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Abstracts / Journal of Biotechnology 185S (2014) S5–S17 S7
endoneurial neo-vessels with tight junctions. In the following timepoints an active process of axonal regeneration was documented;after two months we showed, in the regenerated mid-graft, a nor-mal number of well myelinated axons with normal G-ratio anddefinite signs of reinnervation in the tibial and peroneal branch.No inflammatory cells were evident. Electrophysiological assess-ment showed a motor action potential at 90 days postoperativelydistally, recordable at the paw. Overall the neurophysiological andneuropathological data demonstrate the terrific quality of nerveregeneration using our MD which performs better in comparisonto the already available in clinical practice NeuroGen Integra® col-lagen tubes, used as a control. These results make us confident inproposing our MD in the clinical practice to treat injury of the PNS.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.026
Lysosomal enzyme activities in human brain
Tommaso Beccari 1,∗, Emanuele Persichetti 1,Anna Tasegian 1, Michela Codini 1, Maria RacheleCeccarini 1, Tim Moors 2, Davide Chiasserini 1,Paolo Eusebi 1, Lucilla Parnetti 1, Paolo Calabresi 1
1 University of Perugia, Italy2 Vrije Universiteit Amsterdam, The Netherlands
E-mail address: [email protected] (T. Beccari).
Lysosomal enzymes are involved in macromolecules degradation;impairment of their functionality is the cause of lysosomal stor-age diseases. In recent years, lysosomal dysfunction has also beenshown in neurodegenerative disorders such as Parkinson’s dis-ease (PD). Mutations in the GBA1 gene, coding for the lysosomalbeta-glucocerebrosidase, cause Gaucher’s disease. Interestingly,decreased beta-glucocerebrosidase activity has been described insubstantia nigra of sporadic PD patients, further providing a linkbetween beta-glucocerebrosidase deficiency and PD. This is thefirst study investigating the expression of beta-hexosaminidase,alpha-fucosidase, beta-mannosidase, alpha-mannosidase, beta-galactosidase, beta-glucocerebrosidase and cathepsin E in differentareas of the human brain. Activity assays were performed onputamen, caudate, hippocampus, and frontal cortex from 13 PDpatients and 11 control subjects and substantia nigra from 3 PDpatients and 6 controls. Results revealed a 20% decrement ofbeta-glucocerebrosidase activity in substantia nigra of PD patients.Decreased activity (50%) of cathepsin E was also observed incaudate of PD patients. Interestingly, 21% and 60% increases in beta-hexosaminidase and alpha-mannosidase activities were found incaudate of PD patients. Lysosomal enzymes tested in this studyshowed different activities in different brain areas. These resultsmay contribute to the understanding of the role of lysosomalenzymes in neurodegenerative disorders.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.027
RNA polymerase genetic engineering (RPGE) asa new strategy for drug discovery andimprovement of industrial microorganisms
Pietro Alifano ∗, Adelfia Talà
Dipartimento di Scienze e Tecnologie Biologiche eAmbientali, Università del Salento, Lecce, Italy
E-mail address: [email protected] (P. Alifano).
Actinomycetes are ecologically important bacteria and prolific pro-ducers of a variety of biologically active compounds includingantibiotics, anticancer agents and immune-suppressants, whichare widely used in medicine. Advances in genome research haverevealed that their potential has been underestimated due to thepresence of silent or weakly expressed biosynthetic gene clusterscoding for unknown secondary metabolites. These metabolites mayrepresent a valuable source for new drug discovery. Methods toactivate or enhance the expression of the silent or weakly expressedgene clusters are therefore of the utmost importance during earlystages of drug discovery when enough material is necessary tocharacterize chemically and biologically the new compounds. Avariety of methods have successfully employed including metabolicremodeling, manipulation of pathway-specific regulatory genesand cell-to-cell signaling, ribosome engineering, co-cultivation,rare earth utilization. Here we describe a general strategy for drugdiscovery and improvement of industrial microorganisms, which isbased on RNA polymerase genetic engineering (RPGE). A powerfulRPGE method relies on intergeneric transfer of natural “mutant-type” RNA polymerase �-chain encoding genes rpoB(R) from“rare” actinomycetes with two or more rpoB paralogs to desiredbacteria.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.028
Production of recombinant therapeuticproteins in the milk of transgenic animals
Haydar Bagis
University of Adıyaman, Medical Faculty andMedical Genetic Department, Turkey
E-mail address: [email protected].
A transgenic animal has a piece of foreign DNA stably integratedinto its genome. This foreign DNA consists of a construct contain-ing a specific promoter region, a gene coding and other regulatoryelements. Transgenic animals are used for production of recom-binant proteins for scientific, pharmaceutical, and agriculturalpurposes. The ability of transgenic animals to produce biologicallyactive recombinant proteins in an efficient and economic mannerwas demonstrated a long time ago and has attracted substantialattention and investments. The benefits of transgenic protein pro-duction include safety from human viral contamination, low-cost,high volume production, correct posttranslational modifications,and applicability to a wide range of complex proteins. All of therecombinant proteins produced and marketed to date are pro-duced in bacterial, yeast, and mammalian cell culture systems. Anumber of “new” technologies, including Chinese hamster ovary,insect cell culture and Baby hamster kidney, transgenic plants, andmilk from transgenic livestock, have products in development. Wedemonstrate that hIFN-g was efficiently expressed in the mam-mary gland of transgenic mice under the control of the mWAPgene promoter and exhibits biological properties without anyadverse. The promise of biopharming, that is the actual commercial
S8 Abstracts / Journal of Biotechnology 185S (2014) S5–S17
production of pharmaceuticals and other bioproducts, is nearingfulfillment.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.029
Recombinant production and inhibitor bindingand stability characterization of 12 humancarbonic anhydrase catalytic domains inbacterial and mammalian cells
Daumantas Matulis 1,∗, Vaida Jogaite 1, JurgitaMatuliene 1, Justina Kazokaite 1, AisteKasiliauskaite 1, David Daniel Timm 1, VilmaMichailoviene 1, Aurelija Mickeviciute 1, SandraBaksyte 1, Alexey Smirnov 1, Lena Manakova 2,Visvaldas Kairys 3
1 Department of Biothermodynamics and DrugDesign, Institute of Biotechnology, Vilnius University,Lithuania2 Department of Protein – Nucleic Acid Interactions,Institute of Biotechnology, Vilnius University,Lithuania3 Department of Bioinformatics, Institute ofBiotechnology, Vilnius University, Lithuania
E-mail address: [email protected] (D. Matulis).
Carbonic anhydrases (CA) catalyze carbon dioxide hydration reac-tion into bicarbonate and protons and participate in carbonmetabolism and pH regulation. There are 12 catalytically activeCA isozymes in human body. They vary in tissue expression andcatalytic activity. Isoforms I, II, III, VII, and XIII are cytosolic, VIis secreted, VA and VB are mitochondrial, IV is membrane boundthrough a lipid anchor, and IX, XII, and XIV contain transmembraneand proteoglycan domains. CA inhibitors have been used as drugs totreat glaucoma, epileptic seizures, altitude sickness, and as diuret-ics. However, most of them exhibit poor selectivity and result invarious side effects.
We have recombinantly produced all 12 isoform catalyticdomains and characterized their activities, thermal stabilities,and inhibitor binding thermodynamics by the stopped-flowCO2 hydration assay, isothermal titration calorimetry, and thefluorescent thermal shift assay. The combined use of these meth-ods provided a detailed picture of the intrinsic energetics ofprotein–ligand interactions. Several CA isoforms were crystallizedwith inhibitors providing structural insight into the binding reac-tion. Structure–thermodynamics correlations provide clues in thedesign of compounds that selectively inhibit desired CA isoformsand could be leads towards drugs.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.030
Euglenoid flagellates: Gene organization andexpression, and biotechnological potency
Juraj Krajcovic 1,∗, Matej Vesteg 2
1 Department of Genetics, Faculty of NaturalSciences, Comenius University, 842 15 Bratislava,Slovakia2 Department of Biology and Ecology, Faculty ofScience, University of Ostrava, 701 03 Ostrava, CzechRepublic
E-mail address: [email protected] (J. Krajcovic).
Euglenids are mainly fresh water protists with an extensive capac-ity for usage of a wide variety of alternative carbon sourcesfor growth. Phototrophic euglenids possesses complex greenchloroplasts bounded by three membranes descending from achlorophycean algal symbiont. Some Euglena nuclear genes haveobtained complex presequences required for chloroplast target-ing. We have revealed the presence of short introns either in thepresequence-encoding regions or shortly downstream of them innucleus-encoded Euglena gracilis genes for chloroplast proteins.The sequencing of the E. gracilis chloroplast genome disclosed anunusually high number of introns including twintrons – intronswithin introns. The treatment of E. gracilis by compounds inhibi-ting bacterial replication, transcription and translation leads to thepermanent loss of the ability to form green colonies – bleaching. Theprocess is accompanied by selective loss of plastid-encoded genes,while it has no effect on cell growth and viability. Our observationsstrongly support a suggestion that transcription of both the plas-tid chromosome as well as nucleus-encoded genes for chloroplastproteins are relatively insensitive to environmental changes andthat gene expression is regulated mainly at the post-transcriptionallevel in this flagellate. E. gracilis is a suitable source for the genera-tion of several biotechnologically relevant metabolites.
http://dx.doi.org/10.1016/j.jbiotec.2014.07.031
The two-hybrid system: A powerful tool todissect molecular mechanisms underlyingmembrane traffic diseases
Cecilia Bucci ∗, Laura Cogli
Department of Biological and EnvironmentalSciences and Technologies, University of Salento,Lecce, Italy
E-mail address: [email protected] (C. Bucci).
Alterations of membrane traffic events have important conse-quences on key cellular processes such as signal transduction,proliferation, migration, apoptosis, and mitosis. Thus, it is not sur-prising that mutations in membrane traffic genes often give riseto severe human disorders. Also, in a number of membrane trafficdiseases, mutations actually affect ubiquitously expressed genesbut the defect is restricted to specific cell types, making harder tounderstand the molecular basis of these diseases. In this respect,it is interesting to note that membrane traffic diseases frequentlyaffect the nervous system. The yeast two-hybrid system, a power-ful technology to identify protein–protein interactions, has beensuccessfully used to comprehend the molecular basis of severalmembrane traffic diseases. Indeed, although there are weaknessesas the high number of false positive, it is an easy low-tech methodproviding rapid identification of putative interacting proteins. Weused the two-hybrid technology to discover the molecular basis ofCharcot-Marie-Tooth type 2b (CMT2B). This disease affects periph-eral neurons and it is caused by mutations in the small GTPase Rab7,
