funding.The current Laboratory Directed Research &Development Program reflects the operating stylesand many of the procedures of the earlier pro-grams, which have evolved somewhat informallyover the years. It also encompasses the require-ments of the current DOE Order 5000.4A.

PROGRAM STRUCTURE: The LDRD Programdoes not h2ve any formalized substructure in thatthere are not defined a priori any separate or dis-tinct categories of projects. All projects and theirproposals, large and small, regardless of institu-tional purpose or potential impact, are treatedsimilarly in the Program's administrative proce-dures.

GOALS AND OBJECTIVES: The goals andobjectives of BNL's LDRD Program can be in-ferred from the Program's stated purposes. Theseare to (1) encourage and support the developmentof new ideas and technology, (2) promote the earlyexploration and exploitation of creative and inno-vative concepts, and (3) develop new "fundable"R&D projects and programs. The emphasis isclearly articulated by BNL to be on supportingexploratory research "which could lead to newprograms, projects, and directions" for the Labora-tory.

GENERAL CHARACTERISTICS OF THELDRD PROGRAM: Projects or studies that areappropriate candidates for the Laboratory's LDRDProgram include, but are not limited to, (1) pro-jects, normally relatively small, in the forefrontareas of basic and applied science and technologyfor the primary purpose of enriching laboratorycapabilities, (2) advanced study of new hypotheses,

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new concepts, or innovative approaches to scientif-ic or technical problems, (3) experiments andanalyses directed toward "proof of principle" orearly determination of the utility of new scientificideas, and (4) conception and preliminary technicalanalysis of experimental facilities or devices.


OVERALL COORDINATION: Overall respon-sibility for coordination, oversight, and administra-tion of BNL's LDRD Program resides with theLaboratory's Deputy Director. The Deputy Direc-tor is assisted in the administration of the programby the Office of the Associate Director for Admin-istration, which administers the program budget,establishes the project accounts, maintains summa-ry reports, and reports Program activities to theDOE through the Brookhaven Area Office Manag-er.

Responsibility for the allocation of resources andthe orchestration, review and selection of propos-als, lies with a top-level group, called the Labora-tory Directed Research & Development Commit-tee. The Committee is made up of seven mem-bers. The Laboratory's Deputy Director is thechairperson of the Committee. The other mem-bers are the Associate and Assistant Directors ofthe Laboratory.

Martin BlumeJohn D. AxeSeymour BaronHenry C. GrahnMark SakittMelvin Schwartz

Richard B. Setlow

ChairpersonBasic Energy SciencesApplied ProgramsAdministrationPlanning & PolicyHigh Energy &

Nuclear PhysicsLife Sciences

ALLOCATING FUNDS: There are two types ofdecisions to be made each year concerning theallocation of funds for the LDRD Program. Theseare (1) how much money should be budgeted over-all for the Program; and (2) of this, how much, ifany, should go to each competing project or pro-posal. Both of these decisions are made by top-level management.

Concerning the overall budget, for each upcomingfiscal year the Laboratory Director, in consultationwith the Deputy Director and the Associate Direc-tor for Administration, develops an overall levei offunding for the LDRD Program. The budgetamount is then incorporated into the Laboratory'sLDRD Plan which formally requests authorizationfrom the DOE to expend ftinds for the LDRDProgram up to this ceiling amount.

The actual level of funding available for LDRD,however, may turn out to be much less than thisceiling. The actual level is determined during thecourse of the year and is affected by several con-siderations, including: the specific merits of thevarious project proposals, as determined by Labo-ratory management and the members of the LDRDCommittee; the overall financial health of the Lab-oratory; and a number of budgetary tradeoffs be-tween LDRD and other overhead expenses.





$ in millions








Concerning the allocation of resources to specifictopic areas or to individual project proposals, suchissues are addressed on a case-by-case basis by theLDRD Committee, once specific proposals havebeen received. The Committee meets periodicallyto review and recommend project proposals, andto determine the amount of funding to be madeavailable to the LDRD Program. The require-ments of DOE Order 5000.4A are carefully con-sidered during the selection process to ensure thatproposals are consistent with DOE's criteria.

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REQUEST FOR PROPOSALS: The availabilityof special funds for research under the LDRDProgram is well publicized throughout the Labora-tory. This is done using two methods, one occur-ring at yearly intervals, the other occurring irregu-larly. Each year in May a memo is sent by theLaboratory Director to all scientific staff, issuing a"call for proposals." This memo is accompaniedby an attached document, entitled "Guidelines andProcedures for Developing Proposals via the Labo-ratory Directed Research and Development(LDRD) Program." The other method is by an-nouncement in the Brookhaven Bulletin, theLaboratory's weekly newspaper; but the nature ofthe announcements varies and they appear at irreg-ular intervals. In some years the Bulletin prints anarticle that amounts to a separate call for propos-als. In other years the Bulletin publishes articleson specific research projects which, in effect, helpadvertise the LDRD Program.

The "Guidelines and Procedures" document speci-fies the requirements necessary for participation inthe program. It states the program's purpose, gen-

eral characteristics, procedures for applying, andrestrictions. An application for funding, that is, aproject proposal, takes the form of a completed"Proposal Questionnaire." An application must beapproved up the chain-of-command, by theinitiator's Department Chairman or Division Head,and by the cognizant Associate Director. Plans toensure the satisfactory continuation of the principalinvestigator's regularly funded programs must alsobe approved. The applications are then forwardedto the Chairperson of the LDRD Committee forfurther review and consideration for funding.

The process which solicits and encourages thedevelopment of proposals has evolved into twomodes of operation. Specifically, the ideas forproposal development may originate among thescientific staff in response to the general call forproposals. Alternatively, they may be initiated bytop-level Laboratory management. Eventually,both follow the standard procedure for proposalapproval, up the chain-of-command to the samedecision makers. The fact that all proposals mustbe approved up the chain-of-command permitsBNL managers to consider all ideas together whendesigning the mix of projects for the LDRD Pro-gram.

An initiative from management typically takes theform of a general topic area or item of specialinterest. It is not a directive, nor is it included inthe call for proposals, but the idea is communicat-ed to a group of scientific staff, which is known tobe in a position capable of pursuing and develop-ing the idea in the form of a more formal propos-al.

PROPOSAL REVIEW: Once a proposal is ap-proved by the cognizant line managers, all propos-als are forwarded to the Chairperson of the LDRDCommittee who transmits a copy of all proposalsreceived to the LDRD Committee for review. TheCommittee considers all proposals that have metcertain minimum requirements pertaining to theDepartment's and BNL's LDRD policies. TheAssociate Director for Administration performs areview to ensure compliance with DOE require-ments.

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Lead responsibility for the review of a proposal isthen assigned to thai member of the Committeewho last approved it in the chain-of-command, thatis, the member who oversees and directs the tech-nical area from which the. proposal originated. Allmembers have several weeks to review the propos-al and prepare for the next Committee meeting.During this time, additional reviews, if desired,may be arranged.

Formal peer reviews, consisting of written com-ments by experts outside the normal lines of super-vision, are not usually performed. The membersof the Committee are considered to have sufficienttechnical knowledge so that peer reviews are sel-dom required.

At the next Committee meeting, the Committeemember responsible for the review of the proposalpresents the proposal to the other members of theCommittee. This is done without the membernecessarily becoming an advocate for the proposedproject.

SELECTION CRITERIA: Before proposals canbe considered by the LDRD Committee, they mustbe screened to ensure that they meet a set of mini-mum requirements concerning the Department'sLDRD policies and the Laboratory's own guide-lines.

Minimum requirements of each proposal are: (1)consistency with program purpose; (2) consistencywith missions of BNL, DOE, and NRC; (3) ap-proval by Department Chairman and/or DivisionHead, and cognizant Associate Director; (4) assur-ance of satisfactory continuation of principalinvestigator's regularly funded programs; (5) mod-est size and limited duration; (6) will not substitutefor, supplement, or extend funding for tasks nor-mally funded by DOE, NRC, or other users of theLaboratory; (7) will not require the acquisition ofpermanent staff; (8) will not create a commitmentof future multi-year funding to reach a useful stageof completion; and (9) will not fund constructionline-item projects, facility maintenance, or generalpurpose capital equipment.

The selection criteria used to evaluate and rankindividual proposals are not formally stated orpublished. While the "Guidelines and Procedures"document clearly states that "awards will be madeon a competitive basis," the factors or selectioncriteria to be considered in this competition are notlisted. Nevertheless, selection is based on (I) sci-entific merit, (2) compliance with minimum re-quirements, (3) proposal cost as compared to theamount of available funding, (4) innovativeness,and (5) its potential for follow-on funding. Therequirements of DOE Order 5000.4A are alsocarefully considered during the selection process toensure that proposals are consistent with DOEcriteria.

PROJECT APPROVAL: After all presentationsare heard, the Committee attempts to arrive at aconsensus concerning the highest priority propos-als. Differences, if any, are resolved by theChairperson. Also, a balance is struck betweenthe prevailing financial needs of the Laboratory,which may vary over the course of the year, andthe priorities of the projects considered. Somefunding is held in reserve during the earlier meet-ings of the fiscal year so that funds remain avail-able for proposals submitted at later dates. Thefunding amount requested in any one specific pro-posal may be changed or adjusted during the ap-proval process. The Committee's recommendationis then submitted to the Director for his approval.

The Associate Director for Administration is thennotified, so that a separate Laboratory overheadaccount can be established to budget and collectthe costs for the project. Statistics on the numberof projects approved, compared to those rejected,show an overall approval rate of about 39 percentfor new starts. From inception of the programthrough September 1993, 313 proposals were con-sidered and 122 were approved. All eight scientif-ic departments were represented in the FY 1993LDRD program.

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S39k ;PHYSICS \ / A P f

S271k x


N S L s 'S150k

^ ^ ^ ^ ^ ^ \ / '•'Vs





PROJECT SUPERVISION: Supervision over theactual performance of LDRD projects is carriedout in the same way as other research projects atthe Laboratory. Each principal investigator isassigned to an organizational unit (Department,Division), which is supervised by a manager.

Each manager is responsible for seeing that theobligations of the principal investigator are satis-factorily fulfilled and that the research itself iscarried out according to standard expectations ofprofessionalism and scientific method. The man-ager is kept informed of the project's status,schedule, and progress.

The manager ensures that the work is completed ina timely manner and that annual status reports aresubmitted to the Deputy Director. In addition,LDRD Program activity is reported to the DOEArea Office Manager, including copies of all fund-ed proposals, a LDRD Program data base, and aproject funding and schedule summary report.

PROJECT REPORTING: Routine documentationof each project funded under the LDRD programconsists of a file containing: (1) a copy of thewritten proposal; (2) all interim status reports; (3)notifications of changes in research direction, ifany; and (4) reports on costs charged. Also, aformal Annual Report on the LDRD Program issubmitted to BNL management and the DOE, sum-marizing work progress, accomplishments, andstatus on all projects.

Documentation for the overall Program consists of(1) various program history files; (2) a running listof all proposals with their acceptance/rejectionstatus, (3) funding schedule and summary reportsfor all approved projects, (4) permanent recordson cost accounting, and a data base containinginformation on each funded project (description,funding by fiscal year, status and accomplish-ments, follow-on funding, publications, etc.).

NOTE: Those projects «'h:c!i involve animal ver-tebrates or human subjects have been so identifiedin ihe text.

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road Technical and Scientific Categories

T h e programs targeted for funding by BNL'sLDRD program in FY 1993 fall into the followingbroad technical and scientific categories:

New Directions for Energy Technologies: In thecourse of basic research efforts there are occasion-ally discoveries which hold promise for utilizationin energy technologies. Such research is of signifi-cance, both for the Laboratory and for the DOE.Typical of this category is the LDRD project onMaglev Studies (#92-09) which links the U.S.Maglev program to the demonstrated technicalcapabilities at BNL in superconducting magnets.

Global Change: BNL has had several programs inatmospheric, oceanographic, and mathematical sci-ences, which produce results of relevance to ques-tions of global climate change and acid precipita-tion. LDRD proposals in these areas which movein new avenues are given priority as the Lab triesto make contributions to solution of these problems.One such program is that on calcification of marinealga (Project #93-10).

Radiation Therapies and Imaging: Applications ofthe Laboratory's facilities to the treatment of cancerand in imaging of the human body for diagnosticpurposes, including such techniques as non-invasivecoronary angiography, microplaner x-ray therapy,uranium neutron capture therapy and the use ofother imaging techniques, are a significant part ofthe LDRD effort.

Genetic Studies: This area of research at the Labo-ratory has produced many new ideas and applica-tions which exceed the capacity of DOE for sup-port, and which are too important to remain unde-veloped. LDRD funded projects in this area arevaried in subject matter covering many bio-medicaltopics.

New Directions for the Development and Utili-zation of BNL Facilities: High priority is as-signed to ideas for more efficient utilization andfor new directions for our major facilities. Ideasfor more useful sources of x-rays at the synchro-tron light source, for the utilization of newelectron sources in the chemical study of pulseradiolysis, and new laser systems are all areaswhich are given priority for support.

Other Miscellaneous: Finally, the program isopen to significant and original ideas which donot necessarily fall within the bounds of theaforementioned priority areas. These usuallyinvolve small individual program/projects thatare judged to have high scientific/technical mer-it. Typically, materials development, electro-chemical studies, advanced nuclear concepts andnew bio-chemistry technologies fall into thiscategory.

Brookhaven National Laboratory's FY 1993LDRD Program covered the following Scientificand Technical Areas:


New Directions for EnergyTechnologies

Global ChangeRadiation Therapies and ImagingGenetic StudiesNew Directions for the Development

and Utilization of BNL FacilitiesOther - Miscellaneous


$ 40



TOTAL $ 2,006

These dollars represented 28 individual pro-grams/projects covering all BNL scientific de-partments.

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A new malaria enzyme - a potentialsource for a new diagnostic test formalaria and a target for a newantimalarial drug

Effect of a bacterial spore protein onmutagenesis

Structure and function of adenoviruspenton base protein

High gain harmonic generationexperiment

BNL Maglev studies

Structural investigations of Pt-basedcatalysts

Studies on the cellular toxicity ofcocaine and cocaethylene

Human melonocyte transformation

Exploratory applications of x-raymicroscopy; determination of thehigher ordered structure of eukaryoticchromosomes

Uranium neutron capture therapy(UNCT)

FY 91


































































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Tunneling microscopy studies ofnanoscale structures

Nuclear techniques for study ofbiological channels

RF sources for accelerator physics(CAP)

An EBIS source of high charge stateions up to uranium

Design of a dedicated x-ray source forcoronary angiography

Calcification in marine alga: amolecular approach to a majorpathway of global carbon cycling

Structure-sensitive properties ofadvanced permanent magnetmaterials: experiment and theory

The structure and reactivity ofelectrode/electrolyte interfaces

A very high flux research reactor

Induction and repair of double-strandbreaks in the DNA of humanlymphocytes

Genetics of drug addiction

Cryogenic targets for the productionof PET isotopes

Development of microdialysistechnology
















































































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Investigation of the utility ofmaximum-entropy methods for theanalysis of powder for diffraction data

Accelerator technology

Analysis of structures and interactionsof nucleic acids and proteins by smallangle x-ray diffraction

Enhancement of microplanar radiationtherapy (MRT) of the intracerebral rat9L gliocarcoma by preinfusion with adiiodofluorecein-histone conjugate

Relaxographic MRI and functional MRI





































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Ground Coils

I S I I N TPropulsion


I ST~I 111 I H LJL


N i I S nr

A/.ew Directions for Energy Technologies

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Qn the previous page: A schematic representationof a high speed "Mag-Lev" train utilizing superconductingmagnets.

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NL Maglev Studies 92-09

J. Wegrzyn


ONL is a leader in superconducting magnet devel-opment for high energy physics. The program'sobjective is to further develop and apply this mag-net expertise to maglev technology. Maglev repre-sents a high speed ground transportation system thatuses superconducting magnets to levitate, guide and

propel vehicles over elevated guideways at speedsof 500 miles per hour. The intent of QllS WOTk ISto place BNL in a position to receive funds fortesting prototype superconducting magnets formaglev from the National Maglev Initiative (NMI).The NMI is composed of the Department of Trans-portation, the Department of Energy and the ArmyCorps of Engineers. The deliverable from thiswork is a comprehensive proposal to NMI thatestablishes BNL as an appropriate site for screeningand testing prototype magnets for maglev. Tasksperformed under this LORD program were datagathering, computer simulation and engineeringdesign studies. These studies formed the basis ofthe proposed BNL Maglev Magnet Test Stand. Aunique facility designed to test prototype maglevmagnets.


PURPOSE: The purpose of this program is toidentify directed research opportunities forBrookhaven within the emerging National MaglevProgram. Maglev offers the potential to relieveseveral major problems (traffic congestion, landshortages, pollution and energy use) related to thetransportation infrastructure in the United States.The Intermodal Surface Transportation EfficiencyAct of 1991 (ISTEA) provides the basis from whicha US-designed maglev system can be developed.The key question to be answered by ISTEA is: how

and where can maglev technology be showcasedto demonstrate its full potential? As an example,it is widely recognized that the necessary tech-nology for constructing low temperaturesuperconducting magnets and refrigerators formaglev exists today. This statement does notimply that there exists no need within the maglevprogram for strong engineering programs. Tothe contrary, the maglev program demands

reliable, lightweight, energy efficient andruggedjzed magnets antl cryogenic hardware ill&tcan meet the exact needs and specifications ofpublic transportation systems. These specifi-cations are obtainable only with an extensivefield testing program of prototype magnetics andtheir subsystems. This LDRD program outlinesa flexible, modest cost magnet test stand specifi-cally designed for screening the performance offull scale maglev magnets. The purpose of theBNL Magnet Test Stand is to assist the privatesector in their development of magnets and thegovernment sector in their evaluation of thesedesigns.

APPROACH: The design, evaluation and test-ing of maglev magnet systems grow in impor-tance as the Maglev Program moves forwardinto the hardware development phase. Thecurrent maglev test program calls for the devel-opment of persistent current, air coresuperconducting magnets that are reliable andsafe. These magnets are unique in that they areto operate under both fluctuating electromagneticand mechanical loads. With this LDRD we haveinvestigated the magnet stability problem as itrelates to maglev. In particular we are propos-ing to quantify and qualify the technical chal-lenges of building, testing and successfully oper-ating superconducting maglev magnets for bothEDS and EMS maglev designs.


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One benefit of the proposed magnet test stand is tolower the government's risk factor associated withmaglev development. Cost savings are realizedthrough using the magnet test stand to optimize thedesign/performance of maglev magnets prior to fullscale guideway tests. The hourly cost of laboratorytesting is always significantly less than conductingfull scale field tests. In addition to cost benefits thismaglev magnet test stand approach allows for:

• establishing base-line magnet design specs;

• strengthening government's expertise in relationto contractor's work;

• developing generic subassemblies such as pro-tective circuitry and magnet energizing schemes;

• measuring of EM stray fields, cryogenic heatloads and AC loss mechanisms;

• being able to quantify and qualify magnet's fail-ure modes.

TECHNICAL PROGRESS AND RESULTS- FISCAL YEAR 1993: Three dimensional magnet-ic/guideway reference models were developed as aguide in the design of the magnet test stand. Abrief description of the models and test stand fol-lows:

Stray EMF Model: Since magnetic fields are gen-erated in the neighborhood of circuits or loops thatcarry electric current, shielding strategies need tobe part of a maglev magnet design. The strengthsof these fields (magnetic induction) depend on thelocation, direction and magnitude of the current foreach contributing circuit. A FORTRAN programwas written to determine the magnetic induction forcircuits arranged in a maglev train configuration.An acceptable design has DC fields less than 3Gauss and AC fields less than 0.5 Gauss within thevehicle.

Mutual Inductance/Force Model: The force betweenthe two circuit loops can be obtained by multiplyingtogether their currents with the gradient of theirmutual inductance. A FORTRAN program waswritten to evaluate the mutual inductance and the

force components between two current loops.The mutual inductance between two parallelarbitrarily shaped loops was first obtained byintegrating the vector potential of the first loopalong the path of the second loop. The obtainedgeneral expression for mutual inductance degen-erated into the classical Neuman expression forthe case of two identical, lined-up rectangularloops and thereby verified the program.

Guideway Induced Current Model: When asuperconducting magnet moves over a guidewaycoil an induced current will occur in the trackloop or the guideway coil. This induced currentcan be calculated by solving a first order ordi-nary differential equation involving train-loopcurrent, resistance, self-inductance, and themutual inductance between the train and thetrack loop.

Because of the inductive coupling of the trackloops, simultaneous equations were solved toobtain the induced currents. For a series of dis-crete guideway coils, a FORTRAN program wasdeveloped to compute track currents correspond-ing to a unit train-loop current. The track cur-rents so obtained were then used to evaluate theforce between the train and the track as de-scribed earlier. The model can also be used toevaluate the contribution to the magnetic fielddue to these time-dependent currents.

Multiple Train Loop Model: For the case whenthe coupling between train magnets is small, themethod of superposition can be used to calculatemultiple train magnet/guideway interactions.With proper off-set and scaling, the mutual in-ductance between the train and the track loopswere determined for specified train-loop pitch,size, magnitude and direction of the train-loopcurrent. Induced currents and magnetic induc-tion were similarly computed as for the case of asingle train loop.

BNL Maglev Test Stand: In a functioningmaglev system the guideway coils are excited bythe passage (transit) of the superconducting mag-nets, which are housed in the vehicle. The coilsare passive and dissipate power only during the


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interval of time that the vehicle is above the coil.Essentially, the concept of the BNL Maglev MagnetTest Stand is to simulate the condition of "movingmagnet(s) over a passive guideway" with "fixedmagnet(s) placed over an active guideway". There-fore the testing of superconducting maglev magnetsrequires the development and construction of anactive, truncated guideway.

The active guideway is configured as a flat array often stationary but time-dependent current sources.These current loops are controlled by external pow-er supplies to simulate the conditions oftransversing superconducting magnets. The currentsin the active guideway have been predeterminedfrom computer studies which calculated the inducedcurrents as a function of train speed, magnetstrength, guideway configuration, etc. The teststand also requires an appropriate sized coolingsystem to avoid self-heating of the actively drivencoils. Both the guideway and the magnet/cryogenicsubsystem are instrumented to measure and tocontrol AC and mechanical vibrations, heat loads,magnetic field strengths and dynamic forces.

The test stand's major components are: 2.5 MWelectric power substation, 100KW AC/DC powersupplies, 160 ton chiller, work station, 50 KATguideway coils, superconducting test magnet, EMFshield and magnet suspension system. Since thetest stand's mission is to evaluate prototype maglevmagnets, the facility is designed to accommodateone to five Tesla superconducting magnets of one totwo meters in length with a lift force up to ten tons.The guideway coils consist of hollow soft copper(water cooled) conductors (5 cm x 5 cm) and canbe configured into any shape up to 8 meters inlength.


A presentation detailing the National MaglevInitiative, the Northeast activity in maglev andBNL's maglev role was given at the DOE (Bos-ton Area Office) New England Energy TaskForce (NEETF) meeting.

A Northeast Regional Maglev Forum was heldon Tuesday and Wednesday, Oct 8-9, 1992 inHearing Room C of the New York State Legisla-tive Office Building in Albany, N. Y. TheMaglev Regional Forum focused on coordinatingstate and local government activities on maglevand other forms of high-speed surface transpor-tation proposed for the Northeast region, as wellas discuss federal efforts to facilitate the devel-opment of maglev technology in the UnitedStates. New York State Lt. Gov. Stan Lundineopened the workshop.


A Field Work Proposal (FWP) is in place withthe Maglev Task Force on using BNL as a testsite for maglev magnets. Funding depends onwhether maglev is included in the FY'94 budget.




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Phaeodactylum tricornum0-12h 12-24h 24-36h 36-48h


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lobal Change

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n the previous page: Knowledge of the factors control-ling phytoplankton production is critical to understand theocean carbon cycle. Specific proteins expressed under iron,phosphorus, or nitrogen limitation are candidates formolecular markers that identify controlling environmentalvariables.

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aalcification in Marine Alga: A Molecular Approach to a• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Major Pathway of Global Carbon Cycling

93-10> • • • • • • • •

J. LaRoche


I h e oceanic reservoir of inorganic carbon is 50times that of the atmosphere and it exchanges withthe latter on timescales of months to hundreds ofyears. At the most basic level, the internal cyclingof carbon in the oceans is controlled by marineorganisms through the production and transport ofbiogenic carbon in the form of 'soft' parts (organictissue) and 'hard' parts (CaCO3 shells). The pro-duction of hard parts of biocalcification reducesalkalinity and increase pCO2 opposing to someextent the effect of organic carbon production (de-crease in pCO2 but no effect on alkalinity). Thecoccolithophorid Emiliania huxleii is the most abun-dant and most important organism producingCaCO3 in the oceans and this is due to its ability toform dense blooms in both coastal and open oceans.Based on the restricted information available fromgeological records, the biology ofcoccolithophorids, and current scenarios for globalwarming, it is reasonable to envision positive feed-back involving increased calcification that wouldtend to increase the accumulation of pCO2 in theatmosphere and the global warming potential. It isproposed to study the process of calcification in E.huxleii using molecular biological techniques tobetter understand which environmental factorsregulate the expression of this process. Calcifica-tion is important, yet poorly studied and a combina-tion of physiological, biochemical and molecularapproaches will provide a basic understanding ofthis process. The project is timely given the em-phasis of funding agencies towards the study ofglobally important problems and the use of molecu-lar techniques in environmental research.


PURPOSE: The aim of this work is to identifythe factors controlling calcification in thecoccolithophorid Emiliania huxleii. Previouswork on the physiology of this species indicatethat the ability to calcify has been irreversiblylost in some strains of E. huxleii. This suggeststhat the ability to calcify in this species is inpart controlled at the genetic level. E.huxleii has a complex life cycle and can be ob-served as a naked motile cell, a naked or calcify-ing non-motile cell. Several environmentalfactors such as, nutrient concentrations or lightare also believed to affect calcification rates.

APPROACH: Two strains of E. huxleii areused in the study of calcification. Strain CCMP373 is a non-calcifying strain isolated in I960.The calcifying strain, CCMP 374 is a recentisolate (1989) in which approximately 50% ofthe carbon fixed goes into the production ofCaCO3. A comparison of the two strains at thephysiological, biochemical and molecular levelshould give some indication of the metabolicpathways and enzymes involved in calcification.

TECHNICAL PROGRESS AND RESULTS:The two E. huxleii strains (CCMP 373 andCCMP 374 have been obtained from theBigelow culture collection and are currentlygrown successfully in my laboratory. Cultureconditions and media preparation have beenoptimized to ensure good yield of cells in 8 literculture vessels. The cells are routinely countedusing a light microscope which contain lightpolarizing filters which allow the enumeration ofcalcifying cells. The comparison of the twostrains is in progress. Polyacrylamide gel elec-


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trophoresis has been used to compare total protein An analytical system to measure alkalinity inprofiles and western blots have confirmed the simi- cultures of E. huxleii is currently being assem-larity between the major photosynthetic proteins bled and will be used to routinely measure calci-such as the light harvesting chlorophyll binding fication rates in cultures,proteins, rubisco and Dl in the two E. huxleiistrains. DNA and RNA has been isolated from the LDRD FUNDING:two algal strains and the randomly amplified poly-morphic DNA Fingerprinting (RAPD) techniquebased on the polymerase chain reaction is beingdeveloped to assess the similarity of the genome ofthese two strains. A DNA library of the calcifyingstrain is under construction for differential screen-ing in an attempt to isolate genes involved in theprocess of calcification.



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adiation Therapies and Imaging

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Qn the previous page: Scanning transmission electronmicroscope (STEM) micrograph of uranium-loadedapoferritin. Apoferritin is a natural blood protein with aspherical shell that normally carries iron in its centralcavity. Here it has been loaded with ~800 uranium atomswhich appear as a dense (bright) core surrounded by thelower density (grey) apoferritin protein shell. Seven suchloaded molecules are seen in this microghaph. Full width128 nm.

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tudies on the Cellular Toxicity 92-15• • • • • • • C B

N.D. Volkowof Cocaine and Cocaethylene


l h i s project seeks to measure the effects of co-caine, cocaethylene, alcohol, and alcohol withcocaine on elemental distribution in the cells of theleech ganglia (neurons and glial cells). We hypoth-esize that the effects of cocaine and cocaethyleneare equipotent in their ability to block Na+ con-ductance into the cell and that the combination withalcohol will, in addition, disturb the intracellularconcentration of Cl" and Ca+ + . This projectutilizes electron probe x-ray microanalysis (EPMA)to measure the effects of various doses of cocaine,cocaethylene and alcohol as well as these drugs incombination on cellular elemental distribution in theleech ganglia model.


PURPOSE: The purpose of this study is to investi-gate the contribution of the local anesthetic proper-ties of cocaine to its toxic effects when given aloneor in combination with alcohol. Since local anes-thetic actions of cocaine lead to changes in cellularion concentration through interaction with Na+ andK+ channels, we plan to measure these effectsusing electron probe x-ray microanalysis. Thoughmost of the pharmacological actions of cocaine havebeen related to its sympathomimetic effects, theacute lethality from cocaine has been related to itsanesthetic properties. It has also been shown thatthe combined use of cocaine and alcohol markedlyincreases the lethality of cocaine by eighteen fold.It has been postulated that the increase in toxicity isdue to the production of cocaethylene, a metaboliteformed by the interaction of cocaine and alcohol.We propose, however, that the increased toxicity isbrought about by the differential interference with

ion exchange across membranes by alcohol andcocaine.

APPROACH: Initially we investigated the ef-fects of cocaine and cocaethylene on the rate ofuptake of Rb as a K marker into leech glial cellsand neurons. The leech was chosen as an exper-imental model because its nervous system isrelatively simple and has been extensively stud-ied. Furthermore, it has large easily recognizedneurons and distinctive glial cells making thispreparation ideal for EPMA studies. In ourinitial studies we looked for changes in Rb up-take velocity into neurons and glial cells. Sinceleech glial cells are passive accumulators of K, ifcocaine or cocaethylene affected the rate ofuptake of Rb we could have concluded that thesedrugs might affect the neuron-glial homeostaticrelationship by altering glial K buffering proper-ties. These initial studies however failed todemonstrate any significant effect on Rb uptake,although we observed some curious and incon-sistent effects on Na.

We then began studies to determine if elementaldistribution was altered in neurons and glial cellsin response to dose. Since any effects werelikely to be over an extend time period, wechose to examine the effects of cocaine andcocaethylene dose on elemental distribution after20 min. exposure to these drugs over the rangeof 10 6, 10"5, and 10"4M. Using EPMA offrozen hydrated tissue sections we measured Na,K, Cl, S, P, Ca, Mg and H2O content of leechneurons and giial cells.

TECHNICAL PROGRESS AND RESULTS:As mentioned above, we did not observe aneffect of cocaine or cocaethylene on Rb uptakein neurons or glial cells. Initially, it appearedthat there was a dose dependent effect of cocaineon glial Na but not on neuron Na. Over the


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dosage range studies, cocaine decreased glial Na ina dose dependent fashion after an initial and unex-plained increase over control by the lowest dose;neuron Na remained unchanged except when ex-posed to the highest dose (10~4 M cocaine). Underthose conditions neuron Na decreased nearly 30%(Fig. 1). Interestingly glial Cl content was notaffected by cocaine (Fig.2). Thus, initially, theaffects of cocaine on glial elemental content appearto be nearly entirely on Na. Neuron Cl on theother hand decreased in proportion to the Na de-crease. Water content and elemental content, inparticular K (Fig.3) of leech neurons and glial cells,with the exception of Na and Cl (neuron) was notaffected by cocaine (Figs.l & 2). However, aftercompleting the initial microprobe studies, it appearsthat these affects are likely to be minor and/orsecondary to other affects.

Over the past year we have completed the analysisof cocaethylene effects. These experiments showeda marked effect of cocaethylene on glial, but notneurons, Na and Cl . We presume that the effectcould be primarily on one of these elements(ions) while the other follows passively. Becauseof these surprising findings we extended our studiesto include a dose response series looking at theeffects of cocaine (10~6 to 10~4M) in combinationwith 200 mmol alcohol (Figs. 1-3). This concentra-tion of alcohol was chosen as a typical mid-rangeconcentration. In combination, there appeared tobe little effect on either neuron or glial elementalcontent. Thus in summarizing our results it appearsthat there is actually little if any effect of cocaine orcocaine and alcohol on neuron or glial elementaldistribution. The major effect on elemental distri-bution is associated with cocaethylene (a metaboliteof alcohol and cocaine) on Na and Cl in glial cellbut not neurons.

Because of these findings we have begun someexploratory studies using a patch clamp techniqueto determine if cocaethylene can cause changes onNa and Cl channel function in BC3H-1 mousetumor cells. These preliminary patch clamp studiesare currently underway.


These data suggest that cocaethylene, a metabo-lite of cocaine and alcohol has a significanteffect on Na and Cl distribution in glial cells butnot neurons. Cocaine, cocaine and alcohol, andalcohol alone does not effect elemental distribu-tion in any significant way over the dosage rangetested in either neuron or glial cell. The factthat cocaethylene produced a significant effect onthe CNS elemental distribution is consistent withits know toxicity. This toxicity may be differentthan either the pharmacological properties pro-ducing a toxic response in vivo of cocaine, alco-hol or cocaine and alcohol. That k the effectscocaethylene on a cellular level rray be unrelatedto either the excitatory, depressant, or Na chan-nel blocking (anesthetic) properties of cocaineand alcohol alone or in combination. At thispoint we are uncertain how our observed find-ings relate to the behavioral and physiologicalCNS toxicity effects of cocaethylene. How, forexample does a specific glial effect impair CNSfunction to produce excitability and seizures?There is a growing interest in the neuro-glialrelationship. Our findings are certainly consis-tent with the possibility that cocaethylene CNStoxicity may relate to this interaction. To devel-op a plausible hypothesis we have extended ourstudy to investigate whether or not cocaethylenecan affect channel function. If so then we canhypothesize that our findings are a measure ofchannel function. If this is true then we canconsider as an heuristic model that the CNStoxicity of cocaethylene is caused by interruptionof the nuero/glial relationship. We might alsobe able to develop an hypothesis explaining howthis interruption is caused based upon the cellu-lar mechanism of glial maintenance of elemental(ion) distribution in the CNS. The ramificationsof the elemental changes induced in glial cells bycocaethylene may be significant in affecting glialmetabolism and their homeostatic function in theCNS. We believe that these results may signalearly evidence of a toxic effect on the neuro/glialphysiological relationship.


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No publications have, as yet, resulted from thiswork. However we are encouraged by the interest-ing effects of cocaethylene on glial cells and wefeel that this is new information which may havesignificance to both the action of local anesthetics ingeneral and to the specific problem of cocainetoxicity. We anticipate completing the preliminarypatch clamp studies within a few weeks, and plan todetermine what additional and further studies areindicated at that time to permit us to develop aheuristic model from which reasonable and testablehypothesis can be formulated.

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We anticipate developing a formal NIH proposalupon successful completion of these initial pilotstudies.




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These Figures were obtained from Dr. A.Saubermann (SUNY - Stony Brook)

U ranium Neutron Capture Therapy (UNCT) 92-20

J.F. Hainfeld


Uranium-235 appears to have potential advantagesover boron-10 in neutron capture therapy. Itsfission products have a greater range in tissue,11-18 mm vs. 5-9 mm (for 10B), the energy of thefission products is greater (200 MeV vs. 2.8 MeV),but its neutron capture cross section is 6.6 timesless. Calculations indicate that 235U should be ~ 3times as effective as 10B. For an anti-tumorantibody-directed approach, ~ 300 uranium atoms arerequired to be conjugated to each antibody to deliv-er a therapeutic dose. A recent breakthrough at

BNL has achieved attachment of ~ 800 uraniumatoms per antibody with maintenance of immu-noreactivity. This project will carry this workfurther by developing a uranium assay systemand using it to determine the relative biologicaleffect (RBE) or effectiveness of U-235 fission oncell killing compared to BNCT or toX-radiation. We will also test the in vitrobinding of the uranium -loaded antibody to targethuman tumor cells versus non-tumor cells.Finally, mice with human tumor implants will beinjected with the 238U-antibody complex to deter-mine the uptake of uranium into the tumor andinto other tissues. These studies will explore the


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feasibility of this approach to uranium neutroncapture therapy (UNCT). This proposed workcould lead to significant improvements in clinicalradiation therapy, particularly for central nervoussystem neoplasms.


PURPOSE: The purpose of this work is to evalu-ate the apparent advantage and feasibility of UNCT.More specifically, an antibody-directed approachwill be investigated with our unique method ofattaching — 800 uranium atoms to each antibody. Alist of specific objectives is:

1. Develop analytical methods to accurately mea-sure U-238 and U-235 at the low levels ofconcentration necessary for the various studies.

2. Determine the RBE of U-235 fission products.

3. Improve the stability of the uranium-antibodyconjugate.

4. Test the cell binding of the uranium-antibodyconjugate to representative human tumor andnon-tumor cell lines.

5. Determine the in vivo biodistribution of theuranium-antibody conjugate in nude mice withhuman tumor xenografts.

APPROACH: Boron neutron capture therapy(BNCT) has been investigated extensively over thepast 35 years with recent dramatic success in theuse of Na4B24H22S2 to treat transplanted intracranialgliosarcomas in rats (Joel, et al., 1990) and in theuse of p-boronophenylalanine (BPA) to treat mela-nomas (Coderre, et al., 1990). BPA-mediatedBNCT is now undergoing Phase I clinical trials.Although BNCT holds much promise, there may beserious problems in adapting it to antibody-directedradiation therapy: 1)"'B upon neutron irradiationproduces an alpha particle and 7Li. The effectiveranges of these particles are about one cell diame-ter, so that multicell carcinomas may not be ade-quately irradiated since antibodies do not uniformlyaccess all tumor cells. Cells are typically at amaximum distance of 25-50 mm from the nearestcapiilary (intercapiliary distance is 50-100 mm) and

the 5-9 mm range of "'B products is seen as adisadvantage. Transient vascular permeability toantibodies may, however, be enhanced by irradi-ation of tissue with ~ 1000 rad ( - 10 Gy) ofsubtherapeutic photon irradiation. 2) If antibodytargeting of boron is used (Mizusawa et al.,1982; Alan et al., 1989; Barth et al., 1991), it iscalculated that ~ 1,000 boron atoms or more needto be coupled to each antibody molecule for aneffective tumor dose at neutron fluences readilyachievable in deep-seated (6-8 cm) human tu-mors (Fairchild and Bond, 1985). This has beenimpossible to achieve thus far, despite manyefforts.

The potential usefulness of 2"U implanted into atumor for radiation therapy by slow neutronswas first recognized 50 years ago (Zahl andCooper, 1941). Since then, only a few experi-mental studies (McClintock and Friedman, 1945;Tobias et al., 1948; Steinfield, 1952, Knock,1959) relating to uranium neutron capture thera-py (UNCT) have been reported, and none ofthese was pursued very far. Two of these stud-ies (McClintock and Friedman, 1945; Knock,1959) stressed the importance of linking uraniumto tumor specific antibodies. Although work onUNCT was not continued, BNCT has beenpursued with ever increasing vigor since 1940(Zahl et al., 1940; Slatkin, 1990).

Uranium-235 fissions under neutron irradiationproducing an energy of 200 MeV. The advan-tages of 235U over 1('B are: 1) Each 235U atommay be ~ 3 times as effective as '"B: the thermalneutron cross section is 6.6 times less for MSU,but the energy released is 71.4 times greater. 2)The high energy produced from 215U should havean effective range of about 11 to 18 mm, com-pared to 5 to 9 mm for "'B, thus providing betterdose delivery to solid tumors, especially whencombined with preirradiation, hyperthermia, orother physico-chemical treatments that enhancepermeability of tumor blood vessels tomacromolecules.

Our microdosimetric calculations suggest thatUNCT, unlike BNCT, should be ideally suitedto radiotherapy of malignant tumors using


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tissue-specific monoclonal antibodies. The reasonsUNCT has not been pursued are probably: 1) toxic-ity of compounds and 2) inadequate attachmentmethods to antibodies. Since 235U may be ~ 3 timesmore effective than 10B, it is expected that ~ 300 Uatoms attached to each antibody will be adequate.This is still a formidable task. The focus on use ofantibodies to target to tumors is attractive comparedto simple compounds since higher tumor tonon-tumor (blood, liver, kidney, spleen, stomach,bone, etc.) ratios, e.g., 5 to 50:1, have beenachieved in athymic mice with human tumorxenografts.


Alan, A., Soloway, A.H., Barth, R.F., Mafune,N., Adams, D.M., Knoth, W.H. Boron neutroncapture therapy: Linkage of boronated macromole-cule to monoclonal antibodies directed againsttumor associated antigens. J. Med. Chem. 32,2326-2330(1989).

Barth, R.F., Soloway, A.H., Adams, D.M., Alam,F. Delivery of boron-10 for neutron capture therapyby means of monoclonal antibody-starburstdendrimer immunoconjugates. Jn Proc. FourthInt. Symp. on Neutron Capture Therapy. PlenumPress, in press (1991).

Coderre, J.A., Glass, J.D., Fairchild, R.G., Micca,P.L., Fand, I., Joel, D.D. Selective delivery ofboron by the melanin precursor analoguep-boronophenylalanine to tumors other than mela-noma. Cancer Res. 50, 138-141 (1990).

Diimanian, F.A., Garrett, R.F., Thomlinson,W.C., et al. Computed tomography with mono-chromatic X-rays from the National SynchrotronLight Source. Nucl. Instr. and Meth. B56/57.1208-1213(1991).

Fairchild, R.G. and Bond, V.P. Current status of10B-neutron capture therapy: Enhancement oftumor dose via beam filtration and dose rate, andthe effects of these parameters on minimum boroncontent: A theoretical evaluation. Int. J. RadiationOncology Biol. Phys. J_L, 831-840 (1985).

Joel, D.D., Fairchild, R.G., Laissue, J.A.,Saraf, S.K., Kalef-Ezra, J.A., and Slatkin, D.N.Boron neutron capture therapy of intracerebralrat gliosarcomas. Proc. Nat. Acad. Sci. 87,9808-9812 (1990).

Hainfeld, J.F., Foley, C.J., Srivastava, S.C.,Mausner, L.F., Feng, N.I., Meinken, G.E..Seplevski, Z. Radioactive gold clusterimmunuconjugates: Potential agents for cancertherapy. Nucl. Med. Biol., J7, 2987-294(1990).

Knock, F.E. Perfusion of uranium-antibodycomplexes for the neutron captive therapy oftumors. Surgery, Gynecology and Obstetrics.109. 445-449 (1959).

Mizusawa, E., Dahlman, H.L., Bennett, S.J.,Goldenberg, D.M., Hawthorne, M.F. Neutroncapture therapy of human cancer: In vivo re-sults on the preparation of boron-labeled antibod-ies to carcinoembryonic antigen. Proc. Nat.Acad. Sci. USA. 79, 3011-3014 (1982).

McClintock, L.A. and M.M. Friedman. Utiliza-tion of antibody for the localization of metalsand dyes in the tissues: Preliminary report. Am.J. Roentgenol. 54, 704-706 (1945).

Slatkin, D.N. Boron neutron-capture therapy.Neutron News I , 25-28 (1990).

Slatkin, D.N. A history of boronneutron-capture therapy of brain tumors: Postu-lation of a brain radiation dose tolerance limit.Brain JJ4, 5 609-1629 (1991).

Steinfield, W. Preparation of uranium arsphena-mine, a possible capture compound. In Quarter-ly Progress Report Oct. 1-Dec. 31, 1952.Brookhaven National Laboratory. BNL 2J9(S-16), p.5l (Abstract). (1952).

Tobias, C.A., Weymouth. P.P., Wasserman.L.R., Stapleton, G E. Some biological effectsdue to nuclear fission. Science 107 115-118(1948).


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Zahl, P.A., Cooper, F.S., Dunning, J.R. Some invivo effects of localized nuclear disintegrationproducts on a transplantable mouse sarcoma. Proc.Nat. Acad. Sci. USA 26, 589-598 (1940).

Zahl, P.A. and Cooper, F.S. Physical and biologi-cal considerations in the use of slow neutrons forcancer therapy. Radiology 37, 673-682 (1941).

Long-term objectives once this work is complet-ed. Since this phase can only assess the prelimi-nary feasibility of this approach for therapy, furtherwork, including clinical trials, is required to com-plete appropriate testing. Ultimately, a methodolo-gy for routine clinical use must be developed.

The overall strategy to carrying out the projectwas:

1. Devise a method to attach — 300 uraniumatoms per antibody; conjugate should retainimmunoreactivity.

2. Develop methods to accurately measureU-238 and U-235 suitable for theproposed experiments.

3. Measure RBE of U-235.

4. Test in vitro cell binding ofuranium-antibody conjugate. The bindingof the uranium-antibody conjugate to tumorand non-tumor cells will be measured.

5. Find the biodistribution of the conjugatewhen injected into mice.

6. Test the targeting of uranium in vivo innude mice with human tumors.

7. Perform dosimetry calculations and theo-retical simulations to determine the expect-ed outcome of this form of uranium admin-istration.


1. 800 uranium atoms were attached to antibodies;immunoreactivity was maintained. A methodwas devised using a protein carrier (apoferritin)

to encase the uranium which was crystallizedwithin. Antibody fragments were attached(Fig. 1) and the final conjugate was found tobind nearly to the same extent as native anti-body to its target antigen.

Fig 1 Schematic drawing ol apoterntin loaded with 800 uraniumatoms m its central cavity Fab1 antibody fragments are covalerVyattached to the apoferntm protein shell to target the uranium totumors

2. The stability of the uranium conjugate wasimproved. It was found that the uraniumslowly "leaked" out of the apoferritin carrierunder in vivo conditions (serum, 37°C).Various chemical treatments were exploredto prevent this and one gave exceptionalstability with no uranium loss over a 3 dayperiod. Dr. F. Furuya proposed the stabili-zation method ultimately used.

3. Uranium measurement methods were devel-oped. For this project it will be necessary tomeasure uranium at the 1-100 ppb (parts perbillion) level. Six methods were testedincluding chemical colorometric and electronmicroscopic analysis. Two of the mostsensitive bulk methods were pursued: Neu-tron activation and Inductively CoupledPlasma Mass Spectrometry (ICPMS). Thesegave sensitivities of ~ 1 ppb (parts per billion,i.e., 1 mg U per 1 g of matrix material) forU-238; U-235 was detected by ICPMS at

— lppb or by direct gamma counting (—10 ppb).

4. Preliminary RBE measurements for U-235fission products. Experiments were carriedout at the thermal neutron beam of theBrookhaven Medical Research Reactor(BMRR) to measure the relative biologicaleffectiveness (RBE) of the reaction products


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from the neutron-induced fission of 235U in cellcultures. The uranium compound used in theexperiments was prepared by first dissolving-93%-enriched 235U metal in nitric acid, thenadding ammonium hydroxide and oxalic acid tothe solution. Two different cell lines werestudied: Chinese Hamster V79 cells and 9L ratgliosarcoma cells. The experiments were de-signed to follow the procedures for the RBEmeasurements of IOB neutron capture, used byGabel et al. (1984) and Laster et al. (1991) forV79 cells, and Coderre et al. (1993) for 9L ratgliosarcoma cells.

The 235U concentrations used in the experimentswere chosen to provide approximately the samephysical dose as in the boron experiments. Theseconcentrations were approximately 10, 20, and 40mg 235U/ml of the growth medium for the V79cells, and approximately 10, 25, 40, and 50 mg23SU/ml of the growth medium for the 9L ratgliosarcoma cells. The preliminary results with theV79 cells indicate that the RBE values for the fis-sion products obtained with the 20 mg 235U/mlconcentration are at least 40% higher than those forthe boron neutron capture reaction measured at the30 mg 10B/ml concentration when compared to137Cs gamma rays using an end point of 10% cellsurvival. Data analysis of 9L gliosarcoma cellexperiments is in progress. Cell culture experi-ments were conducted in collaboration with J.A.Coderre, M.S. Makar, B.H. Laster, C.R. Gordon,and L.S. Warkentien.


1. Gabel D., Fairchild R.G., Borner H.G.,Larsson B. The relative biological effectivenessin V79 Chinese Hamster cells of the neutroncapture reaction in boron and nitrogen. Radiat.Res. 98:307-316, 1984.

2. Laster B.H., Kahl S.B., Popenoe E.A., PateD.W., and Fairchild R.G. Biological efficacyof boronated low-density lipoprotein for boronneutron capture therapy as measured in cellculture. Cancer Res. 51:4588-4593, 1991.

3. Coderre J.A., Makar M.S., Micca P.L.,Nawrocky M.M., Liu H.B., Joel D.D., Slatkin


D.N., and Amols H.I. Derivations of rela-tive biological effectiveness for the high-LETradiations produced during boron neutroncapture irradiations of the 9L rat gliosarcomain vitro and in vivo. Submitted to Int. J.Rad. One. Biol. Phys. for publication.

Cell binding of uranium-antibody conjugateto tumor and non-tumor cells. U-238 wasloaded into the apoferritin carrier, and CO17-1A monoclonal antibody to human coloncarcinoma cells was attached. Thisimmunoconjugate was incubated with humantumor cells and non-tumor cells. Afterextensive washing, the cells were dissolvedin acid and the uranium content measured byICPMS. Typical results are shown in Fig.2. The tumor to non-tumor ratio achievedfor this experiment was 23.

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5. Preliminary in vivo biodistributions in micewith human tumors. Uranium(238)-antibody conjugates were prepared andinjected into nude mice with human tumorimplants. Three such experiments wereconducted over the course of this project.After various time periods (24 or 96 hrs),the animals were euthanized and necropsywas performed. The various organs andtissues were dissolved and analyzed foruranium content by ICPMS. All of theseruns were done before in vivo stability of theuranium carrier was accomplished. Thismeant that the uranium most likely becameseparated from the antibody and would not


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be diiected to the tumor. In fact, no tumortargeting specificity was found. The bulk ofthe uranium localized in the liver and spleen, ascould be expected if it was not stabilized orwell attached to the antibody. The injectedamount was 4 mg of U-238 per mouse and typi-cal biodistribution values were (after 24 hrs):


liverspleenfemurkidneyfecal materiallungsstomach and itumorheartmusclebloodbrain

Run 1amount(ng/gm)


intestine 1187621

Run 2amount(ng/gm)



Run 3amount(ng/gm)


not detectable

not detectable

Although these experiments need to be repeatedwith the stabilized uranium, it is interesting that thelevels in the blood and brain are very low andshould contribute low background for NCT of braintumors.

Principal Collaborators: D. N. Slatkin, F. A.Dilmanian, R. Ma, G. Harbottle, M. Lin.

All of the above work was done with the excel-lent technical help of K. Carbone.


Publication:Hainfeld, J. F. Uranium-loaded apoferritin withantibodies attached: Molecular design for urani-um neutron capture therapy. Proc. Natl. Acad.Sci., 89, 11064-11068(1992).

Patent:Submitted to US Patent Office: AUI 91-05, J.F.Hainfeld, "Loading and Conjugating CavityBiostructures".


Proposals for follow-on funding:"Uranium Neutron Capture Therapy for Cancer"submitted to both N.I.H. and D.O.E.


FY 1991FY 1992FY 1993FY 1994

- 0 -97,021

103,952- 0 -

NOTE: This project involves animal verte-brates.

esign of a Dedicated X-Ray Source• • • • • • • • • • • • • • • • • • • • • • • • • • • i

for Coronary Angiography


L. Blumberg and E.B. Blum


W e propose to study the feasibility of building acompact source of 33 KeV x-rays for digital sub-traction coronary angiography. Previous experi-

ments at Stanford Synchrotron Radiation Labora-tory and at the X17 SMERF facility at NSLShave successfully demonstrated the technique ofdigital subtraction angiography using x-rays withan energy near the iodine K-absorption edge for


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imaging the coronary arteries but have been con-ducted on large research synchrotron light sources.We will examine whether a source that is smallenough to be used in an urban medical center canbe built. Such a device can be an inexpensive clini-cal alternative to present methods of non-invasivedigital subtraction angiography, small enough to beemployed in an urban medical center, and couldhave other medical, industrial, and aerospace appli-cations.

The obvious source of tunable, 33 KeV x-rays issynchrotron radiation. Obtaining enough photons tobe clinically useful, requires a combination of astorage ring with an energy near 1.5 GeV and ahigh-field wiggler magnet. The storage ring isbased on proven technology; the wiggler will re-quire considerable development. In this study wewill explore optimizing the design of an electronstorage ring and a companion wiggler to see if theycould be deployed in a hospital environment.

The second component of the study will be anexamination of using Compton back scattering ofinfrared photons from an electron beam to obtain33 KeV x-rays. Compared to synchrotron radiation,much lower energy electron beams are needed toproduce the x-rays. Storage rings have the advan-tage of higher average electron currents than linearcolliders but, since they recirculate the electronbeam after the collision with the infrared photons,they are less tolerant of any beam disturbancecaused by the electron-photon interaction. A prelim-inary estimate shows that we can obtain ten timesgreater photon flux from inverse Compton scatter-ing than is achieved at SMERF but with a largerenergy spread (2.2 KeV compared to 70 eV). Theestimate assumed a storage ring based on the SXLSPhase 1 prototype machine with a 0.5 Amp, 136.1MeV electron beam and a 100 mj pulse of 10.6 fimradiation from an infrared FEL. This part of thework will (1) study the disruption of the electronbeam by the photon-electron collisions, (2) explorethe feasibility of the required high power infraredFEL, and (3) develop preliminary parameters forthe design of an appropriate storage ring and injec-tor. After comparing the synchrotron radiation andinverse Compton scattering sources we will selectthe best approach for further study and prepare a

report to be submitted to a funding agency, suchas the DOE, to solicit funds to carry out a de-tailed conceptual design report.


PURPOSE: One of the most important tech-niques used to diagnose heart disease is coronaryangiography. It is performed using x-rays from aconventional source to follow the flow of acontrast agent containing iodine through thecoronary arteries. The contrast agent is intro-duced by direct injection from a catheter in thecoronary arteries that was threaded through thefemoral artery all the way from the groin to theheart. Coronary angiography is only used whenit is absolutely essential because of the risk offatalities and other serious complications arisingfrom the insertion of the catheter. The techniquealso exposes the patients to large amounts of x-rays.

Research, begun at SSRL [E. Rubenstein, et. al..Nucl. Inst. and Meth. A291 (1990) 80] andcontinued on the X17 beam line at NSLS [W.Thomlinson, et. al.. Rev. Sci. Instrum. 63(1992) 625], demonstrated the feasibility ofimaging human coronary arteries followingvenous injection of the contrast agent. Thetechnique, called digital subtraction angiography(DSA), uses two monochromatic beams of x-rays, one slightly above and one slightly belowthe iodine K absorption edge (33.169 KeV) tocollect simultaneous images. When the twoimages are subtracted, the contrast agent, con-tained primarily in the blood vessels, is revealedand the background that is common to bothimages is suppressed. The images must be col-lected during a single heartbeat to avoid blurringfrom motion of the blood vessels. Conventionalx-ray sources are too weak to provide the intenseflux that is required in the narrow energy band-width of the beams. Only the most powerfulsynchrotron radiation beams from wiggler mag-net sources can provide the intensity required inthe short exposure time.


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Although DSA experiments have shown promise,they have been conducted at large, research syn-chrotron radiation facilities. A small, dedicatedsource will be needed before DSA can be used as astandard medical procedure. The source must pro-vide the intense beam characteristic of synchrotronradiation, but must be small and inexpensiveenough to fit in a hospital.

APPROACH: The most obvious source of radia-tion for DSA is a small storage ring with a strongwiggler magnet. Another possibility, suggested bythe successful LEGS experiment at the NSLS[A.M. Sandorfi, et. al., IEEE Trans. Nucl. Sci,NS-30 (1983) 3083] is Compton back-scattering ofinfrared photons from an electron beam. Thestorage ring based source uses well known technol-ogy but may be too expensive for the medicalcommunity to accept. Compton back-scattering canpotentially provide a smaller source but requires aninfrared photon beam that is more intense than canbe provided by existing technology. We haveexamined storage ring and Compton back-scatteringsources to see if either appears to be suitable for ahospital-based DSA facility.

TECHNICAL PROGRESS AND RESULTS: Twoapproaches for production of narrow-band x-raybeams at the K-edge of Iodine (33.17 KeV) werestudied: Compton back-scattering of infrared pho-tons from an FEL on a storage ring electron beamand synchrotron radiation from a high field wigglerin a storage ring. For Compton scattering the basickinematics and cross-section, together with intensityestimates of x-rays from 10.6 pm photons scatteredfrom 136 MeV electrons into a 2 mrad cone, weresummarized in BNL-47503 (May, 1992). Thisbeam had a large kinematic energy spread (2.2KeV) compared to the 125 eV presently used fordigital subtraction angiography at X-17 and wouldpresumably require monochromatizing - perhapsusing suitably tuned multilayer mirrors. Anothermodel using lower energy (75 MeV) electrons andhigher energy (A=3.2 jim) photons was proposed inBNL-40816 (Nov. 1992) giving a smaller variationof x-ray energy with angle. However, the energyspread here was also large (2.6 KeV) to attain aclinically useful spot size of 12-cm diameter at 30-m from the source. In both cases the computed x-

ray intensity per 0.5 x 0.5 mm pixel for a 4-msec exposure is comparable to that presentlyattained at X-17. However, the design assumedan FEL oscillator within the storage ring and thephoton beam power required from the FEL of400 KW greatly exceeds the Renieri Limit attain-able from an in-ring FEL.

A more attractive approach (NSLS Seminar,May 28, 1992, BNL report to be issued) is alinac-driven FEL outside the storage ring in aring-oscillator configuration which would scatter100-micron photons from a 418 MeV electronbeam. The infrared photon beam power requiredhere is a more modest 57 KW. We are alsoaddressing the problem of excessive dose deliv-ered to the patient per image ( 5.5-rad) at 33KeV; Pharmaceuticals containing higher Z con-trast elements such as Gd (K-edge = 50.2 KeV),Ho (K-edge = 55.6 KeV) and Yb (K-edge= 61.3KeV) are now available and will result in afactor of about 3 smaller absorption coefficientin tissue. The required storage ring energywould increase to 568 MeV for Yb for the exter-nal 100 fim FEL discussed above. A compact(I3-m circumference) ring could still be realizedat this energy. Dose reduction is one of theessential considerations if we are to address therequirements stated in the recent (Dec. 11, 1992)NIH RFA on coronary angiography x-ray sourc-es.

A second approach utilizing a wiggler sourceand a line beam scanned through the patient aspresently used at X-17 was also pursued [BNL-49524, Sept. 1993]. An 8 Tesla superconductingwiggler was designed and it was shown thatadequate intensity can be attained with a storagering of 1.31 GeV electron energy. For higher Zcontrast agents this energy requirement wouldnot increase significantly since synchrotronradiation power at fixed magnetic field is pro-portional to cube of the energy. Such a ring canalso be relatively compact (50-m circumferencesuch as the commercially available machine builtby Maxwell-Brobeck for LSU).


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1. L.N. Blumberg, "Conceptual Design Study ofan Intense X-Ray Source for Coronary Angiog-raphy", BNL-47356 (March, 1992).

2. L.N. Biumberg, "Kinematics of Compton Back-Scattering X-Ray Source for Angiography",BNL-47503 (May, 1992).

3. L.N. Blumberg, "Compton Back-Scattering X-Ray Source for Coronary Angiography", BNL-40816 (November, 1992), presented at the 12th

International Conference on Applications ofAccelerators in Research and Industry, Denton,TX. Nov. 2-5, 1992.

4. L.N. Blumberg and E. Blum, "Simulation ofEmittance Dilution in Electron Storage Ring

from Compton Back-Scattering", BNL-49166(May, 1993), presented at the IEEE 1993 Parti-cle Accelerator Conference, Washington, DC,May 17-20, 1993, proceedings to be published.

5. E. Blum, "Conceptual Design of an 8 TeslaSuperconducting Wiggler for a DedicatedDigital Subtraction Angiography Source",BNL-49524, Sept. 1993.

6. E. Blum, "A Storage Ring Based InverseCompton Scattering Angiography Source?",BNL report to be issued.




$ - 0 -50,000

- 0 -

aryogenic Targets for the Production of PET Isotopes 93-23! • • • • • • •D. Schlyer


l h e r e is currently a critical need for a reliablesource of fluorine-18 for clinical diagnostic proce-dures for brain tumors, heart disease and epilepsy.During the past two years, enriched oxygen-18water (the traditional target material for productionof fluorine-18) has not been available. The purposeof this proposal is to explore an alternative to theenriched water target. More specifically, we willdesign, build and test cyclotron targets which utilizeisotopically enriched gases which are frozen into aconical target maintained at liquid nitrogen tempera-tures. A conical target is used to minimize theamount of enriched isotope which must be used andmaximize the area for heat dissipation. A diagnos-tic target will be built in order to test theoreticalcalculations which suggest that it is possible to usepower densities much higher than those currently inuse. The physical form and density of the solidlayer will be determined in order to evaluate heat

transfer properties. All the equipment is inplace and the money required will be for thefabrication of the diagnostic target and severalprototype targets. This type of cryogenic targetis predicted to reduce the cost of fluorine-18production by a factor of more than four andsignificant! / reduce the loss of enriched isotope.


PURPOSE: The purpose of this proposal is tobuild and test a series of cyclotron targets whichutilize cryogenically cooled enriched isotopegases for the production of PET radioisotopes.The target bodies will be conical in shape andsuspended in liquid nitrogen. The advantage ofthis design is that the recovery of the enrichedisotopes is extremely efficient and easy to use.


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APPROACH: The target designs will be conical inshape in order to increase the effective depth of thetarget material. The targets will be constructed ofcopper or silver to increase heat transfer and sub-merged in liquid nitrogen for cooling. The parame-ters to be studied will be the incline of the cone,target material thickness, number and spacing offins on the rear of the cone, means of deposition ofthe target material onto the surface of the cone andbeam wobbling. The goal is to maximize the sur-face area through which the heat can be conductedand minimize the thickness of the target materialwhile making a target which is practical in terms ofmaking the beam incident over a large fraction ofthe target surface. The number and spacing of finsto be added to the back of the cone is critical inorder to maximize the heat conduction to the liquidnitrogen while allowing enough space for liquidHow along the surface of the fins. The depositionrate of the target material is also an importantparameter since if the target material is deposited asa frost then the thermal conductivity will be verylow and the material will vaporize when the beamstrikes the material. The heat transfer parameterscan be tested with a diagnostic target which willhave thermocouples on the fins, on the entrance tothe cone and on the front foil. The rate of loss ofthe liquid niirogen will be monitored to measure thetotal heat dissipation. The physical form of thetarget material layer will be determined by physicalinspection before irradiation.

TECHNICAL PROGRESS AND RESULTS:The project has been very sucessfu! in demonstrat-ing the usefulness of the cryogenic targets. Quanti-ties of fluorine-18 similar to those produced inliquid targets have been produced in the cryogenictarget and used for the synthesis of severalradiotracers including fluorodeoxyglucose (FDG)the most widely used PET radiotracer. The produc-tion yields is given in Table 1. It was also deter-mined that the shape of the cone would have aneffect on the yield from the target and especialiy onthe amount of gas which could be used in the tar-get. The results from these experiments are givenin Table 2.


1. A Cryogenic Solid Target for the Productionof [18F]Fluoride from Enriched [18O]CarbonDioxide. Mahmoud L Firouzbakht, David J.Schlyer, S. John Gately and Alfred P Wolf. JNucl. Med. 34 1993, 69P. Abstract present-ed at the 40th Annual Society of NuclearMedicine Meeting, Los Angeles, CA.

2. A Cryogenic Solid Target for the Productionof [lsF]Fluoride from Enriched [18OJCarbonDioxide. Mahmoud L Firouzbakht, David J.Schlyer, S. John Gately an d Alfred P Wolf.Appl. Radiat and Isot. 44 1993, 1081-1084.

3. The Yield of F-18 from Different Target De-signs in the I8O(p,n)l8F Reaction on Froz-en[!8O]CO2, Mahmoud L Firouzbakht.David J. Schlyer, and Alfred P Wolf. Ab-stract presented at the Fifth InternationalSymposium Targetry and Target Chemistry.Brookhaven National Lab, USA, September19-23, 1993.

4. The Yield of F-18 from Different Target De-signs in the 18O(p.n)18F Reaction on Frozen[I8O]CO2, Mahmoud L Firouzbakht, DavidJ. Schlyer, and Alfred P Wolf. Abstract pre-sented at the Tenth International Symposiumon Radiopharmaceutical Chemistry. Kyoto,Japan, October 25-28, 1993.

5. Record of Invention filed with theBrookhaven National Laboratory Office ofTechnology Transfer June 17, 1993.


I. Possible CRADA with Accsys TechnologyInc. for development of target systems for asmall linear accelerator for production of PETradioisotopes.




S -610-.0350-


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Table 1

Beam Current(//A)


Fluorine-18 Production from the

Irradiation time(min.)


i18O]Carbon Dioxide Ice Target

Saturation Yield[1sO]Enrichment (mCi///A) Treatment






112211344111111111 (3 liters)1 (1 liter)1 (1 liter)4444444

Treatments:1. Carbon dioxide was expanded into the cone and then the cone brought to liquid nitrogen temperatures.2. Cone was cooled to liquid nitrogen temperature and then the carbon dioxide gas was introduced.3. The target was tilted with the point of the cone down, the carbon dioxide was introduced and then the

cone cooled to liquid nitrogen temperatures.4. Same as 1 except that a 1/2 inch collimator was used instead of 5/8".


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Table 2.

Production for Various Target Designs


Amount ofC 0 2








53±450 + 3

43±244 ±3


Percent ofTheoretical










All runs were carried out at 6//A for 10 minutes at an energy of 17.4 MeV incident on the carbondioxide. Percent theoretical is based on the yield from 99% [180]C02.

evelopment of Microdialysis Technology 93-24

Stephen L. Dewey


In a continuing effort to foster new and innovativeresearch directions that will ultimately provide abetter understanding of the neurochemical organiza-tion of the central nervous system (CNS), wedeveloped an in vivo microdialysis facility that iscapable of measuring specific neurotransmitterconcentrations in freely moving animals. Thismethodology greatly complements our ongoinginitiatives using positron emission tomography(PET). Microdialysis data provides unique andnecessary information concerning the selection ofappropriate therapeutic drugs and their doses aswell as their pharmacokinetic profiles. These stud-ies are used to maximize the design of our neuro-pharmacologic protocols and predetermine thefeasibility of a particular experimental strategy.

The goals of these microdialysis studies are toquantitate endogenous dopamine and serotoninrelease and to examine their modulation byneurotransmitters that have been shown to exertan influence on these systems (i.e., GABA,acetylcholine, glutamate, and opiates). Thesestudies will emphasize several clinically relevantissues that are directly applicable to PET in thefollowing respects: (1) direct measurements ofendogenous dopamine and serotonin levels willbe made following baseline and drug pretreat-ment conditions (pharmacologic agents will bechosen that are under consideration for use inPET studies; (2) studies will be performed intwo different neuroanatomic foci (striatum andfrontal cortex) to assess the differential sensi-tivity of meso-cortical and nigro-striatal dopa-mine systems, respectively, to pharmacologic


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interventions; and (3) studies will be designed in amanner parallel to clinical therapeutic strategies.

These studies will specifically enhance the inter-pretation and design of our PET studies. It is ourintention to further investigate the effects of anes-thetic agents on these neurotransmitter interactionsas our primate PET studies require the use of gen-eral anesthetic agents. To that end, ourmicrodialysis facility can be used in anesthetized orfreely moving animals. These studies will impacton every primate PET study we perform.


PURPOSE: The microdialysis concept was intro-duced in the early 1970's, and is only now emerg-ing as a powerful technique because of the commer-cial availability of the probes and its ideal compati-bility with high pressure liquid chromatography(HPLC) and electrochemical techniques used forneurotransmitter determinations. Microdialysissampling provides several advantages over moreconventional techniques. These include providing ameans of continuous sampling without fluid loss,and the ability to separate drugs from or introducedinto the system during the experiment Further-more, these studies are very useful for examiningthe neurochemical organization of the CNS as theintegrity of the neuropil is not drastically perturbed.In addition, it is possible to measure multipleanalytes by coupling other analytical techniques tothe microdialysis system.

APPROACH: Technically, a hydrophilic tube (adialysis capillary or probe) through which lowmolecular weight substances can easily diffuse issurgically positioned in a predeterminedneuroanatomical foci under anesthesia and stereo-tactic guidance. The perfusion medium, whoseionic strength and pH closely matches those of theextracellular space, is pumped through this tube atprecisely controlled flow rates. This allows drugsto be predictably introduced into or removed fromthe extracellular space by establishment of adiffusional steady state across the capillary wall.

TECHNICAL PROGRESS AND RESULTS:The microdialysis facility was setup and installedin June, 1993. Since that time, we have com-pleted the initial tests that are required for deter-mining the sensitivity and stability of the HPLC.We completed tests on the dialysis pump(CMA/100) and calibrated its ability to reliablyflow at rates between 1.0 -5.0 microliter/min.Upon completion of these initial experiments, webegan a series of studies designed to examine theeffects of different drugs on extracellular dopa-mine concentrations. These drugs include co-caine, d-amphetamine, methylphenidate,altanserin, citalopram, gamma-vinyl GABA,tetrabenazine, naloxone, and morphine. Thesestudies were all performed in freely moving rats.Data is presented in the graphs.

Drugs used in these studies have different mech-anisms of action. Below is a brief description ofthese drugs and their clinical use.

1. Baseline measurements: Initial studies exam-ined the variability of our baseline measure-ments of dopamine in freely moving rats.These studies were essential as they will beused as baseline measurements to which wecan compare our drug treatment studies.These findings indicate that baseline concen-trations of extracellular dopamine remainconstant over 5 hours.

2. Methylphenidate: Known as Ritalin , this drugis used to treat hyperactive children. We havedemonstrated that it raises dopamine concen-trations in a dose dependent manner and thatsequential injections do not produce the sameeffects. This is consistent with a desensitiza-tion of the dopamine system to this drug. Wehave radiolabeled methylphenidate with car-bon-11 and performed PET studies in bothhumans and primates.

3. Citalopram: A drug used successfully inEurope to treat depression, citalopram raisesserotonin concentrations. This study demon-strates that citalopram not only alters serotoninconcentrations, but it can produce markedchanges in dopamine as well. These studies


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have been performed in primates using PET andradiolabeled raclopride (a dopamine D2 selectiveradiotracer).

4. Altanserin: Altanserin is a highly selectiveserotonin antagonist. This drug produces theopposite effects of citalopram as it has beenshown to inhibit serotonin receptors. Thesemicrodialysis studies confirm this finding in thatcitalopram caused a decrease in dopamine con-centrations while altanserin increased dopamineconcentrations.

5. Gamma-vinyl GABA (GVG): Known asVigabatrin, this drug has recently been approvedfor the treatment of childhood epilepsy as it hasbeen shown to greatly elevate GABA levels, theprimary inhibitory neurotransmitter in the CNS.These microdialysis studies demonstrate thatGVG also produces marked decreases in striataldopamine concentrations. We have completed aseries of PET studies using GVG and labeledraclopride to examine the effects of this drug ondopamine release in the anesthetized baboon(Dewey, et al., J. Neuroscience, 12: 3773-3780(1992)). Taken with our work using altanserinand citalopram, these studies demonstrate theusefulness of this technique for assessing themultitransmittereffectsofneurotransmitter-specif-ic drugs. Furthermore, as these studies providepharmacokinetic profiles of the effects of thesedrugs on extracellular dopamine, we can use thismethodology to better design our PET studies.

6. Cocaine: We examined the effects of cocaine onextracellular dopamine concentrations in thestriatum of freely moving rats. Cocaine adminis-tration increased dopamine release in a dosedependent manner. These findings, along withour findings using d-amphetamine, are consistentwith previous studies and were performed pri-marily to demonstrated our ability to observechanges in striatal dopamine.

7. D-amphetamine: Long known for being a drugof abuse, we examined the effects of sequentialinjections of d-amphetamine on extracellulardopamine release. Unlike our findings withmethylphenidate, d-amphetamine produces repro-

ducible effects on dopamine concentrations asevidenced by similar responses in extracellularrelease. These findings are consistent withPET studies in primates using d-amphetamineand labeled raclopride.

8. Tetrabenazine: Used in the 1960's to treatschizophrenia, tetrabenazine is a drug thatrapidly depletes biogenic amine concentra-tions. Our findings, however, represent thefirst evidence suggesting that initially, tetra-benazine causes an increase in extracellulardopamine concentrations. These findingssupport our earlier PET studies demonstratingthat tetrabenazine produces a decrease in "C-raclopride binding in the primate. These find-ings also demonstrate that biogenic aminedepletion takes several hours.

9. Natoxone: Known as Narcan , naloxone is themost frequently used opiate antagonist inhospital emergency rooms for the treatment ofheroin overdoses. Clinically used to treatthese overdoses due to its selectivity andantagonism of the opiate system, we examinedthe effects of this drug on extraceullar dopa-mine concentrations. In our microdialysisstudies, naloxone resulted in a decrease indopamine concentrations. These findings areconsistent with our primate PET studies usingnaloxone and labeled raclopride.

10. Morphine: An opiate agonist, morphine isused clinically for the treatment of intractablepain associated with such diseases as cancer.This action is produced through its ability tomimic the actions of endogenous opiates(endorphins and enkephlins). We examinedthe effects of morphine on extracellulardopamine concentrations. As its mechanismof action is opposite to that of naloxone, wepredicted that it would raise extracellulardopamine concentrations. These findings areconsistent with our primate PET studies usingmorphine and labeled raclopride.

These microdialysis studies using naloxone andmorphine are good examples of how microdialysisstudies can be used to design PET studies. The


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time course of these effects were examined and thepretreatment intervals for the PET studies werearrived at by our findings with microdialysis. Similarmicrodialysis information was used to design ourongoing studies with fenfluramine and labeledraclopride in normal human volunteers.Together, these studies demonstrate the usefulness ofin vivo microdialysis for assessing many differentaspects of drug pharmacokinetics. Furthermore, thesestudies demonstrate that drugs designed to act onspecific neurotransmitter systems often produce effectson other functionally-linked systems. This examina-tion of neurotransmitter interactions complements ourongoing research effort in this area using PET in bothprimate and human subjects. Combined with thesePET studies, we can begin to understand the effectsof anesthesia, drug pretreatment intervals, dosing,and routes of administration on neurotransmitterphysiology. These studies have important clinicalimplications in terms of the development of new andalternative therapeutic treatment strategies.


1. Dewey, S.L., Smith, G.S., Logan, J. et al.(1993) The effects of acute and chronic haloper-idol treatment on GABAergic and serotonergicmodulation of dopamine release measured withpositron emission tomography. Soc. Neurosci.Abstr. 19: 128.7.

2. Dewey, S.L., Smith, G.S., Alexoff, D.L. et al.(1993) Changes in dopamine with PET andmicrodialysis. American Psychiatric Association(Submitted).

3. Smith, G.S., Dewey, S.L., Logan, J. et al.(1993) Opiate modulation of striatal dopamine

release measured with positron emission tomog-raphy (PET) and "C-raclopride. Soc. Neurosci.Abstr. 19: 128.9.

4. Dewey, S.L., Smith, G.S.. Alexoff, A., et al.(1993) Positron emission tomography and invivo microdialysis studies of neurotransmitterinteractions. American College ofNeuropsycho-pharmacology (Submitted).

5. Smith, G.S., Dewey. S.L., Logan, J., el al.(1993) In vivo studies of the serotonin-dopa-mine interaction with positron emission tomog-raphy and microdialysis. American College ofNeuropsychopharmacology (Submitted).


1. P01 NS-15638 "Positron Emitters and PETTin Metabolism and Neurology" Joanna Fowler.Principal Investigator, National Institute ofNeurological Disease and Stroke, renewalapplication submitted, October, 1993,$6,363,239. Project 3, ""PET Studies ofNeurotransmitter Interactions in the Investiga-tions of Typical and Atypical AntipsychoticDrugs", Stephen L. Dewey. Principal Investi-gator.






- 0 -50,15162.000 (est.- 0 -

Note: This project involved vertebrate animals.


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Effects of melhylphenidale ondopanilnr release









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Analysis of Structures and Interactions of Nucleic

Acids and Proteins by Small-Angle X-Ray Diffraction


M.S. Capel


Completion of the Biology Department's time-re-solved small-angle x-ray diffraction facility atstation X12B of the NSLS now provides uniquecapabilities for analyzing nucleic acid and proteinstructures and interactions in solution. Static andtime-dependent measurements of the radial depen-dence of x-ray scattering from solutions of biologi-cal macromolecules provide measures of molecularmass, characteristic size (e.g. radius of gyration,maximum interatomic chord) and, in favorablecases, the ability to experimentally test detailedmolecular models of the involved macromolecularsystems.

Our program will take advantage of the high fluxand tunability of the NSLS x-ray synchrotron

source and the advanced detection technology ofthe X12B endstation to develop methods forinvestigation of the static and dynamical proper-ties of proteins, nucleic acids, and their com-plexes in solution. Projects currently supportedunder this initiative include:

1) Determination of the geometry of the com-plex between molecular chaparones GroESand GroEL. and changes in this system thatoccur during the course of their interactionwith folding substrates and cofactors, viaextreme small-angle solution scattering.

2) Development of kinetic methodologies forobserving protein folding (and unfolding) viasmall angle diffraction. To this end. we aredeveloping a rapid mixing device for obser-vation of millisecond-time scale alterations in


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macromolecular structure induced by mixing ordiluting out a chaotrophic agent from a proteinsolution.

3) Development of energy-dependent scatteringmethods for acquiring specific distance informa-tion in macromolecular systems, including: a)flexure of the long axis DNA duplexes in re-sponse to binding of effector proteins and b)distances separating subunits of oligomeric(multi-subunit) protein complexes.

Here, the organo-gold cluster labeling technologydeveloped by J. Hainfeld (BNL Biology Depart-ment), is used to specifically perturb the electrondensity of subunits of a macromolecule. By mea-suring solution diffraction at a number of energiesone can extract a component scattering functiondominated by scattering from the exogenous goldlabel, and consequently details of the spatial distri-bution of the label and the subunits to which it iscovalently bound, within the target complex.


PURPOSE: New experimental techniques andinstrumentation must be developed to fully realizethe potential of synchrotron x-ray radiation for useas a static and dynamical probe in biological struc-tural studies. Energy dependent (resonant) solutionscattering studies were not possible before theadvent of second generation synchrotron sources,and are still rather rare in the literature. Short timedomain kinetic scattering studies were also impossi-ble outside of the flux regime provided by synchro-tron sources. Aside from instrumental issues thisclass of studies is made problematic by the verylarge quantities of pure protein required (10's ofgrams), and are not feasible without "genetic engi-neering" techniques.

APPROACH: We hope to make significant meth-odological and experimental progress in both areasmentioned above by combining the unique instru-mentation of the X12B station and genetic over-expression methods to produce the large quantitiesof pure biological macromolecules these techniquesrequire.

TECHNICAL PROGRESS AND RESULTS:Solution Small-Angle Diffraction: We are usingsmall ansle solution scattering to study the qua-ternary structure of the molecular chaparonesGroES and GroEL and their complex. Ourresults confirm electronmicroscopic data thatindicate that GroEL is to first approximation ahollow right-cylinder composed of two stackedrings of seven equivalent subunits. Measure-ment of the change in radius of gyration ofGroEL upon binding of GroES (consisting of asingle ring of seven subunits) show that GroESbinds within the central channel of GroEL nearone end. Figure la shows the logarithm ofradial scattered intensity for GroEL and theGroES-GroEL complex (inset shows linearplots). Figures 2a and 2b graphically illustratethe results of Monte-Carlo simulations of twomodels for the GroES-GroEL complex. Theupper traces of the logarithm of scattered intensi-ty vs Q predicted by the two models in theregion Q = 0.05 - 0.15). The lower panelsconsist of the modeled P(r) functions (interatom-ic distance distribution functions) for free GroEL(open circles) superimposed on those from twomodel complexes. Comparison of the uppertraces of Figures 2a and 2b with Figure 1 showsthat the model wherein GroES (black ellipses)binds away from the center of mass of theGroEL system gives a better fit with our experi-mental data.

Kinetics: We have purchased and rebuilt a com-mercial rapid mixing device for compatibilitywith x-ray transmission and the control systemsof XI2B. With this device we can mix a proteinsolution with another buffer resulting in a signif-icant change in the physical state of the solutionwithin a dead-time of 2-4 msec, and shot vol-umes of 10-20 fi). The Figures below displaybench mark tests of the facility. Fifteen ml of a30 mg/ml solution of hen egg white lysozymewas loaded into channel one of the mixing de-vice and buffer loaded in the second. At timezero, 50 y\ of buffer was fed through the flowcell at maximum stepping rate of 3 ml/sec.Seventeen msec after the onset of channel twoaction, the channel 1 syringe delivered 25 fi\ oflysozyme solution to the mixer at I ml/sec.


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Five msec after completion of the mixing sequence,our programmable 2D imaging frame store wascommanded to acquire 25 5 msec, 25 25 msec and25 100 msec duration data frames. The mixing anddata collection sequence was iterated 500 times.The accumulated 2D data frames were corrected fordetector counting nonuniformities and radiallyaveraged about the beam center. A second se-quence with buffer in both mixing channels wassubsequently run. Corresponding buffer radialprofiles were subtracted from the lysozyme solutionprofiles, with corrections for incident flux, sampletransmission and detector and data collection deadtimes. The buffer-subtracted cumulative radialprofiles from the first 5, 25, and 100 msec framesare displayed in Figure 3 (all traces normalized tofixed monitor counts, with arbitrary vertical offset).Figure 4 shows the corresponding guinier analysesfrom the data traces of Figure 1 (with arbitraryvertical offset). This device will be used for kineticstudies of the rate of refolding of urea-denaturedStaphyloccoccal nuclease and engineered mutationsof the nuclease in order to test models of proteinfolding. We have cloned and successfullyoverexpressed the nuclease gene in Escherichia colito obtain gram quantities of pure enzyme.

Resonant Diffraction: J. Hainfeld (BNL BiologyDepartment) has developed two targetable organo-gold clusters as site-specific labels in scanningtransmission microscopy: undecagold containing 11-Au moieties and a 55-Au cluster. Both clusters canbe targeted to a variety of functional groups com-mon to biological macromolecules including: ami-no, carboxyl, sulfhydryl and phosphate functionalgroups. We intend to use these site-specific goldlabels as a means of specifically perturbing thescattering function of DNA in DNA-protein com-plexes in order to observe large scale conformation-al changes in the DNA, induced by protein binding.We will do this by first synthesizing short duplexdeoxyoligonucleotides that are cognate to one ormore DNA-binding proteins (e.g. catabolite activa-tor protein, CAP), with an organo-gold clusterbound to both ends. The radial small-angle scatter-ing function of the modified oligo will be measuredat a number of x-ray energies, spanning the Au Lmabsorption edge (11.919 keV). Subtraction of thescattering profiles obtained at and away from the

L]n edge yields a partial scattering functionwhose parameters are determined by the spatialdistribution of Au with in the oligo. In particu-lar the ensemble average Au-Au distance will bemeasured. The same measurements will berepeated for the protein-oligo complex. Largechanges in the curvature of the long axis of theoligo (thought to occur in the CAP-DNA com-plex) will be manifested as a shortening of theaverage Au-Au distance.

In order to develop and test experimental proto-cols for this type of measurement we attemptedto measure the radius of gyration of the Au coreof the undecagold and Au35 clusters in solution.The radial scattering profiles of Au-cluster solu-tions were measured at 50 eV intervals surround-ing 11.919 keV. Figure 5 shows the "anoma-lous" excess scattering profile obtained as thedifference profiles between 11.769 and 11.919keV for both clusters. Figure 6 is the corre-sponding guinier analysis for both curves inFigure 3. The radii of gyration (Rg) agree ex-actly with those predicted from crystallographiccoordinates obtained from studies with closelyrelated Au-ciusters. This result validates ourresonant diffraction data collection protocols.


1. Flanagan, J. M., Kataoka, M., and M. S.Capel. Investigation of the Interaction ofGroEL with ADP, GroES and UnfoldedPolypeptide by Small-Angle X-ray Scatter-ing. (1993, in preparation).

2. Flanagan, J. M., Shanklin, J.. and M. S.Capel. CIpP is a Dodecamer With a CentralAxial Pore. (1993, in preparation).




$ - 0 -42,27698.00048.000



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0.05 0.15

rcEi-GroEs \ /

r ft)


Figure 1 Figure 2a

0 (A )

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Figure 30 (A )


Rg - 4 .66

Rg - 3.09

Figure 4

o • Au ,, complex

• • Aus5 complex









A 5 msec

Tfi~ B 25 msec*• 7 *^W c 100 msec -



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Q2 ( A " 2 )



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B nhancement of Microplanar Beam Radiation Therapy (MRT) 93-33

of the Intracerebral Rat 9L Gliocarcoma by Pre-infusion with a Diiodofluorecein-Histone Conjugate

D.N. Slatkin,M. Miura and

P. Spanne


i he original statement of work for this project

includes the following:

i) To synthesize histone H3 (cysteine 110)-acetylamino (4 \5 ' diiodo) fluorescein(H3AADIF).

ii) To infuse H3AADIF into brain-irradiated andnormal mice and gerbils.

iii) To assay the uptake of the diiodofluoresceingroup by tissues, especially by the centra)nervous system (CNS) tissues of these animals.

iv) To analyze, using Monte Carlo photon/electrontransport codes, the radiophysical parameters ofthe MRT irradiation protocol shown by ourexperiments to be effective in palliating the 9Lintracerebral gliosarcoma of rats. The analysiswill focus particularly on the values and spatialdistribution of the minimum absorbed dosesbetween microplanar beams.

v) To devise microplanar beam irradiations ofmice and of gerbils that will yield absorbeddose patterns in the brains of mice and gerbilsthat mimic to the greatest extent feasible thera-peutically effective absorbed dose pattern ingliosarcoma-bearing rats.

vi) To determine experimentally the enhancement,if any, of microplanar beam irradiation damageto the brains of mice and gerbils by prior infu-sion of H3AADIF.

This progress report shall focus on the follow-ing:

i) To locate and analyze the older and the morecurrent scientific literature regarding endocy-tosis and nuclear importation of histones andbasic polypeptides in vitro and in vivo.

ii) To devise a system for testing the endocyto-sis and nuclear importation of histones andbasic polypeptides by proliferating and non-proliferating cells in vitro that could appro-priately model these effects in vivo.

iii) To survey the physical and chemical proper-ties of elements other than iodine which mayprove more suitable than iodine for Augereffect-enhancement of MRT, especially withrespect to the effects of the various energiesof wiggler-enhanced x-ray beams presentlyavailable and foreseen at the NSLS X17Bbeamline with and without beam filtration byGd or Cu.


PURPOSE: To achieve some progress duringFY 93 in those aspects of the proposed workabout which there seemed to be few uncertaintieswith regard to overall research strategy andfeasibility within our available resources.

APPROACH: Dr. Per O. Spanne, a co-inventorof MRT, took the opportunity afforded him bythis LDRD project to embark on a thoroughevaluation of the Sandia National Laboratoryreport entitled "Integrated TIGER series ofcoupled electron/photon Monte Carlo transport


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codes", by J.A. Halbleib et al.. (SAND-91-1634;Radiation Shielding information Center, Oak RidgeNational Laboratory, 1992) as it relates to theINHOM (EGS4) code used in the report on NiRTentitled "Microbeam Radiation Therapy" (Slatkin etal.. Medical Physics 19, 1395-1400, 1992).

Not only was it not feasible for Drs. Miura andSlatkin to begin the synthesis of H3AADIF prompt-ly, it was also judged better to resurvey the choiceof H3 adducts most likely to enhance MRT usingthe present XI7B beam and using a proposed high-er-tesla wiggler with an increased (= 2.8GeV)electron storage ring energy. As a result of ourresurvey. we have decided to add Gd as a prospec-tive combined MRT/NCT/MRI adduct to the his-tone moiety of chromatin.

Specifically, we may be able to synthesize an ana-logue of iodoacetylamino (4',5' diiodo) fluoresceinthat bears an amino group, so as to enable an amidelinkage to be made with one of the peripheral car-boxyl groups of diethylene triamine pentaacetic acid[DTPA]. The DTPA moiety, in turn, will be ableto chelate gadolinium-157, the thermal neutroncapture cross section of which is 255,000 barns,nearly two orders of magnitude greater than that ofboron-10. Thus, chromatin-associated gadolinium-157 could be effective as an NCT agent via theefficiency of Auger electron-induced DNA damage.This strategy leaves intact the introduction of iodineinto chromatin but adds another physical reaction,NCT, for possible use in radiation therapy. In bothoptions [F or I + Gd], the histone H3 sulfhydryl ora suitable basic polypeptide would be derivatizedvia the iodoacetylamino group of the synthetictarget moiety. Moreover, Gd. like I, would aug-ment the efficacy of MRT via Auger electrons.

TECHNICAL PROGRESS AND RESUI.TS:i) Experiments MRT 2. MRT 4 and MRT 5 were

analyzed and their results correlated and sum-marized in a manuscript shortly to be submittedto Dr. E.P. Cronkite for possible transmissionto the Proceedings of the National Academy ofSciences.

ii) To simulate MRT of the intracerebral ratgliosarcoma using the integrated TIGER series

(ITS) of photon/electron transport codes.Drs. Spanne and Slatkin devised the follow-ing model of a rat head:

a) Anterior to the mid-interocular trans-verse plane, a cone with a 26 mm diam-eter circular base (the "interocular"plane), truncated at a 7.7 mm-diametercircle »front of "nose". The length(height) of the truncated core is 27 mm.

b) Posterior to the "interocular" transverseplane, a 31 mm-long cylinder in whichthe "interaural" traverse plane is as-sumed to be 21 mm posterior to the"interocular" transverse plane. Theanteroposterior beam used for MRT of abrain tumor centered 4 mm to the rightof the sagittal planes beam is assumed tobe "parasagittal," 12 mm high and 25^mwide. The lower edge of the array of100 irradiated slices (each 25//m wideand each separated from its adjacent sliceby 100|tm, center-to-center) is along thehorizontal midplane of the cylinder. Themost medial of the 100 irradiated slices

is 1 mm to the left of the vertical mid-plane of the cylinder. The mid-verticalaxis of the central (= 50th) antero-poste-rior slice intersects the surface of thephantom at a point (on the "nose") thatis =* 37mm anterior to the center of a4mm-diameter spherical "tumor", 6mmbeneath the surface of the cylinder half-way between the "interocular" and"interaural" traverse planes. With theseconstraints. Dr. Spanne has found thatthe XI7B beam, additionally filteredwith 0.25mm Gd, yields a peak/valleyabsorbed dose ratio of = 22:1 at thetumor, and that a 625 Gy-entrance ab-sorbed dose yields peak doses of about250 Gy at the center of the tumor.

These novel computations enable us for the firsttime to surmise the minimum interbeam dose inthe tumor zone. The 625 Gy-entrance dosesshown by us to be non-damaging to the rat brainafter one month are associated with minimum


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interheam absorbed doses of = 12 Gy around thecenter of the cerebrum. Our doubling the entranceabsorbed dose to 1250 Gy doubles the interbeamdoses at the center of the brain to = 24 Gy. More-over, 1250 Gy-entrance doses were associated withresidual brain damage in some rats so irradiated.These results are remarkable and the Monte Carlotechniques used to derive them are necessary forthe future development of clinical MRT becausethey will enable one to correlate dose/effect rela-tionships from macroscopic beams with those frommicroplanar beams. Indeed 12 Gy would be ex-pected to be relatively safe compared with 24 Gy ifeach were given in one irradiation session to amacroscopic field of the rat brain. Many moredetailed computations and model experiments willbe required before MRT dose/effect relationshipscan be predictably correlated with dose/effect rela-tionships in the human brain using macroscopicradiation fields.


1. An invention entitled "Microbeam RadiationTherapy", by D.N. Slatkin, P.O. Spanne, andFA. Dilmanian. was filed by DOE on April27. 1993 (S.N. 052.927). Certain aspects ofthis invention are described in Slatkin et al.,Microbeam radiation therapy. Med Phys. \9,1395-1400 C1992).

2. A manuscript entitled "Subacute Neuropatho-logical Effects of Microplanar Beams of XRays from a Synchrotron Wiggler" by D.N..Slatkin, P. Spanne, F.A. Dilmanian, J.-O.Gebbers, and J.A. Laissue, was prepared forpossible submission to PNAS under the aegisof Dr. E.P. Cronkite.

3. .Some of the scientific literature regardingendocytosis and nuclear importation ofhistones and basic polypeptides was searchedand studied by M. Miura and D.N. Slatkinwith the object of refining the strategies ofsynthesis and assay to be pursued under thisLDRD project.


FY 1992FY 1993FY 1994FY 1995

S - 0 -42.63898.000 (est.)- 0 -

NOTE: This project involves vertebrate ani-mals.

•fc elaxographic MRI and Functional MRI 93-35• • • • • • • • • • • •Joanna S. Fowler


JL his LDRD Project has been underway for Imonth. It is a collaboration between CharlesSpringer, Chemistry Department. SUNY, StonyBrook. Joanna Fowler and Alfred Wolf, ChemistryDepartment, BNL and Nora Volkow, Medical

Department, BNL. ft addresses functional MRIstudies in laboratory animals using CharlesSpringer's high field animal MRI instrument atStony Brook. It provides support for CharlesSpringer to spend a sabbatical at Brookhaven inwhich two parallel efforts will be undertaken: (I)to understand the quantitative and mechanisticaspects of the functional MR image and (2) to


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establish the groundwork for expanding the presentPET program at Brookhaven to include functionalMR! including the design of an NMR laboratoryand the aquisition of a high field (4Tesla) humanNMR instrument (proposal submitted to DOE).


At the time of this report, the Project has gotten offto a very productive start. We have undertakenstudies of photic stimulation of the visual cortex inthe mouse using functional MRI. Mr. Wei Huang,a USB Chemistry Graduate student, has made twofundamental discoveries with regard to the function-al response to photic stimulation in the anesthetizedmouse. The first is that the intensity of the re-sponse depends upon the duration of anesthesia andmaximizes just prior to awakening. The responsealso depends on the duration of the stimulus. Theresponse dies away after more than eight seconds ofcontinuous stiumlation but there is always an im-pulse response at the end of the long stimulus. We

are in the process of quantifying these responsesand increasing their precision. The interpreta-tion of these findings is being guided by Dr.Nora D. Volkow in the Medical Department.

We have also begun to delineate the specifica-tions and other information required for thepurchase of a high field human MRI instrumentfor the proposal to DOE and have met withrepresentatives of Siemens and General Electric.The preparation of documents required to pro-ceed with an order is underway in consultationwith OHER officials in Germantown.




$ - 0 -8,675



NOTE: This project involves animal verte-brates.


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Qenetic Studies

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Qn the previous page: Malarial parasites, the whitespheres, inside red blood cells. That the parasite is whiteindicates it has a proteinase activity on its surface thatcleaved a fluorescent substrate.

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New Malaria Enzyme - A Potential Source

for a New Diagnostic Test for Malaria and a Targetfor a New Antimalarial Drug


W.F. Mangel


A s classical drug treatment of malaria becomesless effective because of resistant parasites and withthe difficulties in producing successful vaccines, thedevelopment of new antimalarial agents becomesincreasingly important. Our objective is to test thehypothesis that a highly specific proteinase whoseactivity is required for reinvasion of erythrocytes byPlasmodium falciparum is a legitimate target forrationally designed drugs. The hypothesis will betested by synthesizing equally specific proteinase in-hibitors that should inhibit reinvasion but not nor-mal physiological processes.


PURPOSE: The purpose of the research is to char-acterize a cell surface proteinase activity on P.falciparum. Using our fluorogenic substrates, wehave preliminary evidence for the existence of acell surface proteinase activity. We also havepreliminary evidence that bovine pancreatic trypsininhibitor (BPTI) inhibits reinvasion of red bloodcells.

Long term objectives include the development ofsecond generation proteinase inhibitors and attemptsto develop an antimalarial vaccine against the re-combinant proteinase or fragments of it, either sur-face sequences or sequences of the active site. Ifsuccessful, the technology we devise can easily beextended to the treatment of other parasitic diseas-es.

APPROACH: This year we asked whether the cellsurface proteinase activity we assay with ourfluorogenic substrates is from the same enzymewhose activity is required for reinvasion of redblood cells and whose activity is inhibited by BPTI.

TECHNICAL PROGRESS AND RESULTS:With prima facie evidence for an unusual cellsurface proteinase activity that interacted with(Ile-Pro-Arg-NH)2-Rhodamine, we focused onfinding proteinase inhibitors of reinvasion.Although we had planned eventually to mutateBPTI into becoming a highly specific inhibitor ofa proteinase required for reinvasion, there wasno reason to believe that wild-type BPTI wouldinhibit reinvasion. However, it does. BPTI wasadded to a culture of red blood cells containingPlasmagel-purified schizonts from P. falciparum.After 18 hours at 37'C, the parasitemia wasmeasured. The control cultures contained 10.8.8, 12, and 10% ring-infected erythrocytes.Two cultures with 10 /tM BPTI contained4.8,and 5.2% ring-infected cells. Two cultureswith 40 iM BPTI contained less than 0.05%ring-infected cells, i.e. we could detect no in-fected cells.

We then asked whether inhibition of reinvasionby BPTI was reversible or irreversible andwhether BPTI inhibited the binding ofmerozoites to the surface of red blood cells.Purified Plasmodium chabaudi merozoites wereincubated in PBS plus or minus inhibitor for 15min at room temperature. The cells werewashed and resuspended in PBS and then mixedwith normal mouse red blood cells. The percentof trophozoite-infected red blood cells and thebinding of merozoites to red blood cells wereevaluated on Giemsa stained smears after 6hours at 37°C. In the absence of BPTI, I % ofthe cells contained trophozoites, and this numberwas reduced to 0.2-0.3% in the presence of 10pM BPTI. In the absence of BPTI, 13-20% ofthe merozoites were attached to red blood cells,and this number did not decrease in the presenceof BPTI. DFP, a potent serine proteinase inhibi-tor, gave similar results when present at 10 fiM.Chymostatin, a reversible inhibitor of chyrno-


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trypsin-like proteinases, did not affect binding norreinvasion, at 10 /xg/ml. However, if it was notwashed away before reinvasion, its presence re-duced the number of trophozoite-infected red bloodcells from I % to 0.3-0.5%. Thus, BPTI is anirreversible inhibitor of reinvasion of red bloodcells.

To determine whether the proteinase that cleaves(Ile-Pro-Arg-NHj^-Rhodamine is inhibited by theproteinase inhibitors that inhibit reinvasion, weincubated mature P. falciparum schizonts with vari-ous proteinase inhibitors for 30 min., added Rhoda-mine-based substrates to a concentration of 40 fiMand. after 1.5 hours, looked at the schizonts in thefluorescence microscope. The data indicated thattwo proteinase inhibitors inhibited the proteinasethat cleaves flle-Pro-Arg-NH):-Rhodamine- BPTFand chymostatin.

In conclusion, we have shown that the proteinaseon the cell surface that cleaves ('(le-Pro-Arg-NHj-,-Rhodamine also interacts with BPTI. The activityof this proteinase is required for reinvasion of redblood cells as reinvasion is prevented by the pres-ence of BPTI. Thus, this is the target we havebeen searching for, a cell surface enzyme requiredfor reinvasion. And since we have an assay and aninhibitor of the enzyme, we should be able to puri-fy the enzyme in the next phase of this project.


1. Barale, J . -C, Langsley, G , Mangel, W. I .and C. Braun-Breton Maiarial ProteasesAssignment of Function to Activity. Re-search in Immunology 142: 672-681 (1992).

2. Kirz, J., Ade, H , Fu. J., Jacobsen. C\.Lindaas. S., Oehler, V., Van't Hof. J.. Wil-liams, S., Wirick. S., McGrath, W. J.,Mangel, W. F., and X. Zhang. ScanningTransmission X-Ray Microscopy at theNSLS. In X-Rav Microscopy IV. A. I.Erko, ed., Bogorodsky Pechatnik,Chernogolovka. H993, in press).


The writing of an NIH grant is almost done. Allthat is needed to complete the grant is to com-plete the section on the purification of the pro-teinase.




S - 0 -52.76355,87558.956

- 0 -

sffect of a Bacterial Spore Protein on Mutagenesis 91-21

J.K. Setlow


IMutagenesJs and killing caused by a spore proteinfrom Bacillus sublilis that drastically alters UNAconfiguration are being studied in the bacteria lisch-erichia roli and Haemophilus influenzae and in vitrowith purified protein and DNA.


PURPOSE: Since we have found that mutagenesis is substantially increased when DNA hecomes more negatively supercoiled. the projectinvolves investigation of details of the medianisrn of this effect on mutation. Thus eventuallywe plan in vitro UNA replication using DNAwith various degrees of negative supercoilinj?. as


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well as purified DNA polymerase and associatedcompounds necessary for the reaction. Then wewill measure the mistakes made by the polymeraseas a function of the degree of supercoiling, with aphage assay developed by S. Ripley's laboratoryconvenient for in vitro mutagenesis studies.

APPROACH: The cloned gene coding for theprotein from B. subtilis on a plasmid in E. colireadily mutagenizes itself (thus we call this systema "suicide plasmid"). Sequencing of relevant partsof the plasmid in strains that have lost the ability tomutagenize E. coli is expected to provide clues tothe mutagenic mechanism.

Putting the B. subtilis gene into the H. influenzaevector constructed in this laboratory and then intoH. influenzae cells has not yet been achieved, prob-ably because the B. subtilis protein made in H.influenzae is deleterious to the cell. Low copyvariants of the plasmid vector are expected to solvethe problem. The importance of the H. influenzaeconstruct is that this bacterium, unlike E. coli, isnot normally readily mutagenized, and thus may bea better system for investigating the mechanism ofmutagenesis caused by DNA supercoiling.

TECHNICAL PROGRESS AND RESULTS: (a)Mutagenesis and killing were investigated as a func-tion of number of cell divisions following isolationof the cloned B. subtilis gene in E. coli. The re-sults showed conclusively that these two biologicaleffects involve different causal factors, since themutagenic ability of the clones is lost much morerapidly than the lethal effect.

(b) We now have 50 different clones containing the6. subtilis gene that have lost some or all of themutagenic ability in E. coli. The coding sequencefor the B. subtilis protein has been sequenced infifteen of these, and in all cases has been foundidentical to the original sequence in B. subtilis.Thus we conclude that there must have been chang-es in the sequence of the regulatory regions of theplasmids that control the protein, and these are nowbeing sequenced.

(c) There are strains of B. subtilis which lack thespecific protease which has been shown to be re-

sponsible for initially destroying the DNA-alter-ing protein in spores prior to DNA replicationwhen the spores germinate. We have investigat-ed whether there is a change in spontaneousmutation of the cells when they germinate fromspores with and without defects in this protein.We expected to find an increase in mutation inthe strains lacking the specific protein, and weresurprised to find the opposite. The reason mayinvolve the fact that the wild-type strain beginsDNA replication earlier in germination than thedefective strains, so that there is some replica-tion in the presence of the binding protein thatalters DNA configuration. The delayed replica-tion in the mutants may allow other (non-specif-ic) proteases to eliminate this protein beforereplication begins.

(d) Using a somewhat different method than weused earlier, we have shown that the pure B.subtilis protein bound to pure H. influenzaetransforming DNA causes mutations in loci closeto the antibiotic resistance marker of the trans-forming DNA, following selection oftransformants resistant to that antibiotic. Thedata imply that the protein is dragged into thecell when it is complexed with transformingDNA, because it must be present when thenewly integrated DNA is replicated in order tocause mutagenesis in adjoining loci.

(e) We earlier showed that H. influenzae muta-tions were increased by a mutant we isolated thatincreases DNA supercoiling and decreased byanother of our mutants that decreases thesupercoiling, as shown by in vitro assay. Wealso showed that these mutants were alterationsin the gyrase B locus. We have now conclusive-ly demonstrated that the mutations caused by theincreased DNA supercoiling are close to thegyrase B gene, by isolation of 41 of ihe mutantsand analysis of single and double transformationsfrom their DNA.

(f) The effect of the B. subtilis protein on re-combination has been measured with H.influenzae transforming DNA. Since the effectswere rather small, 37 assays were done with 9different genetic markers determined singly and.


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where there was linkage between them, double andtriple transformations. The single markers wereaffected differently by the protein, from a 20% lossup to a 52% loss in frequency of transformation.The double and triple transformations showed thatthe bigger the piece required to be integrated intothe chromosome of the recipient ceil for transfor-mation to occur, the bigger the protein-induceddecrease in frequency, suggesting that the protein tosome extent interferes with the integration of largerfragments of DNA into the recipient's chromosome.

fg) The plasmid containing the coding gene for theB. subtilis protein has a lac promoter so that syn-thesis of the protein may be induced by isopropyl-/3-D-thiogaIactopyranoside (IPTG). Two differentE. coli K-12 strains which are Rec+ and contain theplasmid became considerably more UV-resistantwhen treated with IPTG, and so did an isogenicRec strain. A K-12 B E. coli hybrid which weshowed was extraordinarily prone to mutation whenthe plasmid was present was made UV-resistant bythe plasmid whether or not there was IPTG treat-ment. These effects are due to the change in DNAconfiguration of the E. coli cells caused by the B.subtilis protein, so that the photochemistry is drasti-cally changed: fewer pyrimidine dimers are formed

compared to the number formed in the hybridstrain without plasmid or the K-12 strains with-out IPTG induction.


A manuscript entitled, "A DNA-bindrng proteinfrom Bacillus subtilis spores which alters DNAconformation protects the biological activity oftransforming DNA against ultraviolet inactiva-tion" has been submitted for publication to theJournal of Bacteriology.


A National Institutes of Health proposal entitled,"Mutation by DNA Supercoiling and DNA AConformation" was submitted in June 1993.




S - 0 -87,461


- 0 -

structure and Function of Adenovirus Penton Base Protein 91-23

P.I. Freimuth


Ihe goal of this research is to investigate howadenovirus penetrates cell membranes to initiateinfection. Viruses such as HIV or influenza arebounded by a cell-derived membrane and enter cellsby fusion of the viral and cell membranes.Adenoviruses, picornaviruses, and papillomavirusesare members of a large group of viruses that haveno membrane; the mechanism by which these hy-drophilic particles pass through lipid bilayers is notknown. Farlier studies indicated that adenovirus istaken into cells by receptor-mediated endocytosis

and that endosomes containing virus particles be-come leaky. This led to the hypothesis thatsome component of the virus particle lysesendosomal membranes resulting in the release ofthe endosomal contents into the cell cytoplasm.Detergent-like properties would be expected of aprotein with membrane lytic activity, and earlierstudies indicated that one of the viral surfaceproteins, the penton base subunit, partitionedinto a hydrophobic phase under conditions thatmimic the chemistry of endosomes (e.g. pH 5-6j. In addition, penton base has long beenknown to cause adherent cells to round up anddetach. Together, these properties suggest that


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penton base interacts directly with cell membranes.Our project was designed to study the role ofpenton base during adenovirus infection using somerecently developed technologies in genetic engi-neering.


PURPOSE: The objective of this research was todevelop systems in which functions of penton basecould be studied both in the context of the purifiedprotein and in the context of virus reproduction incultured human cells. This was an important issuesince the existing evidence for penton base functionin endosome lysis outlined above was based pre-dominantly on properties of the isolated subunitwith little or no supporting data of a virologicalnature. In the first part of the study, we planned todetermine whether the cell-detach ing and detergent-like properties of the native penton base proteinwere reconstituted in the recombinant protein ex-pressed in bacteria. If so, then regions of the pro-tein that are important for these activities could beidentified by site-directed mutagenesis. In the sec-ond half of the study, mutations that interfered withfunctions of the recombinant penton base would betransferred into the viral chromosome to determinethe effect on viral reproduction.

APPROACH: To express penton base in bacteria,we copied the penton base gene from viral DNAusing the polymerase chain reaction (PCR), andcloned it in the bacteriophage T7 expression vectorsystem developed by Dunn and Studier of the BNLBiology Department. The resulting construct di-rected the expression of large quantities of recombi-nant penton base in bacterial cells, and although theprotein was insoluble in the cells, it could be recov-ered by denaturation and refolding. Deletion mu-tants were constructed by truncating the gene atseveral natural restriction sites. Point mutationswere constructed by annealing mismatched syntheticoligonucleotides to single-stranded copies of thepenton base gene and conversion to double-strandedDNA in an in vitro reaction. To transfer mutationsinto the viral chromosome, we took advantage ofthe fact that purified adenoviral DNA is infectiousif it is introduced (transfected) into cultured human

cells either as a calcium-phosphate precipitate orby electroporation. Furthermore, intact infec-tious viral DNA can be assembled in transfectedcells by recombination between overlapping,subgenomic DNA fragments. Therefore, wetransfected cells with plasmid DNA containingmutated copies of the penton base gene andoverlapping fragments of viral DNA.

TECHNICAL PROGRESS AND RESULTS:We first characterized the renaturation of recom-binant penton base. About 20% of the proteinwas pentameric, and this species could be sepa-rated from other products by ion exchange chro-matography. Refolded pentamers assembledquantitatively with purified viral fiber subunits,indicating that the penton base protein was re-folded into a conformation that closely resem-bled that of the native subunit. Analysis ofrefolded pentamers in the BNL STEM supportedthe conclusion that the protein species indeedwas pentameric. Alignment of penton basesequences from different viral serotypes suggest-ed that the protein has two highly conservedstructural domains that are separated by ahypervariable linker region. Each domain wasexpressed independently in bacteria. Thecarboxy-terminal domain but not the amino-terminal or the hypervariable domains precipitat-ed from solution below pH 6.5; this regionprobably accounts for the partition of pentonbase into a hydrophobic phase that was observedin earlier studies. Neither the intact protein norany of the fragments could permeabilize cells ina standard 51Cr-release assay, and fluorescein-conjugated penton base accumulated in intracel-lular vesicles but was not detected in either thecytoplasm or nuclei. We concluded that pentonbase has no Iytic activity, at least not in thecontext of the isolated subunit.

The recombinant penton base also has a cell-detaching activity that is indistinguishable fromthat of the native capsomer. Analysis of deletionmutants showed that this activity did not requirethe intact protein, and permitted the mapping ofthis activity to a 20 residue fragment within thecentral, hypervariable region of penton base.The 20 residue fragment contained the sequence


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arg-gly-asp (RGD), a motif that is specificallyrecognized by a family of receptors in the cellmembrane known as the integrins. Integrins medi-ate adhesion of cells to an insoluble matrix com-posed of proteins like fibronectin that are rich inRGD sequences. We predicted that the penton baseRGD competed with the extracellular matrix forintegrins, resulting in cell rounding and eventualdetachment from the matrix-coated substratum.This was confirmed by site-directed mutagenesis ofthe penton base gene to change the RGD sequenceto several unrelated sequences. All the point mu-tants had lost the cell detaching activity.

The possibility that the penton base in virions mightbind to integrins did not seem reasonable sinceadenoviruses attach to cells through the fiber sub-unit, and there was no precedent for any singlevirus using multiple distinct receptors. Further-more, the RGD sequence is not conserved in thepenton base proteins of all adenovirus serotypes,and functional RGD sequences can be demonstratedin many purified proteins that have no physiologicalinteraction with integrins in their normal context.To resolve whether the interaction between pentonbase and integrins is merely an artifact associatedwith the purified protein or actually is physiologi-cally significant, the penton base gene in the viralchromosome was mutated to change the RGD se-quence to four different unrelated sequences that intheory should not be recognized by integrins. Allfour viruses were infectious and reproduced to highyields in cultured cells, but the kinetics of infectionwas delayed by up to 10 hours depending on thetype of cells that were infected. Fn contrast to theendocytosis of wild-type virus, which essentially iscomplete within 30 minutes of virus attachment, themutant viruses attach normally but are endocytosedat a much slower rate. These and other experi-ments support the conclusion that adenovirus em-ploys two receptors, one that recognizes the fibersubunit and mediates attachment of the virus to thecell membrane, and an integrin which recognizesthe penton base RGD sequence and mediates virusendocytosis. In future research we will investigatethe interaction between these two receptors, wheth-er this dual receptor mechanism also is used by theadenovirus serotypes in which the penton base RGD

sequence has not been conserved, and the rela-tionship of this mechanism to viral host range.


1. Freimuth, P. and C. W. Anderson. HumanAdenovirus Serotype 12 Virion PrecursorspMu and pVI Are Cleaved at Amino-Termi-nal and Carboxy-Terminal Sites That Con-form to the Adenovirus 2 EndoproteinaseCleavage Consensus Sequence. Virology193: 348-355 (1993).

2. Bai, M., Harfe, B., and P. Freimuth. Muta-tions That Alter an Arg-Gly-Asp (RGD) Se-quence in the Adenovirus Type 2 PentonBase Protein Abolish its Cell-Rounding Ac-tivity and Delay Virus Reproduction in FlatCells. J. Virology 67: 5198-5205 (1993).

3. Bai, M., Campisi, L., and P. Freimuth.The Adenovirus-12 Penton Base Binds toIntegrin av/33 and Assembles In Vitro withAdenovirus-2 Fibers. J. Virology (1993,submitted).

4. Bai, M. and P. Freimuth. Penton Base-Integrin Binding is Sufficient for Infection byAdenovirus-2 (1993, in preparation).

5. Bai, M. and P. Freimuth. The RGD Se-quence in the Adenovirus-2 Penton Base isConstrained in an Active Conformation(1993, in preparation).


1. A National Institutes of Health proposal enti-tled, "The Dual Receptor Mechanism ofAdenovirus Infection" was submitted inOctober 1993.




$ - 0 -64,623

138,87764,920- 0 -


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H uman Melanocyte Transformation 92-17

B.M. Sutherland


JWIalignant melanoma is an invasive, often lethalcancer of human skin, fn spite of its increasingincidence and devastating human consequences, thecritical DNA a)teration(s) in melanoma are un-known. We have devised a scheme for a transfor-mation assay for human melanocytes which willidentify partial and fully oncogenic transformantsand their gene alterations. It is proposed to developthis system, use it to follow transformation ofhuman melanocytes, and to isolate transforminggene(s) from human melanomas.


PURPOSE: To develop cellular and molecularmethods for studying the response of human prima-ry melanocytes to radiation. Although malignantmelanoma is a disease of severe human conse-quence, the responses to UV of human primarymelanocytes are poorly understood, in large part aconsequence of their limited growth in culture,difficulty of in vitro growth, poor colony-formingability, and complications of molecular measure-ments due to their pigmentation. We plan to workout solutions to these problems, allowing assess-ment of the responses of primary humanmelanocytes to UV at the molecular and cellularlevels.

APPROACH: Primary cultures of human cells,including fibroblasts, keratinocytes and melanocytesare initiated from the foreskins of healthy neonates.Cells are exposed to UV of different wavelengthregions [254 nm, broad spectrum UVB (290-320nm), or broad spectrum UVA (320-400 nm)], andthe levels of pyrimidine dimers determined by thealkaline agarose gel method developed in my labo-ratory using the electronic imaging system of J.

Sutherland; in additions, survival of cells undernon-growth conditions (to simulate the situationin human skin) is determined by a new methodwe have developed using electronic imaging.

TECHNICAL PROGRESS AND RESULTS:We have developed a system for neoplastictransformation of primary human melanocytes:cells are exposed to UV or ionizing radiation,allowed to grow to express radiation-inducedmutations, and plated in soft agar under condi-tions in which unirradiated melanocytes can notgrow. Figure 1A shows a photograph of anelectronic image of unirradiated melanocytesplated in soft agar, which is non-permissive fortheir growth, while Figure IB shows a similarphotograph of UV-irradiated, neoplasticallytransformed melanocytes growing in soft agar(photographs were taken at the same magnifica-tion).

We have quantitated the frequencies of transfor-mation by ultraviolet radiation administered tothe melanocytes in a single dose, or the samedose administered to the cells in three incre-ments. Figure IC shows that the split dose re-gime gives much higher frequencies of neoplas-tic transformation than does the single dose forboth broad spectrum UVB (290-320 nm) radia-tion and for broad spectrum UVA (320-400 nm)radiation.

We measured the transformation frequencies inthree melanocyte strains, derived from skin ofthree normal individuals, and found that theydiffer strikingly in their responses: Strain 923shows only a low frequency of small, highlymelanized colonies after exposure to UVA orUVB, and no or only a slight increase if eitherUV regime was divided into three daily treat-ments. In Strain 926, no soft agar colonies overbaseline were detected in a single UVA or UVB


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Anchorage Independence Response ol UVIrradiated Normal Melano^yte Strain 928




vmlet Treatment

A. Untreated human melanocytes in non-permissive soft agar. B. UV-irradiatedhuman melanocytes in soft agar; fouranchorage-independent colonies are shown.C. Frequency of agar colonies inunirradiatcd human melanocytes. in cellsexposed to UVB in one exposure orfractionated into three exposures, and incells exposed to 1JVA in one exposure orthree exposures.

Figure 1


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treatment, but high frequencies (for UVA, ~ 60; forUVB, - 170/105 viable cells plated) of non-mela-nized colonies were observed in cells treated withthe same UV doses fractionated into 3 daily treat-ments. Strain 928 showed significant frequencies(~ 50/105 cells) of melanized colonies after treatmentwith single doses of UVA or UVB, and greatlyincreased frequencies (for UVA, ~ 130 for UVB,— 240/105) in the fractionated regime.

We also investigated the result of combined UV and7 irradiation of melanocytes. First, we found that asingle dose of 12.5 or 25 Cgy of 7 radiation effi-ciently transformed human melanocytes. AlthoughUVA also transforms the cells, the total transforma-tion frequency in cells exposed first to 7 radiationthen to UVA is less than that expected from the twotypes of radiation given separately. These dataindicate some interaction of responses to ionizingand UV radiation at the cellular or molecular leveland suggest that exposure to low doses of ionizingradiation does not increase the risk of melanomainduction in humans.


I. "Differential Response of Normal HumanMelanocytes in Culture to Single and Multi-ple Exposures of UV Radiation," Bennett, P.V. and Sutherland, B. M., Accepted byAmerican Society for Photobiology NationalMeeting, Chicago, IL June 26-30, 1993.


1. "Human Cell Transformation: UV-lonizingInteractions?", B. M. Sutherland, submittedto NASA in October 1993 for funding in FY1994.




$ - 0 -81,46390,757

- 0 -

NOTE: This project involves both human sub-jects and vertebrate animals.

€ xploratory Applications of X-Ray Microscopy;

Determination of the Higher OrderedStructure of Eukaryotic Chromosomes


J. Van't Hot


A x-ray microscope has the potential of providingstructural information bridging that obtained byneutron diffraction and electron microscopy. Withthis in mind, the scanning transmission x-ray micro-scope (STXM) at the NSLS was used to investigatethe structure and radiation sensitivity of metaphasechromosomes while simultaneously determiningwhat improvements, if any, were needed to bringthe microscope to a state of being generally usefulto biological structural investigations. Metaphasechromosomes are ideal objects for this purposebecause they have structure at all size scales and athickness matched to the absorption length of x-rayphotons.

Recent work concerned radiation damage toViciafaba chromosome structure, defined asmass loss, for unstained specimens in both thewet and dry states. Dried specimens remainundamaged after either single or multiple imagesat doses up to 2400 Mrad at wavelengths of 3.15or 3.64 nm. In contrast, wet specimens aredamaged irrespective of the imaging protocol.The damage induced by multiple exposures isgreater than that seen in a single exposure of thesame total dose. Thus, the rate of data collec-tion is greater than or equal to the rate of dam-age. The damage during multiple exposures ofwet chromosomes is influenced by several fac-tors. First, the fixative used influences theextent of radiation damage. Wet chromosomesfixed with glutaraldehyde are more resistant than


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those fixed with formaldehyde or osmium tetroxide.A second factor is ionic strength. Damage to wetchromosomes increases if the ionic strength de-creases below that at which chromatin undergoes aconformational transition. The mass of wet and drychromosomes is the same, consequently quantitativemeasurements can be made on wet specimens.Such measurements give a DNA mass fraction of39 +/- 8% for V. faba chromosomes.


PURPOSE: Scanning transmission x-ray microsco-py provides a new means to determine physical andchemical characteristics of metaphase chromo-somes. All eukaryotic chromosomes contain DNAand protein but they differ in size and shape. Afactor that may contribute to these differences is themass fraction of DNA per chromosome. Thispossibility is testable by measuring chromosomes ofspecies known to have genome sizes that differfrom that of V. faba.

APPROACH: The test consisted of measuring theisolated chromosomes of two species, Pisumsativum and Lathyrus odoratus. Each species has achromosome number of n = 7 and, respectively,genomes of 4.6 and 8.5 pg DNA. The averageamount of DNA per chromosome for the species is:V. faba = 2.2, L. odoratus = 1.2 and P. sativum= 0.6 pg. These values cover nearly a 4-foldrange which is broad enough to detect a differencein DNA mass fraction, if it exists.

TECHNICAL PROGRESS AND RESULTS: Theresults obtained from the experiments indicate thatthe DNA mass fraction of chromosomes of L.odoratus and P. sativum is about 39%, a valueidentical to that obtained for V. faba.

The finding that the DNA mass fraction per chro-mosome among species is a constant and indepen-dent of the amount of DNA per chromosome repre-sents a significant unifying step in biology. Further

steps now can be taken regarding the distributionof mass along the chromosome axis and the na-ture of the internal structure of the chromosomalcoil itself. To take these further steps, however,two requirements must be met. First, the reso-lution of the scanning transmission x-ray micro-scope must reach 20 - 30 nm and second, amethodology is needed to reduce radiation dam-age to the sample. These additional steps areinter-related, since x-ray dose (damage) increasesroughly as the sum of the square of resolutionand the square of the signal to noise ratio.


1. Williams, S., Jacobsen, C , Kirz, J., Lamm,S., and J. Van't Hof. Scanning Transmis-sion X-Ray Microscopy of Hydrated MitoticChromosomes. In X-Rav Microscopy III.A. Michette, G. Morrison, and C. J.Buckley, eds., pp. 108-112, Springer-Verlag, Berlin, 1992.

2. Williams, S., Zhang, X., Jacobsen, C ,Kirz, J., Lindaas, S., Van't Hof, J., and S.S. Lamm. Measurements of Wet Meta-phase Chromosomes in the Scanning Trans-mission X-Ray Microscope. J. Microscopy170: 155-165(1993).

3. Kirz, J., Ade, H., Fu, J. Jacobsen, C ,Lindaas, S., Oehler, V., Van't Hof, J., Wil-liams, S., Wirick, S., McGrath, W. J.,Mangel, W. F., and X. Zhang. ScanningTransmission X-Ray Microscopy at theNSLS. In X-Rav Microscopy IV. A. I.Erko, ed., Bogorodsky Pechatnik,Chernogolovka, (1993, in press).




$ - 0 -74,78379,973- 0 -


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uclear Techniques for Study of Biological Channels 92-27

K.G. Lynn


I he purpose of this project is to apply the instru-mentation of high energy physics to the study ofionic channels in biological membranes. The signalby which we study ionic channels is rectangularcurrent pulse, with unknown rise time ( < 10 ftsec),ranging in duration from the minimal resolutiontime to many seconds, and in amplitude from im-measurable (i.e., less than 1 pA) to nearly inA.Such signals share characteristics with the signalsroutinely detected in high energy physics. In acrude sense, many of the particles of physics arealso pulses of current, as seen by a detector. Thus,we propose to apply for the first time two standardtechnologies of high energy and nuclear physics torecording ionic channels.


PURPOSE: The purpose of this project is to ad-vance the patch voltage clamp technique for thestudy of the picoampere range currents flowingthrough single ionic channels in the cell membrane.The techniques of Physics and Physiology are ap-plied to the project. Two broad areas of instrumen-tation are addressed, i.e., (1) automated analysis ofsingle channel data, and (2) reduction of noise aris-ing from the electronics and electrodes, etc. of thepatch clamp; the available bandwidth of the elec-tronics will also be increased. In addition, we havebegun to use improved techniques developed tomeasure the time required for transitions betweenthe open and closed conformation of a channelprotein. We believe that such data will providevaluable information about the molecular eventsassociated with open-close transitions and placeimportant constraints of structural modeling of thechannel protein.

APPROACH: Real Time Analyzer (RTA).Analysis of the open-close kinetics of singleionic channels is presently carried out after-the-fact by first digitizing the data and subsequentlyanalyzing digitized records with automated ormore often semi-automated software. This pro-cess is very time consuming, requires largeamounts of data storage, and means that in gen-eral several days pass between gathering dataand seeing meaningful results. We have begunto develop a Real Time Analyzer (RTA) whichwill detect open-close transitions and perform theanalysis data as it is collected. It will distill theincoming stream of data into amplitude andduration information thereby vastly reducingdata storage requirements and, more important-ly, it will present meaningful results as the datais collected.

The software is a MS-Windows based applica-tion. The interface from the computer to thehardware is via GPHB bus. All the settings inthe hardware are placed by the software, throughdifferent levels of menu-driven commands. Thesoftware also is responsible for correcting thebase level drift and reading the collected data.These activities are all interrupt driven to insurethat no loss of data would ever happen. In thetime-slice in between, the software will histo-gram the data in a way preset by the user, andgraphically plot them on the screen. When run-ning with the stimulation cell voltage, the soft-ware traces the transit waveform first. The finaloffset transit waveform can be either the oneaveraged over a number of recorded transitwaveforms or drawn by the user using the draw-ing capability in the software, or both. Possibledata analysis is presently being added to thesoftware.

Low Noise Electronics and Improved ElectrodeTechnology. Noise is arguably the single great-


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est limitation of the patch voltage clamp technique.Since the signals to be measured are small - oftenless than 1 pA - and the duration of single channelevents is usually very brief, measurements requireextremely low levels of noise at relatively largeband widths. We believe that it is possible to re-duce the noise at a given bandwidth by a factor of3-4 below the best levels that are presentlyachieved. Such a reduction in noise can also beused to significantly extend the bandwidth (and thusimprove time resolution) while preserving adequatesignal to noise ratio. To achieve these reductionsof noise requires attention to every detail of thetechnique. We have initiated the development ofelectronics with greatly reduced noise and are in-vestigating various materials (selected for lowdielectric loss) for the construction of input connec-tors, electrode holders, and the electrodes them-selves. In the frequency range of greatest impor-tance to most patch clamp recording situations (DCto about 20 kHz), dielectric noise from the electron-ics, connectors, holders and electrodes is a veryimportant - often dominant - noise source. We aretheoretically and practically addressing this sourceof noise and further developed the present under-standing of charge sensitive preamplifiers.

Channel 'Rise time' Measurements. For these mea-surements we have selected a channel (VDAC) withan extremeiy high open state conductance; singlechannel currents larger than lnA can be measured.At the highest bandwidth, which allows an adequatesignal to noise ratio, we measure the rise time ofcurrent at open-close and close-open transitions.This rise time is compared to the system rise time;if measurable differences can be resolved they canbe attributed to the time involved in the channelprotein molecule undergoing the conformationalchanges involved in gating. Ultimately, we believethat we will be able to use bandwidths of 1-2 MHzin these measurements (about 50 times greater thanused previously) and achieve a time resolution ofabout 100 nsec. If our time resolution is adequateto measure the phenomenon, we will investigate avariety of experimental interventions. This will bethe first real attempt of measuring the rise time of acell opening.

TECHNICAL PROGRESS AND RESULTS:Real Time Analyzer (RTA): Development of asecond generation prototype of the RTA hasbeen completed during the past year. Softwaredevelopment for this instrument is continuing atthe time of this writing, and is nearing comple-tion. Our testing of this prototype has conclu-sively verified the feasibility of our approach.The present prototype utilized comparators todetect threshold crossings of up to seven differ-ent current levels (different channels orsubconductance levels of the same channel).The timing of threshold crossing is measuredwith 1 /xsec resolution, which is. therefore, theaccuracy of measured channel event durations.Up to 1,000,000 channel events per second canbe continuously analyzed; this rate exceeds themaximum event rate for any channel type stud-ied to date. The RTA is capable of acquiringdata continuously or in pulse-mode, i.e., onlyduring selected relatively brief intervals definedby experimental interventions (usually stepchanges in the voltage across the patch mem-brane). Software algorithms (and appropriatehardware) have been developed that effectivelycorrect for slow baseline drift and which candetect more catastrophic fault situations andappropriately interrupt data collection. Thisinstrument will be checked by a couple of biolo-gy labs to determine its general use, under vari-ous experimental conditions.

Two of these prototypes have been completed toallow more rapid laboratory testing of the RTA.The results of these tests will be used to deter-mine enhancements to the hardware and softwarewhich will better suit the diverse needs of theexperimental community. We have also begunto explore the feasibility of developing a digital-signal-processor (DSP) based version of theRTA. Such an instrument will be more versatilethan the present hardware solution, although themaximum rate of event detection and the timeresolution might be somewhat reduced. In rela-tion to such a DSP-based instrument, we are alsoinvestigating other detection methods which mayallow operation at lower signal-to-noise ratiosand more reliable detection of subconductancelevels.


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Low Noise Electronics and Electrode Technology:Theoretical work on the origins, characterization,and reduction of all noise sources involved in thepatch voltage clamp technique has continued duringthe past year. Further improvements in the noisecontribution from electrodes have been accom-plished, which have shown this previously dominantnoise source can be reduced to levels that are onlya fraction of those arising from the electronicsthemselves. Thus, our present emphasis is oncontinued reduction of electronic noise. New JFETinput stages are under design, which more appro-priately match their input capacitance to otherunavoidable capacitance (stray capacitance, thefeedback capacitor, plus capacitance associated withits reset mechanism, and, of course, the capacitanceof the electrode and its holder). Our work withdielectric noise sources has shown that the appro-priate input capacitance arising from the JFET stageis significantly less than the total of the capacitancejust listed throughout most of the frequency rangethat is typically important to this application. (DCto more than 100 kHz). Low temperature (80-150"K) operation of the input state is also beinginvestigated to reduce noise. Our work has beenprimarily aimed at the specific needs of the patchvoltage clamp application; nevertheless, many ofthe results will probably prove useful for the devel-opment of ultra-low noise charge amplifiers com-monly used in various detectors.

Channel Risetime Measurements. The major exper-iment project that has grown out of this work is aneffort to measure the time required for conforma-tional changes between the open and closed statesof an ionic channel with an exceptionally high sin-gle channel conductance (VDAC). To date, wehave achieved a time resolution of about 1 /xsec,which is an order of magnitude better than has beenachieved previously. As already noted, we believethat with full utilization of the improved electronicsand techniques we are developing , we will eventu-ally be able to achieve 100 nanosecond time resolu-tion. Cooling of the channel protein significantlyslows its gating kinetics. It has been shown that thepatch and its channels can be successfully cooled to-40"C in solutions containing ethylene glycol. Wewill use such cooling to effectively enhance ourtime resolution by as much as 2-3 orders of magni-

tude. Together these techniques should provideunprecedented resolution of the open-close con-formational changes of this channel protein.

In order to dependably study the VDAC channel,it has proved necessary to produce the channelprotein ourselves. Thus, techniques to preparemitochondrial VDAC protein from rat liver havebeen developed at Rush Medical Center. Theliver of a single 250 gram rat can yield enoughprotein for roughly 6 months of experiments.Once the protein was isolated, it was necessaryto develop methodology to reconstitute theVDAC channel into liposomes (large phospholip-id vesicles). This methodology has just recentlybecome routine at Rush. Patch electrodes (tipdiameter = 1 /tm) readily seal 30-50 fim diame-ter vesicles. A continuing problem is thatVDAC channel activity usually disappears withina minute or so following seal formation andvoltage activation of the channels when thepreparation is bathed in high (typically 3 M)concentrations of KC1. Such concentrations arerequired to maximize the current through theopen channel, thereby providing the best signal-to-noise ratio at a given bandwidth. Even so, aconsiderable amount of data has been accumulat-ed during the past year.

Transitions between the open and closed states ofionic channels have traditionally been viewed asinstantaneous. We believe that our efforts tostudy these transitions themselves will add a newdimension to the investigation of the conforma-tional rearrangements of these molecules, and isnecessary for structural modeling of the channelprotein. Our preliminary results at room tem-perature show that the channel molecule is capa-ble of sub-microsecond transitions between theopen and closed state, but that slower processesare also involved which can be interpreted as amore gradual (~ 10 /tsec) stabilization of a newconformation following a transition.


We have presently written a letter in collabora-tion with the Medical Department to requestfunding from OHER. Hopefully, a proposal


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which is already drafted will be funded through thispart of DOE. Other avenues of funding are alsobeing explored as two companies are interested inmarketing the RTA Instrument. Work on this pro-ject will continue into FY 1994 by internal fundingby Rush Medical College.




$ - 0 -68,39868,033

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I nduction and Repair of Double-Strand! • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Breaks in the DNA of Human Lymphocytes


M.A. Bender


I n January of 1992, an LDRD Proposal was fund-ed to determine the numbers of DNA double-strandbreaks and the rate of their repair in lymphocytesafter irradiation of whole blood samples from alarge population of healthy BNL volunteers. Theobjective was to perform enough preliminary exper-iments to select suitable protocols and demonstratefeasibility for submission of a proposal to the DOEfor continued OHER support.

We have been able to test and evaluate six potentialcandidates for the required protocol for DNA ex-traction, five of them were available as "kits" fromcommercial suppliers. One in particular, basedupon lysis of whole blood with the cationic deter-gent dodecyltrimethlamonium bromide and precipi-tation of the DNA with cetyltrimethylamoniumbromide, is especially promising, through somevariability in the precipitation of DNA from irradi-ated blood remains an issue. Agarose gel electro-phoresis of DNA, and molecular weight determina-tion have been assessed, and feasibility demonstrat-ed, though more samples need to be evaluated toobtain a better measure of sample-to-sample vari-ability.

This project allows for additional developmentalwork to be performed in order to strengthen thedata necessary for future support work.


PURPOSE: In order to directly measure theability of peripheral blood lymphocytes fromhuman subjects to repair radiation-induced DNAdouble strand breaks, a simple and quick methodis needed to extract and purify DNA in reason-ably long and uniform pieces. For the agarosegel electrophoresis technique, we have found thatDNA of the order of several hundred kilobaseslong is suitable. During FY 1993, we had in-vestigated a number of methods, including "con-ventional" ones involving phenol extraction, andhad found a cationic detergent method involvinglysis of whole blood with dodecyltrimethy-lamonium bromide and selective precipitation ofthe DNA with cetyltrimethy-lamonium bromideto be particularly convenient, especially as itavoids phenol and also requires no repetitivehandling of blood fractions that could bebiohazardous. The only real problem with thismethod is that some blood specimens, especiallythose from subjects with low white counts, tendnot to precipitate properly, especially if the cellsare irradiated beforehand as is required for theproposed measurement. During FY 1993, weundertook to find a suitable solution to theseerratic recoveries, as well as to do repetitiveagarose gel runs to optimize running and analy-sis parameters.

APPROACH; One of the companies that mar-kets the cationic detergent DNA isolation method


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in the form of a kit (GENOMIX, Washington Bio-technology, Inc., Bethesda, MD) announced atabout the end of FY 1992 that it would introduce adevice for automatically processing up to eightblood (or other cell) samples that would produceimproved and less variable DNA yields. As itappeared that the device used filtration to processand wash the samples, we decided to try that ap-proach, though manually, ourselves.

TECHNICAL PROGRESS AND RESULTS: Us-ing various filters of different materials of the typesused for sterilization filtration of water, media,etc., in various disposable and reusable syringe-typeholders, we found that we could indeed recovergood yields of DNA even from extractions forwhich no visible DNA precipitate formed uponcetyltrimethylamonium bromide addition. Somedifficulties were experienced with back-flushing thefilters to remove and redissolve the DNA (mechani-cal problems because none of the filter holdersavailable included any support for the "top" of thefilter since they were not designed with back flush-ing in mind). It should be no problem to design afiltering apparatus to obviate these difficulties, how-ever, should we find funding for the DNA doublestrand break repair project.

We were able during FY 1993 to test the newGenomix automated extraction device. We foundthat it did, indeed, recover DNA in high yields asadvertised, even from low white count and irradiat-ed samples. However, the cost is high (~ $30,000),and we believe it more sensible to design our ownfilter holders and process samples manually.

We were also able to evaluate the agarose gel-auto-matic analysis system we proposed to utilize for ourdouble strand break repair measurement during FY1993. We have adopted 0.5% agarose gels asbeing the lowest concentration that can be reason-ably handled without inordinate risk of breakage.

With the fairly large size of the DNA from thecationic detergent method, we found that contin-uous field electrophoresis allowed so much "pil-ing up" of the larger DNA near the origin thatthe automated reader designed by Dr. JohnSutherland (Biology) was sometimes unable toobtain a stable molecular weight estimate. Wetried two things which have corrected this diffi-culty. First, we added an additional, larger sizemarker, selecting bacteriophage T4 at 170 Kb.Second, at the suggestion of Dr. Sutherland, weexperimented with using a pulsed unidirectionalelectrophoresis field to "expand" the spacing ofthe higher molecular weight molecules in the gellane. We have adopted a regimen of 0.5 sec,100V pulses spaced every 10 sec. for a total runtime of ~ 16 hr. This gives good, consistent re-sults. For human peripheral blood lymphocytes,we have found that unirradiated samples averageabout 100 Kb (length average MW), and that adose of 100 Gy of l37Cs gamma rays reducesthis by about half, and that a two-hour recoveryperiod restores about 80% of the original molec-ular weight, which seem quite acceptable num-bers for the sort of repair capacity survey wecontemplate.


None has yet been obtained. It is hoped to in-clude this new measure of repair capacity in anew study that will be proposed for funding inFY 1995.




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NOTE: This project involves both human sub-jects and vertebrate animals.


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enetics of Drug Addiction 93-22

C. Ashby


1 he purpose of the current proposal was to use thetechniques of single cell recording andmicroiontophoresis to examine the function of pre-and postsynaptic dopamine (DA) receptors in themesocorticolimbic system of anesthetized Fischerand Lewis rats. Specifically, the specific experi-ments to be conducted for this proposal were asfollows: 1) determination of the number, as well asthe firing rate and firing pattern, of spontaneouslyactive A9 and A10 DA cells; 2) examination of theresponse of spontaneously active A9 and A10 DAcells to intravenously administered cocaine (0.25-8mg/kg), 7-OH-DPAT (D3 agonist, 0.5-8 Mg/kg),(±)-apomorphine (D2 autoreceptor antagonist, 1-32/ig/kg) and methamphetamine (0.1-3.2 mg/kg); 3)examine the response of spontaneously active or L-glutamate-activated neurons in the nucleusaccumbens (NAc), striatum and medial prefrontalcortex (mPFc) to the iontophoresis (2.5-80nanoamperes) of DA, GABA, cocaine and the D1and D2 receptor agonists SKF 81297 andquinpirole, respectively; 4) the response of 5-HTneurons in the dorsal raphe to 8-OH-DPAT (0.1-2/tg/kg i.v.) and cocaine will be determined 5); theresponse of mPFc and NAc cells to DA, SKF81297, quinpirole and 5-HT will be ascertainedafter the acute and chronic administration of co-caine and 6) use the technique of in vivomicrodialysis in freely moving rats to measureextracellular levels of DA in the NAc and mPFc.

The original statement of work stated that a stimu-lating electrode would be placed in the A10 area, toisolate cells in NAc and mPFc with a glass micropi-pette and quantitate the effects on neuronal firingrate and pattern. However, it was decided to omitthis as recent data indicate that the stimulation ofthe VTA region produces the release of DA, 5-HTand other neurotransmitters in the NAc and mPFc,

thereby making it difficult to interpret the re-sults, ft was decided to use the technique ofmicrodialysis, which allows the exact measure-ment of extracellular levels of neurotransmittersin the brain of awake, freely moving animals.Thus, the interpretation of the results is morestraightforward.


PURPOSE: Previously, it has been shown inanimals and humans that there are significantdifferences between individuals regarding vulner-ability to self-administer addicting drugs. Inaddition, numerous studies indicate that this vul-nerability has a strong genetic component. Re-search over the past three decades has demon-strated that the rewarding or reinforcing effectsof addicting drugs is mediated by dopaminergicneurons in the mesocorticolimbic areas. Conse-quently, one might hypothesize that the differ-ences between individuals regarding drug abusemay be related to differences in the function ofDA neurons in the mesocorticolimbic system,which may be genetically controlled. For obvi-ous ethical reasons and technological, this hy-pothesis can't be readily tested using human sub-jects. However, recent studies have shown thatthere are marked differences between two strainsof rats, F344 (Fischer) and Lewis rats, with re-gards to their propensity to self-administer drugsof abuse and their behavioral response to variousdrugs. Furthermore, these strains of rats aregenetically distinct and have been inbred forover 80 generations.

Thus, based on the above information, it wasdecided to use the techniques of in vivo extracel-lular single cell recording and iontophoresis andmicrodialysis to examine the responsiveness ofpre- and postsynaptic DA receptors in the


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mesocorticolimbic system. In addition, as a con-trol, the responsiveness of DA receptors in thenigrostriatal system, which does not play a signifi-cant role in mediating the rewarding effects ofdrugs of abuse.

The long term objective of this project is to system-atically examine the dopaminergic function in themesocorticolitnbic system of F344 and Lewis ratsusing behavioral, biochemical and electrophysio-logical techniques. Data obtained from this propos-al will be used as an aid to write a grant to NfDAto obtain additional funding.

APPROACH: The techniques listed above will beused to examine dopaminergic function in themesocorticolimbic system in the F344 and Lewisrats.

TECHNICAL PROGRESS AND RESULTS: A.Effect of spontaneously active DA cells and firingrate: Overall, the Lewis rats had 70% fewer spon-taneously active DA cells in both the A9 and A10regions compared to the F344 rats. However, therewas no difference in the mean baseline firing rateof the DA cells isolated. The data regarding thefiring pattern of the cells is currently being ana-lyzed.

B. Response of A9 and A10 DA cells to cocaine,apomorphine, 7-OH-DPAT and methamphetamine:Overall, there was no difference between the F344and Lewis rats regarding the response of A10 DAcells to cocaine, methamphetamine and apomor-phine. However, the A10 DA cells in the Lewisrats were 2 times more sensitive to 7-OH-DPAT.There was no difference between the F344 andLewis rats concerning the sensitivity of A9 DAcells to apomorphine and 7-OH-DPAT. However,the A9 DA cells in the F344 rats were significantlymore responsive to cocaine compared to thoseisolated in the Lewis rats.

C. Behavioral Studies: In a series of tests de-signed to measure "emotionality" or anxiety, itwas observed that Lewis rats were significantlymore emotionally responsive or anxious com-pared to F344 rats.

D. Microdialysis Experiments: I also examinedthe effect of cocaine (3, 10 and 30 mg/kg i.p.)on extracellular DA levels in the NAc of F344and Lewis rats. Overall, there was no signifi-cant difference in DA levels between the strainsafter 3 or 10 mg/kg i.p. of cocaine. However,following a 30 mg/kg i.p. dose of cocaine, theincrease in extracellular DA levels measured inF344 rats was significantly greater than in Lewisrats.


1. Strecker, R.E., Eberle, W.E. and Ashby,Jr., C.R. (1993) Cocaine-induced changes inextracellular dopamine (DA) levels in thenucleus accumbens of Fischer and Lewisrats. Soc. Neurosci. Abstr. 19: 1825 (Ab-stract no. 745.2)


1. Based on preliminary data obtained from thisproposal, a grant was submitted to NIDA onJune 1, 1993. This grant will be reviewedon Oct. 12-15, 1993.




$ -6887



NOTE: This project involves vertebrate ani-mals.


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ew Directions for the Development

and Utitilization of BNL Facilities

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Qn the previous page: A section of a SuperconductingWiggler for the High Gain Harmonic Generation Experiment.

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igh Gain Harmonic Generation Experiment 92-07


1 he purpose of this proposed research is to devel-op the technique of frequency multiplication in aFree-Electron Laser (FEL). The electron beam ofthe BNL Accelerator Test Facility will be used in awiggler assembly to triple and amplify the seedradiation provided by a CO2 laser. This technique isan essential element in the UV-FEL User's Facilitybeing proposed by the NSLS department. BNL willbe in a better position to substantiate the technologyrequired for such a facility.

A successful demonstration of the high gain har-monic generation principle will be an important steptowards the realization of high power, tunable veryshort wavelength radiation sources. FELs based onthis principle fall into the category of fourth genera-tion synchrotron radiation sources and will play asignificant role in photochemistry, atomic andsurface physics, biology and other sciences. There-fore this proposal supports the mission of the BasicEnergy Science Division of the Department ofEnergy.


PURPOSE: The seeded single pass FEL has manyadvantages over other FEL concepts. The outputbandwidth is controlled by the input seed, limitedonly by the pulse length, and a bandwidth of 10"4 ispossible. Similarly, the frequency stability is alsocontrolled by the seed; hence the electron beamenergy stability influences only the output intensityfluctuations, and the requirement on the energystability is largely relaxed. Another obvious advan-tage is that the mirror loss and damage problems ofFEL oscillators are eliminated. In addition, there isno need for a long train of micropulses. The elec-tron beam can consist of single micropulses with

I. Ben-Zvi, S. Krinsky,and L.H. Yu

the high repetition rate available from asuperconducting linac. Thus, it is possible toachieve very good energy stability and highaverage power.

There are powerful, high repetition-rate tunablelasers operating in the IR and visible frequencybands that may be harmonic-multiplied into theVUV and used as seed lasers for the FEL ampli-fier. The interest in harmonic generation inFELs stems from the limitations of conventionallaser harmonic generation techniques, such aslow conversion efficiency, susceptibility to dam-age and limited tunability.

The generation of harmonics followed by expo-nential growth and wiggler tapering has beenproposed and studied in detail as the basis for aUV-FEL User's Facility at BNL. However, thecomplete process of generating the harmonics byprebunching in the fundamental and amplifica-tion in a wiggler tuned to the harmonic has notbeen demonstrated experimentally yet. Thepurpose of the High Gain Harmonic

Generation Experiment is to pursue this studyexperimentally.

APPROACH: In the proposed harmonic gener-ation experiment, we will demonstrate thebunching of a 30 MeV electron beam by a CO2laser of about 1 MW input power. We will studythe super-radiant growth of the third harmonic ata wavelength of 3.47 microns, the exponentialgrowth regime, and finally a tapered wigglerFEL amplifier section. We would like to verifyour theoretical models and to answer importantquestions such as the effect of electron beamparameters on the coherence of the FEL, theeffect of wiggler and alignment errors, and thehigher harmonic contents of the FEL output as afunction of the level of saturation.


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The harmonic generation experiment is proposedfor the BNL Accelerator Test Facility (ATF). Wehave selected the parameters of the harmonic gener-ation experiment to match the electron beam param-eters that have already been demonstrated experi-mentally at the ATF. These include a normalizedrms emittance of 4 p mm mrad at a peak current of130 amperes, an energy of 30 MeV, a CO2 oscilla-tor with a power of a few MW and solid-stateoptical chopping of the CO2 laser co 10-100 pico-second long pulses.

We use an electromagnet wiggler constructed ofmany short sections that may be powered indepen-dently. The technology of a wiggler suitable for thispurpose has been developed at the National Syn-chrotron Light Source at BNL under a previousLDRD project. We use a ferromagnetic yoke ma-chined out of a solid block of low carbon steel. Asuperconducting NbTi coil is wound continuouslyalong the yoke, with the winding direction alternat-ing every half period. The magnetic field of thisundulator is very uniform even for operation abovesaturation.

TECHNICAL PROGRESS AND RESULTS: Wehave done a detailed magnetic design of the variouswiggler sections. This included 2D simulations ofthe peak field, load curve and saturation behaviorof the wiggler, 3D simulations of the parabolic polefaces and more. For the dispersion magnet, wehave analyzed the magnetic design, determined thefocussing properties, and considered the effect ofdepartures from ideal design on the FEL gain. Inthe modulator and radiator wigglers we have ana-lyzed the effects due to the ends of the wiggler anddeveloped methods for their :orrection. In addition,the localized field produced by a trim coil for hori-zontal beam steering has been investigated.

We have assembled a few wiggler sections with theparameters of the radiator wiggler of the High GainHarmonic Generation Experiment, that is a wigglerperiod of 1.8 cm, a gap of 8 mm and parabolicpole face focussing. The length of each section isabout 25 cm. To test these wiggler sections wehave developed a cryogenic wiggler magnetic mea-surement system. The system scans the wigglerwith a computer controlled Hall-Probe cluster in a

liquid helium environment to determine the fieldquality.

In cryogenic test we have determined that thewiggler prototype sections performed to the peakfield predicted by the load curve calculations andhave begun a study of the wiggler errors. Inparticular we have identified two error sourcesthat have been introduced in the manufacturingand testing process and we are working on theirreduction.

The beam line of the experiment has been de-signed, detailed measurements and improvementsof the seed laser have been made, electron beamdiagnostics and optical diagnostics have beendesigned and are under construction. Supportstands have been designed and built for thevarious beam line elements (dipoles,quadrupoles, steerers, diagnostics, vacuum etc.)The quadrupole magnets have been magneticallymeasured. The beam line magnets have beenplaced in position and surveyed. The beam dumphas been built.


1. I. Ben-Zvi, A. Friedman, C M . Hung, G.Ingold, S. Krinsky, L.H. Yu, I. Lehrmanand D. Weissenburger, Design of a Harmon-ic Generation FEL Experi- ment at BNL,Nucl. Instr. & Meth. in Phys. Res. A318,(1992).

2. X. Zhang, I. Ben-Zvi, G. Ingold, S. Krinskyand L.H. Yu, Analysis of thesuperconducting Wiggler Magnets for theATF Harmonic Generation Experiment,Nucl. Instr. & Meth. in Phys. Res. A331,689 (1993).

3. G. Ingold, I. Ben-Zvi, S. Krinsky, D.Lynch, J. Sheehan, L. Solomon, M.Woodle, L. Yu, X. Zhang, W. Sampson, K.Robins, I. Lehrman, R. Heuer, J. Sheehan,D. Weissenburger, A Superconducting ShortPeriod Undulator for a Harmonic GenerationFEL Experiment, Proceedings 1993 Particle


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Accelerator Conference, Washington, DC, May17-20, 1993.

4. J. Sheehan, R. Heuer, I. Lehrman, D.Weissenburger, G. Ingold, L. Solomon, I. Ben-Zvi, L. Yu and M. Woodle, Design and Fabri-cation of the Harmonic Generation FEL, Pro-ceedings 1993 Particle Accelerator Conference,Washington DC, May 17-20, 1993.

5. L. Solomon, G. Ingold, I. Ben-Zvi, S. Krinsky,L.H. Yu, X. Zhang, W. Sampson, M. Garberand K. Robins, Magnetic Field Measurementsof a Superconducting Undulator for a HarmonicFEL Experiment at the NSLS, Proceedings1993 Particle Accelerator Conference, Wash-ington, DC, May 17-20, 1993.

6. I. Ben-Zvi and Xu Z. Qiu, High precision beamalignment of electromagnet wigglers, SPIEConference Proc. Vol. 2013, Electron BeamSources of High-Brightness Radiation (1993)

7. G. Ingold, I. Ben-Zvi, S. Krinsky, D.R.Lynch, J. Sheehan, L. Solomon, M.H.Woodle, L.-H. Yu, X. Zhang, W. Sampson,K. Robins, I.S. Lehrman, R. Heuer, J.R.Sheehan and D. Weissenburger,Superconducting Short-Period Undulator fora Harmonic-Generation FEL Experiment inthe Infrared, SPIE Conference Proc. Vol.2013, Electron Beam Sources of High-Brightness Radiation (1993)




$ - 0 -100,000100,000- 0 -

unneling Microscopy Studies of Nanoscaie Structures 92-22

M. Strongin and B. Ocko


Scanning Tunnelling Microscopy (STM) hasemerged as an important technique in vacuum andelectrochemical surface science since it provides anatomic, real space view of surfaces. The applica-tion of STM could have significant impact onexisting scientific programs at BNL and, in ourview, such a device was necessary to augmentmany programs in Solid State Physics. The prima-ry purpose of the present program was to applySTM to problems in low temperature physics andthe electrodeposition of metals on gold surfaces.The low temperature measurements required thedevelopment of an ultra stable low temperaturemechanical scanning head and, at present, this has

not been successfully done. The electrochemicalstudies have been carried out with a commercialinstrument.

The low temperature STM was to be used forexperiments on the Coulomb blockade which canbe observed at low temperatures, and is due tothe charging energy when electrons tunnel ontosmall grains. It is interesting that the inverse ofthis effect is seen in photoemission core levelstudies on small grains where an energy shiftoccurs due to the charge left behind on an isolat-ed grain after the photoemission process.

The electrochemical STM studies were carriedout to complement ongoing x-ray scattering andconventional electrochemical studies of surfaces.


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Whereas the x-ray scattering is an extremely power-ful technique when surfaces are well-ordered, STMoffers significant advantages when surfaces do nothave long range order. Additionally, the nucleationand growth mechanism can be studied.


PURPOSE AND ACCOMPLISHMENTS: Vacu-um STM Instrumentation: A home built STM wasdesigned and assembled which incorporated anelectrical remote control scheme for the coarse tip-approach. The unique feature of this design is thecompact and rigid STM body which has the poten-tial to adapt to different operating environments.The performance has been tested down to 5.2"Kwhere atomic imaging was obtained. However,further tests and improvements are needed forreliable operation at low temperature. In FY-94 thecollaboration with Professor K. Likharev on Cou-lomb blockade effects in clusters will be continuedat SUNY-Stony Brook. Likharev and co-workershave proposed using this effect for a new genera-tion of electronic devices.

Electrochemical STM Instrumentation: One of themost critical limitations of STM is the mechanicaldrift of the images. Until recently, this drift hashampered our ability to carry our precise measure-ments. In collaboration with Digital Instruments, itwas determined that the primary drift was due tomechanical creep of the tip. A new tip holder wasdesigned and the drift has been significantly re-duced. The electrochemical measurements in basesolutions require the use of a Pt/Ir tip and the tech-nology required for these experiments was devel-oped.

Moire Pattern on a Graphite Surface: A hexagonalsuperlattice on graphite has been observed with aperiod of 66 A. Direct measurement of the anglebetween lattice vectors confirmed that thesuperlattice is a Moire pattern caused by a 2.1degree rotation of the topmost (0001) plane withrespect to the bulk. The STM corrugation of 2.6A is not due to physical buckling, but to differencesin the electronic structure between AA-stacked,normal AB-stacked, and rhombohedral ABC-

stacked graphite. The high tunneling current ofAA-stacked regions is in agreement with thehigh density of states at the Fermi level calculat-ed for AA graphite. Our observation provides abasis for a comparative study of surface elec-tronic structures with different subsurface layerconfiguration.

Atomic-images Near Steps of Graphite Cleavedin Stearic Acid: Acid treated HOPG is a popu-lar substrate for STM studies of organic mole-cules, biological materials and clusters. Wefound that HOPG samples cleaved under stearicacid [CH3 (CH2) 16COOH] solution frequentlyexhibit a f/3xV3) R30" superstructure togetherin coexistence with the underlying honeycombgraphite lattice. In contrast, a triangular latticeis commonly observed for AB-stacked graphite.These structures depend on the bias voltage.After preliminary analysis of our data, we con-clude that the superstructure originates from ionsintercalated under the top (0001) graphite sheetthrough step edges. The STM option is due toan electronic modulation from ordered ions.

C60 Thin Film Modified by Au Overlayers: Wehave carried out STM studies of C60 adsorbedon polycrystalline gold films. The STM imagesof the gold films shows large (— 1000A) atomicflat regions. These are Au(l 11) facets whichcan be identified from the images by their atom-ic lattice structure. 1.7 M.L. of C60 was addedby evaporating C60 powder at 500"C. STMimages show well ordered close-packing C60molecules on both the first C60 layer and for thesecond layer. After additional 2 A of Au wasevaporated we found that the first Au layerforms clusters on top of the C60 film and thatthe underlying ordered C60 structure remainedunchanged. However, the second C60 layerchanged to a granular structure with an averagesize of 80 A. Additional Au clusters were alsofound on top of these C60 grains. These obser-vations indicate that the C60-Au interaction isstrong enough to destroy the C60 bulk crystals,however, monolayer C60 films are relativelystable. We are trying to understand the mecha-nism associated with the second C60 layer.


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STM Study of M-DLN: We utilized STM to studymetal doped diamond-like carbon (M-DLN). Thisstudy complements a CRADA between BNL andDFD Corporation. Undoped DLN's, due to theirpoor conductivity, have not been directly imaged bySTM. However, M-DLN has a much higher con-ductivity and this facilitates STM imaging. Wecould image the morphology of M-DLN from themicron scale down to the atomic scale. Atomicstructures of these materials can also be imaged.We have studied M-DLN samples with differentdoping materials (i.e., Pt, Cr and W), differentdoping concentrations, different substrates anddifferent annealing processes. Comparing theseimages yields valuable information which may leadto optimize material properties. On the atomicscale, we found a graphitization induced by anannealing process. We were able to provide, forthe first time, atomic structure information on M-DLN in real space.

TI Electrodeposited on Au((l 11):Surface x-ray scattering and STM studies haveshown that at a sufficiently negative potential,electrodeposited Tl forms a rotated, hexagonallyordered adlayer on the Au(l 11) surface in both acidand alkaline solutions. Within this potential range,the STM images exhibits a Moire pattern due to therotation of the adlayer. The desorption of the Tladlayer proceeds differently in these two solutions.In acid solutions a rapid partial desorption of Tltakes place reducing the coverage by more than50% and leading to the loss of long-range order.The order-disorder transition is reversible in acidsolutions. This allows us to infer that the disorder-ing of the surface is caused by Tl oxidation ratherthan by gold surface oxidation.

Bi Electrodeposited on Au(l 11):An understanding of cation adsorption on oxidesurfaces is particularly important for oxygen andchlorine electrocatalysis, and on theelectrodeposition of metals. X-ray scattering andvoltametric studies on Au(l 11) single crystal elec-trodes have shown that the presence of bismuthcations shifts the oxide formation potential positive-ly. In addition, bismuth cations shift the oxidereduction potential negatively by about 100 mv.Our STM studies have confirmed the presence of Bi

species on the Au(l 11) surface in the potentialregion positive to a well ordered bismuth mono-layer and negative to O2 evolution. The shift ofthe oxide formation and reduction potentials wasalso observed in the STM imaging. Moreover,STM imaging clearly shows that the oxide sur-face is considerably rougher in the presence ofbismuth cations than for the intrinsic gold oxidesurface.

Au(100) Surface Reconstruction:Au(100) surface is the best electrocatalyst for O2

reduction in alkaline solutions. A particularfeature of the reaction on Au(100) is, that the 4ereduction occurs only in the potential regionwhere OH" chemisorption takes place with aconsiderable charge transfer, yielding aAuOH(12)- layer. At more negative potentialsOH" adsorption does not occur and the 2e-reduc-tion occurs as on the other Au low-index planes.A change from the unreconstructed to a recon-structed structure has been considered as a causeof the 4e-2e change in the reaction mechanism.


1. "Electronic effects in scanning tunnelingmicroscopy: Moire pattern on a graphitesurface", Rong, Z.Y. and Kuiper, P. Phys.Rev. B48 (Dec. 15, 1993—in press).

2. "Direct observation of the Au(100) recon-struction during the course of O2 and HO 2

reduction in alkaline solutions", Polewska,W., Vitus, CM. , Ocko, B.M. and Adzic,R.R. (J. Electroanal. Chem.— accepted forpublication).

3. "Adsorption of bismuth cations on oxidelayers of gold single crystal electrodes: Anvoltametric, x-ray reflectivity and scanningtunneling microscopy study", Wang, J.,Powleska, W., Ocko, B.M. and Adzic, R.R.(in preparation).


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z F Sources for Accelerator Physics 92-28! • • • • • • •R. Palmer


X h e Cluster Klystron is being developed as an11.4 GHz pulsed RF power source for future linearcolliders. One of the major goals of the project is toachieve high efficiency through the use of multiple,low current beams, through the use of a modulatedanode, and by not requiring RF pulse compression.The proposed experiment involves the fabrication ofthree magnetron injection guns (MIGs) enclosed ina common solenoidal magnetic field. The work iscurrently being done in collaboration with SLAC,Varian Corp., and MIT.


PURPOSE: Deeper understanding of the funda-mental principles of particle physics will require theconstruction of new accelerators capable of reachingthe TeV energy scale. Linear electron-positroncolliders, where the beams from two acceleratorscollide head on, are considered by many to offerthe best hope of reaching this new energy regime.The current SLAC electron linac is powered byklystrons that operate at a frequency of 2.86 GHz.Scaling studies have indicated that it should becheaper to build a linac that operates at a higherfrequency. Hence, there are currently a number ofprojects worldwide investigating sources capable ofproducing power around 11 GHz (X-band). Thesource must be capable of producing power outputsaround 100 MW, with a pulse length around 1

microsecond, and with the highest electrical effi-ciency possible.

For a given accelerating voltage in a klystron-type source there is an inverse relation betweenthe efficiency and the current. In a normalklystron, however, low current would imply alow power output. The basic idea of the clusterklystron concept is to get both high power andgood efficiency by using many low currentbeams in parallel.

The first clustered klystron device was construct-ed around 1960. However, because of the choiceof electron gun and the use of individual magnet-ic fields for guiding the beams through the de-vice, it resulted in a complicated, bulky assem-bly. The cluster klystron idea was resurrected in1989 by Palmer. Herrmannsfeldt, and Eppley.They proposed to use compact magnetron injec-tion guns as the electron sources and to put thewhole assembly inside one common solenoidalfield. This results in a more flexible, compactdesign.

If the cluster klystron project is successful insimultaneously achieving all the required physicsparameters, it could be a leading candidate forthe power source for s» future linear collider.However, another important question for anypotential source is economics. This includes boththe cost of constructing the source and the costof operating it. There is reason for hope withthis concept in both regards. The cluster klystronuses a mod- anode, so it does not require the


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expensive pulsed modulator used with conventionalklystrons. In addition, the mod-anode determinesthe length of the RF pulse directly, so expensivepulse compression systems are not required. Theexpected high efficiency should result in the lowestpossible operating costs.

APPROACH: Work on the cluster klystron projecthas been divided into four phases. The first is tobuild and test the operation of the high currentdensity, magnetron type guns at potentials up to125 kV. The initial arrangement will use two beamsand a single 4-ceII klystron cavity. This will allowus to test many of the fundamental properties of theultimate device including stability of the MIG,switching the current with the mod-anode and accel-eration of the beam off the symmetry axis of theelectric and magnetic fields.

The second phase will test the clustering concept byusing three beams with three 4-cell klystron cavi-ties. The third phase would involve increasing thedc potential up to 400 kV and lengthening thesolenoid to accommodate the 16-cell klystron cavi-ty. This would provide the first tests of achievinghigh efficiency. The fourth phase will test a com-plete cluster of three beams with three high effi-ciency klystron cavities.

TECHNICAL PROGRESS AND RESULTS: Theoriginal proposal was reviewed by three expertsselected by the DOE. The reviewers identified anumber of issues requiring further study.

The solenoid magnet was assembled in our experi-mental area in building 901. The solenoid wasconnected to a power supply obtained from theACS and a deionized water supply. The magneticfield profile was measured using a Hall probe.Good agreement was found with computer predic-tions. The good field region of the present solenoidis large enough for the initial phases of the experi-ment.

Detailed theoretical studies were done of the elec-tron beam behavior in the region of the MIG. Therange of potential, magnetic field, temperature andcurrent giving stable operation was identified.

Considerable progress was made in refining theengineering details for the phase 1 experiments.General layouts have been prepared for all therequired mechanical assemblies and a detailedcost estimate has been generated. A diagnosticschamber has been designed to measure the prop-erties of the electron beam produced by theMIG.

A preliminary design of the electrical systemshas been completed and some of the requiredpower supplies have been purchased.

Computer designs have been made of a set ofdiagnostic RF cavities for initial testing, as wellas a set of high efficiency cavities for powergeneration.

David Yu Associates, with whom we are collab-orating, has received a phase I SBIR grant tostart work on RF output couplers and combiners.These devices will be required to combine thepower from the three beams in the cluster.

We are now funded only by the LDRD funds,but are receiving equipment, both used andpurchased, provided by our SLAC collaborators.

ACCOMPLISHMENTS: Three presentationswere made at the International Workshop onPulsed RF Power Sources for Linear Colliders atDubna, Russia in July, 1993. A request forproject funding for FY 1994 and beyond hasbeen submitted to the Department of Energy.The proposal has been sent out by DOE for asecond round of outside review.




S -9894_



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An EBIS Source of High Charge State Ions up to Uranium 92-30


I h e objective of this project is to gain the neces-sary experience in a new area of applied scienceand technology, and to prove that the design of anElectron Beam Ion Source for RHIC applications isfeasible. The project will concentrate on the devel-opment of a prototype source of heavy ions, capa-ble of satisfying present and future programs. AnEBIS of high charge heavy ions, coupled to aradiofrequency quadrupole accelerator (RFQ) and asuperconducting linac, has long been considered.Such a system would offer many advantages: aspectrum of all ions up to uranium, a possibility toallow for future needs for a higher luminosity, anda simpler and more reliable operation requiring lessmaintenance, with substantially reduced manpower.

EBIS is an acronym of Electron Beam Ion Source.This type of source is primarily used to producehighly stripped ions, as high as fully stripped xenonand helium-like uranium. In a typical EBIS, a highcurrent density electron beam is launched along themagnetic axis of a high field solenoid. Coaxialwith the magnetic axis are positively biased cylin-drical electrodes (drift tubes), which together withthe magnetic field form a gated ion trap. Lowcharge state ions (or in some cases neutral atoms)are introduced into the trap and are then subjected,by the electron beam, to successive ionization byelectron impact. These ions are confined radiallyby the negative potential well due to the electronbeam space charge, and axial ly by an appropriatepotential distribution impressed on the electrodes.When the desired ion charge state is reached, theaxial trap potential is modified so as to expel theions from the source, i.e., one gate is opened.Throughout the whole cycle, the electron beam iscontinuously injected into one end of the trap, and

A. Hershcovitch, J. Alessi,and K. Prelec

collected at the ion extraction region outside theopposite end of the trap.

We fabricated, refurbished, and modified variouscomponents of an EBIS with a superconductingmagnet. From its test performance with severalion species, including uranium, we plan to testthe scaling laws for EBIS sources. These exper-iments and analysis will serve as a basis to even-tually design an EBIS with parameters compati-ble with machines such as RHIC. As a basis forthis project, we will use an almost completesuperconducting EBIS, called SuperEBIS, whichwe obtained from Sandia Laboratories on a long-term loan. The support authorized covered thefabrication of the stand to mount the source, aminor repair of the superconducting magnet, andfabrication and purchase of several componentsfor the source, including the fabrication of aMeVVA ion source for primary injection ofheavy metallic ions.


PURPOSE: The objective of this project is togain experience in the design and operation of anEBIS for production of high charge state heavyions. This includes the study of scaling laws forion production in an EBIS. From these results,we will then determine if this type of sourcecould fulfill the requirements of an injector forRHIC. Long-term objectives, if initial resultsare promising, would be to study improvementsto the design of such a source to obtain higherion intensities. These could include improve-ments to the electron beam intensity and studiesof performance with ion injection into the EBIS.This could ultimately lead to a proposal for anew injector for RHIC, in which an EBIS-RFQ-superconducting linac combination could serve


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as a compact injector for heavy ions, located veryclose to the Booster.

APPROACH: What made this project feasible wasthe fact that we were able to obtain on long-termloan from Sandia Laboratories, most components ofa state-of-the art, superconducting EBIS. Althoughabout one million dollars was invested in the devel-opment of this source at Sandia, it was never as-sembled due to the cancellation of the associatedatomic physics program. Our strategy was to fabri-cate the remaining components required for thesource, assemble and test it in stages, and thenproceed with experiments using the source. Al-though originally designed to produce low intensi-ties of almost fully stripped heavy ions for atomicphysics experiments, we planned to operate thesource in a regime more suited as an injector,attempting to produce higher intensities but lowercharge states (e.g. U4 6 +). This relaxes some of theparameters of the source-lower magnetic fields,lower electron beam energies, while requiringenhancement of the total electron beam current.Additionally, we planned to study methods for opti-mizing the charge states that are of interest to us.

TECHNICAL PROGRESS AND RESULTS: Anew test stand was fabricated and assembled. Thesuperconducting magnet, its liquid helium reservoir,and the helium recovery line were plumbed in. Themagnet was successfully energized, and magneticfield measurements were performed with a comput-erized magnetic field measuring set up that wasborrowed from W. Sampson's group. The maincoil was energized up to a field of 1.65 Tesla (inour planned near-term experiments, only one Teslais needed). Magnetic field measurements indicatethat the magnetic field is of excellent quality, as itcan be seen from the figure below, which is a plotof the magnetic field strength versus axial position.

In addition to the main coil, the outer coils wereenergized and their magnetic fields measured, aswell as the various possible combination of coils.We had a pleasant surprise in our ability to operatethe end coils bucking the main coil, to reduce thefringe field outside the main coil. Previous at-tempts to do so failed [R. Schmieder, et al., RSI6i , 259 (1990)].



0 50

• f

• /

/ Mainy


' • •

At 40 A



60 60 100 120

Axial Position - cm110 160 160

SuperEBIS was designed for its own unique elec-tron gun, which had never been tested. Wefabricated this electron gun and corrected somedesign errors. The gun was then mounted onthe top flange of SuperEBIS, and most of thevacuum chamber was assembled (a temporarybottom flange with a rubber O ring was used tocomplete the vacuum enclosure). Duringpumpdown and cooldown, a base pressure of 3 x10"9 Torr was reached (without baking the vacu-um system and with a large rubber O ring!).Based on these results, we are confident that thevacuum system can reach and even surpass abase pressure of 5 x !0 !0 Torr, which a RHICEBIS preinjector will need.

Following vacuum attainment, the electron gunwas successfully tested. With the superconduct-ing magnet energized, an electron beam waslaunched from the electron gun and detected ona split plate detector located at the opposite endof the superconducting magnet. Preliminaryresults are indicative of good alignment betweenthe electron gun and the magnetic field, as wellas good compression of the electron beam.

The drift tube section was assembled (some newparts fabricated), and installed in the vacuumchamber. The collector, ion extractor, andvarious lens were assembled. Some diagnosticswere fabricated and installed.

A MeVVA (metal vapor vacuum arc) ion source,which will be used for injection of heavy metal-lic ions into the SuperEBIS trap, was fabricatedand successfully operated with a total output ofabout 10 mA of titanium and aluminum ions


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(orders of magnitude above what is needed to injectinto SuperEBIS).

Due to the vertical mounting of the superconductingmagnet, and the absence of a magnetic shield,conventional time-of-flight (TOF) measurement, todetermine charge state distribution of the EBISgenerated heavy ions, is extremely difficult in ourpresent setup. To overcome these difficulties, wefabricated and have successfully done initial tests ona novel (in USA) TOF spectrometer that was in-vented by B.A. Mamyrin from Russia. In addition

to enabling us to perform on-line TOF measure-ments (due to its length of 46 cm versus about 2m of a conventional TOF spectrometer), thisdevice should have a higher resolution thanconventional TOF schemes.




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T-he Structure and Reactivity

of Electrode/Electrolyte Interfaces


J. Wang


Ihe objective of this research program is to devel-op a structural understanding of electrochemicalprocesses on an atomic scale by applying surface x-ray scattering (SXS) techniques for in situ structuraldetermination. Although many electrochemicalreactions are known to be highly sensitive to thestructure of electrode surfaces, the relationshipbetween the reactivity and the surface structureremains unknown for most processes. The initialeffort in applying SXS as in situ structural probefor identification and understanding of surfacereactions has been very fruitful. It is believed thatSXS will give a major impact to the field in nearfuture. All SXS measurements were carried out atthe National Synchrotron Light source at beam lineX22B and X22C in collaboration with RadoslavAdzic (Department of Applied Science, BNL) andBenjamin Ocko (Department of Physics, BNL).


PURPOSE: The intent of this project is to bring thestate-of-the-art x-ray scattering methodology intothe electrocatalytic studies on single crystal elec-trode surfaces. This will be followed by further

extension of our in situ techniques, such asscanning tunneling microscope (STM) and Ra-man spectroscopy. The combination of thesetechniques with conventional electrochemicalmethods will enable us to achieve a comprehen-sive understanding of the relationship betweenthe structure of an electrode surface and itsfunction in an electrochemical processes.

APPROACH: SXS measurements were firstcarried out at some well-understood simplesystems to establish appropriate sample prepara-tion-transfer methods and data analysis proce-dures. Then several generally interested systemwere studied in detail. The results demonstratedthe feasibility and the power of this in situ struc-tural probe.

TECHNICAL PROGRESS AND RESULTS: Insitu x-ray scattering techniques have been em-ployed for the first time in the determination ofthe structure of foreign metal adatoms on plati-num single crystal electrodes which are the bestcatalysts for many electrochemical reactions.Because of its high chemical reactivity, thepreparation and transfer of a clean andwell-ordered single crystal electrode into the cellfor x-ray scattering measurements is very diffi-cult. A method for protection of clean platinum


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surface from contamination during the transfer hasbeen developed. It utilizes the irreversible adsorp-tion of cations on the surface which prevents theadsorption of organic species in air during a sampletransfer after annealing in hydrogen flame andcooling in hydrogen gas. The cations can be elec-trochemical ly desorbed in the cell after the transfer,or a formation of monolayer of metal adatoms atunderpotentials can be studied with an addition ofthe same cations into the solution.

Using this method, in situ SXS techniques havebeen successfully applied to structural characteriza-tion of Pb, Bi and Cu monolayers on three lowindex platinum electrodes. Results from fitting thex-ray specular reflectivity curves yield informationon adatom coverage and their spacing from thesubstrate. An ordered structure with a rectangular4Pb-(3 xV3) unit cell has been determined byx-ray glancing angle incident diffraction forelectrodeposition of a Pb monolayer on Pt(l 11) inHC1O4 solutions. Formic acid and intermediates inits oxidation cause a modification of this structure.No incommensurate structures has been observed atelectrodeposited adlayers on Pt single crystal sur-faces; however, most adlayer structures found onAu and Ag are incommensurate. The unfilledd-electron shell of Pt may play an important role inthe formation of commensurate adlayer structure.Further studies with Pt single crystal electrodesshould be carried out, especially forelectrocatalytically interested systems.

As proposed, research was also carried out to studythe role of Pb, Bi and Tl adatoms on the low indexgold surfaces in the reduction of O2 and H2O2. Theswitch from two-electron to four-electron reductionof O2 corresponds to the structural phase transitionfrom a close-packed high coverage phase to a lowcoverage phase. For Tl in alkaline solutions, analigned-hexagonal phase was found at low Tl cover-age. This is the first example of an incommensuratephase formed with coadsorption of OH" anions inthe electrodeposited metal adlayers thereby provid-

ing a direct structure-reactivity correlation.Furthermore, the formation of a well-definedmetal oxide adlayer and metal cation adsorptionin the gold surface oxidation potential regionwere established by the combination of x-rayspecular reflectivity, STM and electrochemicalmeasurements.


1. "The electrodeposition of Pb monolayers onlow index Pt surfaces: an x-ray scattering andscanning tunneling microscopy study", Adzic,R.R.; Wang, J.; Vitus, C M . and Ocko,B.M., Surf. Sci., 293 (1993), L876.

2. "Electrochemical deposition of Tl monolayeron Au(l 11) in alkaline solution: rotationalphase transition and coadsorption of OH"anions", Wang, J.; Adzic, R.R. and Ocko,B.M., in preparation.

3. "Adsorption of Bismuth cations on oxidelayers of gold single crystal electrodes: anvoltammetric, x-ray reflectivity and scanningtunneling microscopic study", Adzic, R.R.,Wang, J.; Polewska, W. and Ocko, B.M., inpreparation.

4. "Structures and catalytic properties of Bimonolayer on Au(100), (110) and (111) elec-trode surfaces", Wang, J.; Adzic, R.R. andOcko, B.M., in preparation.

FOLLOW-ON FUNDING: "Structure andFunction in Electrochemistry", R. Adzic and J.Wang, funded by DOE, Chemical Science Divi-sion (KC-03-02-02) - FY 1994.


FY 1992 $ - 0 -FY 1993 98,834FY 1994 - 0 -


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I nvestigation of the Utility of Maximum-Entropy• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • a

Methods for the Analysis of Powder Diffraction Data


D.E. Cox


JLhe application of the Maximum-Entropy(MaxEnt) method to the detailed analysis of powderdiffraction data is being explored. MaxEnt will firstbe applied to high-resolution synchrotron x-ray dataas a means to locate light atoms in inorganic com-pounds containing heavier ones, which is a verycommon problem in structural studies ofpolycrystalline inorganic materials such as complexhigh Tc oxides and heavy metal oxide catalysts.Other goals are to use MaxEnt to provide details ofthe bonding electron density distribution, and forthe ab-initio solutionof unknown structures. Experi-mental work will be aimed at developing optimumstrategies for the collection of suitably accuratepowder data at beam-line X7A at the NSLS inorder to get the best possible structural information.


PURPOSE: The development of high-resolutionsynchrotron x-ray powder diffraction techniques atbeam-line X7A at the NSLS over the past few yearshas demonstrated the utility of such data for thedetailed structural characterization of a wide rangeof inorganic materials, including many high Tc

systems and complex oxides of interest in catalysis.These studies have generated growing interest inthe application of powder data to the ab-initio solu-tion of unknown crystal structures, traditionally atechique requiring suitably-sized single crystals.However, because of limitations inherent to powdercompared to single crystal data, such as poorercounting statistics, peak overlap and inferior peak-to-background discrimination, the informationcontained in a set of structure factors is generallyless complete and of lower accuracy. Fourier mapsderived from these data tend to be quite noisy, and

information about light atoms, thermal fluctua-tions or bonding effects may be obscured.

Most generally, MaxEnt is the non-parametricmethod of choice to retrieve information fromsparse and noisy data sets. The great enhance-ment possible with the use of MaxEnt stemsfrom its ability to reduce truncation and noiseeffects by orders of magnitude. The methodworks best when the data relate linearly to thephysical information sought. Some recent papersin the literature have demonstrated that MaxEntcan be a very powerful tool in the analysis ofpowder data, for it is the least-biased with re-spect to missing data and may reveal details notseen on Fourier maps. The purpose of the pres-ent work is to apply the MaxEnt method to high-resolution synchrotron data collected at X7A andassess to what extent it can enhance the structur-al information about the complicated systemstypically encountered in materials research.

APPROACH: There are two difficulties to befaced in structure determination from powderdiffraction data; the general problem of non-linear phases and the specific problem of over-lapping of Bragg peaks. The use of MaxEntto solve the former is a topic of much currentinterest, although phasing the data can frequentlybe accomplished with standard techniques. Theoverlap problem can be reduced via a prelimi-nary MaxEnt deconvolution to eliminate theinstrumental resolution function.

In the first stage of the project, MaxEnt will beapplied to the analysis of data collected fromselected reference materials as a benchmark testof the level of detail that can be extracted forsimple systems. Particular attention will begiven to the minimization of systematic errors inthe data collection process, and to a realistic


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evaluation of the esd's. Subsequent applications willbe to more complex heavy metal oxides containingBi and Mo, for example, to superconducting sys-tems, to the thermal fluctuations of alkali-metalatoms in fullerene super- conductors, and perhapsto the analysis of order-disorder transitions involv-ing deuterated groups based on neutron powder datacollected at the HFBR.

TECHNICAL PROGRESS AND RESULTS: Although funding for this project was initially ap-proved for January, 1993, it took some time to findan individual with experience of MaxEnt methods.This has now been accomplished with the arrival ofDr. Robert Papoular on September 1st in a VisitingScientist position on leave from CEN, Saclay,France, and we are fortunate to have someone ofhis calibre for this work. Dr. Papoular was here fora three week visit in May, 1993, and made consid-erable progress towards adapting his software forpowder problems; a preliminary analysis of a dataset from a heavy metal catalyst showed promisingresults.

This visit proved to be very valuable in planningsubsequent work, and appropriate computing equip-ment and the latest MaxEnt software has now beenpurchased and installed. A software package has

been written which permits 3D-density recon-structions from powder data, based on a previ-ous one used for the analysis of polarized neu-tron data. This package runs on a 486 PC-com-patible microcomputer and is currently beinginstalled on a more powerful Alpha-DEC main-frame computer.

The compound LaB6 was chosen as a suitablereference material for the first stage of the pro-ject. La has about 20 times the scattering powerof B, and should provide an excellent test of theability of MaxEnt to reveal the B positions andpossible covalent bonding effects. An accuratedata set was collected at X7A with particularattention paid to the elimination of systematicerrors, and data analysis is underway. Data werealso collected from CuO, in which the electrondensity distribution will be of primary interest inview of the role played by Cu in the high Tc





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A ccelerator Technology 93-30

A.G. Ruggiero


It is important to investigate new methods andapplications of the Accelerator Technology in orderto resolve limitations of the present state of the artand to explore advanced technologies which may beavailable in the near future. The purpose of thestudy is to determine the feasibility of projects ofdifferent nature and application and yet possiblyinterconnected to each other. More specifically ourinterest is focussed on four major topics: - A

large-energy Muon Collider for the search of theHiggs boson; - The properties of a CrystallineBeam and the conceptual design of a verylow-energy storage ring where to attempt form-ing, to observe and to experimentally investigatethe Crystalline Beam; - Methods of producingand controlling Nuclear Energy by Fusion ofLight Ions, like proton and boron, in clean andsmall-size environments; - Feasibility Study ofan intense Spallation Neutron Source obtained byimpinging an intense medium-energy protonbeam on a target.


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The first item deals with a topic of High EnergyPhysics which is of paramount importance and thatwill play an important role during the next ten ortwenty years. The second is basic research whichconsiders the creation of a very cold state of matterto be obtained with very sophisticated techniques ofbeam cooling and diagnostics. The production ofnuclear energy in a clean and modest size environ-ment is the goal of the third topic which considersmethods of colliding beams different from thoseused or being proposed for the magnetic or inertialconfinement. Finally, the last topic of researchexplores the methods of accelerating proton beamsto the energy of several GeV with an average inten-sity two orders of magnitude larger than presentlyachieved in conventional accelerators.

Each of these studies have crucial accelerator phys-ics and technology issues which are important to beinvestigated analytically and numerically. TheMuon Collider project requires the demonstration ofproduction, acceleration and cooling over a periodof time of a couple of milliseconds to avoid deple-tion of the beam intensity due to the short particlelifetime. The Crystalline Beam requires study ofcontrolling space-charge and intrabeam scatteringeffects as well advancing the limits of coolingtechniques. The production of Nuclear Fusionenergy by colliding beams requires also the genera-tion of very intense beams and thus the experimen-tal demonstration of controlling space-charge effectsby gas-neutralization, of electron cooling at verylow energies and of thermalization techniques. Thestudy of intensity limitations and of the. methods tocope with space-charge effects is the common de-nominator, together with the cooling techniques, toall our projects including the feasibility study of theSpallation Neutron Source. In the latter we explorethe technical capability of the components, like rfand magnet systems, of fast cycling Synchrotrons.


PURPOSE: We describe herewith the purpose ofresearch for each of the four topics:

- Determination of a conceptual scenario for amuon collider based on the production of ^

particles with an intense beam of protonswhich could be available with an upgrade ofthe BNL-AGS facility. Also determination ofthe need, requirements and limitations of avery fast stochastic cooling technique to reducethe beam betatron emittance at the collisionpoint.

- Investigation of the fundamental properties andstructure of the Crystalline Beam in the ulti-mate state and the requirements of the magnetlattice of the storage ring where it is to circu-late.

- Conceptual design of very low energy storageand collider rings where the collision head-onof two intense beams of protons and of ions ofboron completely stripped takes place. Calcu-lation of the requirements on the beam intensi-ty and dimensions for an efficient productionof Nuclear Fusion energy around a 1 MWlevel.

- Preliminary feasibility study of a 5 MW Spall-ation Neutron Source based on the use of a600 MeV Linac and two fast cyclingSynchrotrons for the acceleration of protons to3.6 GeV.

APPROACH: The conceptual study of theMuon Collider has been done in collaborationwith about a dozen scientists in the USA. Theinterest on the collider was renewed during theWorkshop on New Methods of Acceleration heldin Port Jefferson during July 1992. Later a smallgroup met again at Napa Valley during Decem-ber 1992 and then again at Los Alamos duringFebruary 1993. We investigated a possible sce-nario and the requirements of stochastic coolingboth analytically and numerically. To study thefeasibility of Crystalline Beams, we have takenadvantage of a collaboration with the LaboratoriNazionali di Legnaro in Italy that has an interestin building a storage ring dedicated to the dem-onstration of such a beam. German and Russianscientists also take part in the collaboration. Inthe USA, a small group of scientists from Berke-ley, Argonne and Brookhaven laboratories havemet in few occasions to discuss the same topic of


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common interest. We performed analytical andcomputational study. Both the Crystalline Beam andthe Muon Collider have recently been importanttopics of discussion at the Montreux Workshop(Oct. 4-8) where we had an opportunity to assessthe state of the studies of the moment. We believeto be the only proponent of the use of collidingbeams for the production of Nuclear Fusion. Wehad several opportunities to check and to debate ourideas on the project with a wider audience in acouple of conferences. We benefited from discuss-ing the project with other scientists and could devel-op refined storage ring and beam design. A BNLstudy group was appointed earlier this year with thetask of exploring the feasibility of the SpallationNeutron Source. As a member of the study groupwe had the opportunity, through a sequence ofregular meetings, to collaborate for the preparationof a preliminary design of the facility and the studyof the beam performance and of some of the com-ponents of the accelerators. We had also an oppor-tunity to test for this study a software that we haveprepared in the meantime for the design of acceler-ation cycles in circular machines.

TECHNICAL PROGRESS AND RESULTS: Anintense proton source of the energy of few tens ofGeV with an average current of a hundred andmore microampere has been proven feasible. Thisresult is required for the production of muon parti-cles for the Muon Collider and for the SpallationNeutron Source. In particular, methods for upgrad-ing the present AGS facility have been studied andproposed. These may require a new Linac of ener-gy as low as 600 MeV and as large as 2.3 GeV,and a Stretcher Ring. Also the research of nuclearphysics would greatly benefit from this upgradebecause the facility could be used as a factory ofkaons and other exotic particles.

In the search of various scenarios of MuonCollider, it was determined that some type of beta-tron cooling is required with a very large coolingrate. A scenario based only on the use of ionizationcooling could yield a luminosity close to 1030 cm"2

s"1 at the energy of 100 GeV per beam. Yet thefeasibility of ionization cooling still remains to beproven experimentally. Betatron stochastic coolingin a single-pass mode has also been proven feasible

for very short bunches of muons provided thatthe Schottky signal is being regenerated with thehelp of powerful non-linear magnets. A seriouslimitation has emerged due to the finite tempera-ture of the pickup devices and of the preamplifi-ers. Further studies to overcome this problemare required. In the meantime the possibility ofstochastic cooling at laser frequencies has beenproposed but it would require development ofpickups and kickers at the same large frequen-cies. It has been noted that a scenario whichrelies on stochastic cooling has a different modeof operation when compared with only ionizationcooling. Because only few particles per bunchare allowed, a method of bunch stacking justprior to collision has also been proposed.

During the study of Crystalline Beams, we haveencountered three fundamental issues: the Equi-librium Configuration, the Confinement Condi-tions and the Stability Conditions. The beamparticles are distributed in a equally spacedconfiguration, made of strings, zig-zags andshells, such that the resulting space-charge forcestake exactly a linear dependence with the particledisplacement so to be matched with externalrestoring and focussing forces. It has been deter-mined that the storage ring is to be made ofalternating gradient functions where the beamenergy is below the transition enrgy of the stor-age ring. Moreover, it is found necessary thatthe magnet lattice be made of periods as short aspossible, with little bending interdispersed. Inparticular, it was determined that a weak focus-sing structure, like a betatron magnet, is notsuitable for the stability of the beam. CrystallineBeams represent configurations with very highand rigid beam densities of interest for the en-hancement of luminosity performance inlow-energy colliders. They represent extremecases where space-charge and intrabeam scatter-ing effects are ultimately kept under control oreffectively cancelled.

As a consequence of the above study we haveperceived the relation between Crystalline Beamsand the low-energy collider rings where NuclearFusion can be produced by colliding a beam ofprotons (56 keV) with a beam of ions of boron


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(619 keV). The storage ring is of relatively smalldimensions having a circumference of about 3.3meter. The most serious limitation is of course thespace charge effect. Yet, we have determined that,once the required density level for the two beamshas been reached, with low-energy electron cooling,space-charge effects should disappear, because theparticles would be completely screened from theelectromagnetic action of each other. Thisressembles indeed the configuration of CrystallineBeams from which we infer the relation betweenthe two projects.

As we have seen, a relation between the studies ofa Muon Collider and of a Spallation NeutronSource has been evidenced, since both require anintense proton source of about the same energy andintensity. We have found that a facility based on a600 MeV Linac followed by a pair of 3.6-GeVfast-cycling Synchrotrons is indeed technicallyfeasible. This scenario requires the investigation ofthe rf capture during injection, the design of the rfcavity system for the acceleration and the analysisof ramping the guiding field at a large rate.


1. A. G. Ruggiero, "Issues in n/j. Colliders",Proceedings of the Mini-Workshop on fi+ fiColliders: Particle Physics & Design, UCLAReport, Napa Valley, California, Dec. 9-12,1992.

2. A. G. Ruggiero, "Combined Ionization andStochastic Cooling", Proceedings of the MuonCollider Workshop, LA-UR-93-866, LosAlamos, New mexico, Feb 22, 1993

3. A. G. Ruggiero, "Stochastic Cooling Require-ments for a Muon Collider", BNL-49553(Sept. 1993). A reduced version of this reportwill appear also in the Proceedings of theWorkshop on Beam Cooling and Related Top-ics, Montreux, Switzerland, Oct. 4-8, 1993.

4. A. G. Ruggiero, "Derivation of aFokker-Planck Equation for Bunched Beams",Proceedings of the Workshop on Beam Cool-

ing and Related Topics, Montreux, Switzer-land, Oct. 4-8, 1993.

5. A. G. Ruggiero, "Confinement and Stabilityof a Crystalline Beam", BNL-49090 (May1993). A reduced version of this report willappear also in the Proceedings of the 1993Particle Accelerator Conference, Washing-ton D.C., May 17-20, 1993.

6. A. G. Ruggiero, "Demonstration of NoFeasibility of a Crystalline Beam in a Beta-tron Magnet", BNL-49529 (Sept. 1993). Areduced version of this report will appearalso in the Proceedings of the Workshop onBeam Cooling and Related Topics,Montreux, Switzerland, Oct. 4-8, 1993.

7. L. Tecchio, ..., A.G. Ruggiero, "A Dedi-cated Storage Ring for Ion Beam Crystalli-zation", Proceedings of the Workshop onBeam Cooling and Related Topics,Montreux, Switzerland, Oct. 4-8, 1993.

8. A. Dainelli, A. G. Ruggiero, ..., "TheBeam Cooling System of the ADRIA Pro-posal", Proceedings of the Workshop onBeam Cooling and Related Topics,Montreux, Switzerland, Oct. 4-8, 1993.

9. A. G. Ruggiero, "Nuclear Fusion of Pro-tons with Boron", BNL-AD/AP-48 (Sept.1992). Contribution to the Conference onProspects for Heavy Ion Inertial Fusion,Aghia Pelaghia, Crete, 26 Sept-1 Oct.1992.

10. A .G. Ruggiero, "Nuclear Fusion of Pro-tons with Boron", Proceedings of the 1993Particle Accelerator Conference, Washing-ton D.C., May 17-20, 1993.

11. A. G. Ruggiero, "Nuclear Fusion of Pro-tons with Boron", Proceedings of the Inter-national Symposium on Heavy Ion InertialFusion, Frascati, Italy, May 25-28, 1993.Accepted for publication in the special issueof II Nuovo Cimento A.


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12. A. G. Ruggiero, "Nuclear Fusion of Protons LDRD FUNDING:with Ions of Boron", talk given as AGS De-partment Seminar on July 9, 1993.

13. A. G. Ruggiero, A. van Steenbergen, "Base-line Design of a 5 MW Spallation NeutronSource", BNL-49246 (June 1993).



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8,6 CH OD






ather - Miscellaneous Areas

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{ /n the previous page: Conceptual configuration of aParticle Bed Reactor (PBR) fuel element tube design. ThePBR shows promise as a very high flux research reactorconcept.

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Mo/C - Ex-situ Mo-L2 3 and K-edge XAFS experi-ments were carried out on a series of Mo/C cata-lysts during September 1993. An analysis programwas developed to analyze the Mo-K edge XANESspectra. The data are being analyzed. The in-situXAFS measurements will also be analyzed.

Cu/ZSM5 - In-situ XAFS measurements were car-ried out during August 1993. The data are beinganalyzed.


1. "EXAFS Study of Metal-Coated particles Pro-duced by Ball Milling", S.M. Heald,J. Jayanetti and K.I. Pandya, Jpn. J. Appl.Phys. Vol 32 (1993) Suppl. 32-2, pp. 499-501.

2. "EXAFS Investigations of Pt/NaY Zeolite Cata-lysts at Industrially Relevant Concentrations",K.I. Pandya, S.M. Heald, J.A. Hriljac, L.PetraKis and J. Fraissard (Submitted to J. Phys.Chem.)

3. Comparison of Metal Particle Size Derivedfrom EXAFS and I29Xe-NMR Spectrosco-pies, J. Fraissard, K.I. Pandya and L.Petrakis (Manuscript in Preparation).

4. Structural Investigations of Pt-Au/Al2O3

Catalysts: K.I. Pandya, L. Petrakis,D. Rouabah and J. Fraissard (Manuscript inPreparation)


FY 1991 $ - 0 -FY 1992 68,334FY 1993 73,017FY 1994 - 0 -

tructure-Sensitive Properties of Advanced Permanent

Magnet Materials: Experimental and Theory


D.O. Welch andM. Suenaga


I h e goal of this program is to achieve an under-standing of the properties and mechanisms whichcontrol the coercivity, remanent magnetization, andmaximum energy product of advanced permanentmagnet materials. These characteristic propertiesdetermine the practical usefulness of materials aspermanent magnets in applications such as electricmotors, sensors, and scientific instruments. Theseproperties are extremely sensitive to the microstruc-ture and crystal lattice defects on a scale ofnanometers and are thus amenable to improvementby appropriate materials processing methods.

Substantial improvements are possible in princi-ple: for example, the coercivity of one of themost advanced magnetic materials, Nd2FeuB, isonly about a third of its theoretical upper limit,which indicates considerable scope for improve-ment in the properties of even magnetic materi-als for which the properties have been optimizedby processing methods which are not consideredas the state-of-the-art.

This study, which is being done in collaborationwith Carleton Fuerst et al., at the General Mo-tors Research Laboratories, focuses on the rela-tionships between nanometer-scale defect struc-ture and the structure-sensitive magnetic proper-


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ties (coercivity, remanence, and magnetic viscosity)of advanced permanent magnet material, exempli-fied by the R2M14X system, where B is a rare-earthelement (Nd. Pr, .. .), M is a transition metal (Fe,Co, . . .) , and X is a metalloid (B, C, ...). This re-search program has both experimental and theoreti-cal components and coexists symbioticaily with theexisting research programs on superconducting ma-terials in the Materials Science Division of the De-partment of Applied Science, since there are manynatural overlaps, especially the focus on the effectof crystal lattice defects on magnetic flux structuresand properties, as well as a concern with the mate-rials science of complex intermetallic compounds,in the research on both classes of materials.


PURPOSE: The specific purpose of this work is tounderstand how various materials processing condi-tions lead to the development of crystal lattice de-fects organized in a nanometer-scale microstructureand how these microstructural elements control thestructure-sensitive magnetic properties of an ad-vanced permanent magnet material such asNd-,FeHB. Understanding of this nature shouldlead to the development of methods for producingsubstantially improved permanent magnets for usein more efficient electric motors, for example. Thetwo groups performing this research bring comple-mentary strengths to the endeavor: the GeneralMotors group was the discoverer of Nd2Fe]4B (in-dependently and simultaneously with a group atSumitomo in Japan) and has pioneered the use ofrapid-solidification-based processing methods forthis material; the BNL Materials Science group hassubstantial experience in microstructural and elec-tromagnetic characterization of complexintermetallic compounds and superconductors, aswell as in the theory of lattice defects and theirproperties in such materials. Another purpose ofthis work is to establish a productive working rela-tionship between these groups of industrial andnational laboratory researchers.

APPROACH: Samples of R-,M]4X (e.g.Nd2Fe14B, Nd2Co14B, Nd2FeMC, Pr2Nd2B, etc.)are fabricated by Carleton Fuerst and his colleagues

at GM and supplied to the BNL group (M.Suenaga, L. Henderson Lewis, et al.) who thencharacterize the defect microstructure using ad-vanced transmission electron microscope (TEM)techniques, as well as atomic force microscopy(AFM), and the magnetic properties are mea-sured using superconducting quantum interfer-ence device (SQUID) magnetometry. Theoreti-cal models of lattice defects and their interactionwith the magnetic domain structure are devel-oped by the BNL group (D. O. Welch, et al.).A theoretical analysis of the utility of neutrondepolarization and/or small-angle scattering forthe characterization of magnetic domains is alsoplanned.

TECHNICAL PROGRESS AND RESULTS: Acomparative study has been made of the micro-structural and magnetic properties of Nd2Fe,4Band Pr2FeuB, as well as their alioys with Coand Ga. The materials were fabricated usingmelt-spinning/hot-working methods and theirroom-temperature coercivity, remanence, andmaximum-energy-product were measured at GM.as well as some characterization of the micro-structure done by low-resolution TEM. High-resolution TEM studies were made at BNL. Thelatter revealed significant differences betweenPr2Fe]4B and Nd2Fe14B in the structure of grainboundaries and intergranular phases, whichhelped to understand the differences between themagnetic properties of the two materials. Ana-lytical electron microscope studies are underwayat BNL to measure the location and concentra-tions of the Co and Ga alloying elements in themicrostructure. The elevated temperaturecoercivity and rate of magnetic flux creep in thetwo materials were measured at BNL and the re-sults reveal some differences in the properties ofthe Pr-and Nd-based materials. Preliminarystudies at BNL of the microstructure made usingatomic-force microscopy were quite promising,providing images of the grain boundary structureover a range of scales from micrometers toatomic resolution. An effort is now underway tomake a magnetically-sensitive AFM tip to imageboth domain boundaries and microstructuresimultaneously, a capability which will be verypowerful in the study of domain-wall/defecl


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pinning interactions, which are the key to under-standing coercivity. Theoretical studies during thisperiod featured work on the development of embed-ded-atom models of cohesion with which to esti-mate the properties of lattice defects and to help un-derstand the location of alloying elements in theR2M14X structure.


1. "Die-Upset Pr2Fe,4B-Type Magnets from Melt-Spin Ribbons," Fuerst, C. D., Brewer, E. G.,Mishra, R. K., Zhu, Yimei, and Welch, D. O.,in preparation (to be submitted to J. Appl.Phys.).


FY 1992FY 1993FY 1994FY 1995

- 0 -99,186108,000 (est.)

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TTVery High Flux Research Reactor 93-20

J. Powell and H. Hudewig


Earlier studies carried out at BNL of a Very High

Flux Research Reactor based on the Particle BedReactor (PBR) concept indicated that thermal neu-tron fluxes well in excess of 2.0xl016 n/cm2-s ap-pear achievable. Such flux levels are approximatelyfive times greater than that of the proposed Ad-vanced Neutron Source reactor, and a factor of 15greater than the High Flux Beam Reactor. Since theoriginal proposal was made (July 1992) the DOEand BNL have begun scoping studies of an intensespallation neutron source. Moreover, the demise ofthe Space Nuclear Thermal Propulsion (SNTP)project has made it impossible to carry out the orig-inally proposed experimental program with the re-sources available. Accordingly,with concurrencefrom BNL the work scope has been altered. PBRtechnology is now being investigated for applicationto the design of a high power density spallalionneutron source. Data from the SNPT/PBR program,and other sources is being applied to this design.

The baseline parameters of such a source arethat it operate in a pulsed mode (10Hz-50Hz),and with a proton beam power of 5MW. Inorder to ensure the most intense source possiblefor a given target material, it must occupy thesmallest volume consistent with efficient heat re-moval, overall neutron production, and availabil-ity of neutrons for experiments. These require-ments can best be satisfied by a paniculate bedtarget arrangement, if the target is to remainsolid, for the following two reasons.

1) Particle beds have the highest heat transferarea per unit volume of any practical target ar-rangement, and

2) The cyclic stresses due to the pulsed natureof the source will be minimized in the case of aparticulate bed compared to any other geometricarrangement, thus minimizing fatigue failures.

The above two properties of particle beds havebeen confirmed experimentally in the SNTP pro-gram, and these basic results will be used in thedesign of a spallation neutron source. Targets


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operating at a maximum power density of 10 MW/Iare well within the capability of such target designs.


PURPOSE: The generation of extremely highneutron fluxes requires that the source of the neu-trons be dimensionally as small as possible, andthat it operate at as high a power as is possiblewithout causing damage. The source of neutronscan either be a reactor or an appropriate target,driven by an accelerator. In both cases the basicproblem is one of removing the heat rapidly enoughto ensure mechanical stability of the source. Due tothe large heat transfer area available in randomlypacked particle beds, fuel elements or targets de-signed using this concept result in the optimumcombination of source power and size. The purposeof this work is to optimize a spallation neutronsource which operates at a power of 5 MW, and anenergy of 3.6 GeV. The accelerator operates in apulsed mode, which is optimal for neutron diffrac-tion experiments. In order to maximize the flux, thetarget arrangement will be based on the particle bedconcept. Experience gained in the SNTP programin the design and operation of high power densityparticle beds will be applied to the design of thetarget. The overall goal of the design will be togenerate a neutron flux of approximately 2.0-5.0xl015 n/cm2-s on the target surface. The longterm objective of the work will be to complete apreliminary design of the target. This design will becombined with the accelerator design to form aproposal which will be submitted to DOE.

APPROACH: The overall strategy to carry out thiswork will combine physics, fluid dynamics, heattransfer, and stress analyses in an iterative manner.The first step consists of a conceptual design in-cluding component sizes and nuclide number densi-ties. This step is followed by a physics analysiswhich predicts the expected neutron yield and thespatial heat deposition. This data is used in the fluiddynamic and heat transfer analysis to determine thepressure drops and temperature distribution withinthe target. Finally, the temperature distribution isused to determine the thermal stress distribution.

The thermal stress is combined with the mechan-ically induced stresses to determine componentstress levels. If at any of the above steps thetarget is either unacceptable or violates a materi-al physical property the design will be rejected.

The above steps are applied to various tradestudies of the target which maximize the neutronflux, while simultaneously making it as safe andinexpensive as possible.

TECHNICAL PROGRESS AND RESULTS:The work to date has concentrated on investi-gating different target materials, target configu-rations, different structural materials, and cool-ant types. Both tungsten and uranium-238 wereinvestigated as the primary target materials. De-spite the superior neutron production capabilityof uranium it was rejected in favor of tungsten,because of the higher melt temperature of tung-sten, and the absence of fission products. Boththese reasons are safety and cost related, and thechoice of tungsten is thus considered correct.The arrangements considered were three differ-ent configurations of particle beds, and the re-quired frits which contain the beds. Finally, onseveral of these targets three different coolantswere investigated. These coolants included liquidlead and sodium, pressurized helium, and heavywater. Heavy water was chosen as the coolant ofchoice, due to its low parasitic neutron absorp-tion, and benign interaction with hydrogenousmoderators should an upset occur. The currenttarget arrangement is shown on Figure 1. It con-sists of a series of particle beds of differentthickness arranged at right angles to the beam,with coolant flowing through the beds parallel tothe proton beam. The particle bed thicknessesare varied in such a manner as to flatten theneutron flux and the heat deposition. The parti-cles in this target are 500 micron diameter tung-sten particles contained between frits manufac-tured of stainless steel 2 mm thick. The targetassembly is contained in a stainless steel pressurevessel. This target is being optimized with"wing", "slab", and "back scatter" moderatorarrangements in mind.


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Two abstracts describing this target concept havebeen submitted to the November 1993 AmericanNuclear Society meeting:

1. M. Todosow and H. Ludewig, "Description ofa Spallation Neutron Source Based on a ParticleBed", Proc. of American Nuclear Society, Win-ter Meeting (1993).

2. H. Ludewig and M. Todosow, "Safety Is-sues Related to a Particle Bed Pulsed Spall-ation Neutron Target", Proc. of AmericanNuclear Society, Winter Meeting (1993).




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Coolant PIFrits

Particle Bed


Inlvt Target1 Coolant

Outlet TargetCoolant

I |Window Target



Coolant.. Coolant

Window "TargetCoolant Coolant

Figure 1 Schematic Illustration of Spoliation Target

Based on Particle Bed Technology