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Vitro Synthesis (vitro + synthesis)
Selected AbstractsIn Vitro synthesis and activity of reporter proteins in an Escherichia coli S30 extract system: An undergraduate experiment,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2005Pamela J. Higgins Abstract This undergraduate laboratory experiment integrates multiple techniques (in vitro synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (,-galactosidase and luciferase) within an Escherichia coli S30 transcription/translation extract. Comparison of the data suggests that luciferase is the more suitable reporter for this specific in vitro extract system. Simple modifications in the experimental design allow for flexibility in the use of materials and the time required to perform the study. Furthermore, extension into additional experiments and alternative techniques are also discussed. [source] MS and clinically isolated syndromes: Shared specificity but diverging clonal patterns of virus-specific IgG antibodies produced in vivo and by CSF B cells in vitroEUROPEAN JOURNAL OF NEUROLOGY, Issue 10 2009G. Skorstad Background:, Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type-1 (HSV-1) is a characteristic feature multiple sclerosis (MS). Methods:, We have used isoelectric focusing-immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV-1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease. Results:,In vitro synthesis of IgG by PBL was not detected in any patient. In contrast, in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo. However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus-specific IgG produced by CSF cells in vitro. Conclusion:, Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B-cell repertoire in CSF samples is only partially representative of the intrathecal B-cell repertoire. [source] Specific interaction between the classical swine fever virus NS5B protein and the viral genomeFEBS JOURNAL, Issue 19 2004Ming Xiao The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3, untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3, UTR and that the NS5B protein was also able to bind the minus-strand 3, UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3, UTR more efficiently than the plus-strand 3, UTR. Further, we observed that the plus-strand 3, UTR with deletion of CCCGG or 21 continuous nucleotides at its 3, terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3, UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3, terminal. Therefore, it is indicated that the 3, CCCGG sequence of the plus-strand 3, UTR, and the 3, CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3, 21 nucleotide terminal site of both the 3, UTRs. [source] Cellulose structure and biosynthesis: What is in store for the 21st century?JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 3 2004R. Malcolm Brown Jr. Abstract This article briefly summarizes historical developments in fundamental research related to the structure and biosynthesis of cellulose. Major advances concerning the structure of cellulose include the discovery of a new suballomorph of cellulose I, the lattice imaging of glucan chains showing no fringe micelle structure, parallel chain orientation in cellulose I, and the discovery of nematic ordered cellulose. Major advances in biosynthesis include the discovery of the terminal synthesizing complex, the isolation and purification of cellulose synthase, the in vitro synthesis of cellulose I, and synthetic cellulose assembly. This article focuses on recent advances in molecular biology with cellulose, including the cloning and sequencing of cellulose synthase genes from bacteria, cyanobacteria, and vascular plants; proof of the terminal synthesizing complex as the site of the catalytic subunit of cellulose synthase; cellulose and callose synthase expression during growth and development; and phylogenetic aspects of cellulose synthase evolution. This article concludes with thoughts about future uses for the accumulating genetic information on cellulose biosynthesis for textiles and forest products and discusses possibilities of new global resources for cellulose production. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 487,495, 2004 [source] Decreased expression of heparin-binding epidermal growth factor,like growth factor as a newly identified pathogenic mechanism of antiphospholipid-mediated defective placentationARTHRITIS & RHEUMATISM, Issue 5 2010N. Di Simone Objective Heparin-binding epidermal growth factor,like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL),mediated defective placentation. Methods HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal ,2 -glycoprotein I,dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. Results Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. Conclusion These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding. [source] In Vitro synthesis and activity of reporter proteins in an Escherichia coli S30 extract system: An undergraduate experiment,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2005Pamela J. Higgins Abstract This undergraduate laboratory experiment integrates multiple techniques (in vitro synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (,-galactosidase and luciferase) within an Escherichia coli S30 transcription/translation extract. Comparison of the data suggests that luciferase is the more suitable reporter for this specific in vitro extract system. Simple modifications in the experimental design allow for flexibility in the use of materials and the time required to perform the study. Furthermore, extension into additional experiments and alternative techniques are also discussed. [source] Simultaneous in Vitro Protein Synthesis Using Solid-Phase DNA TemplateBIOTECHNOLOGY PROGRESS, Issue 6 2004Mary Kate W. DiTursi In vitro protein synthesis is rapidly becoming an accepted tool in functional genomic analysis. We have demonstrated the in vitro synthesis of firefly luciferase on solid-phase template DNA, bound to wells in 96-well plates, using simultaneous transcription and translation in a wheat-germ extract system. The bound DNA template was stable and did not release during transcription. Coupled translation resulted in ca. 1.2 ng/,L luciferase synthesized, which is ca. one-fifth of that synthesized using conventional solution-phase coupled transcription and translation. Reuse of the DNA template was influenced by the complexity of the wheat-germ extract, which resulted in fouling of the transcription surface and reduction of protein synthesis after extended use. The approach developed in this study may enable the development of high-throughput, microscale protein synthesis platforms for use in functional genomic analysis. [source] Cell-free Protein Synthesis through Solubilisate Exchange in Water/Oil Emulsion CompartmentsCHEMBIOCHEM, Issue 8 2004Adriana V. Pietrini Dr. Abstract This work is aimed at finding conditions under which synthetic compartments used as cell models can fuse with each other and allow reagents contained in the different compartments to react. This goal seems to be best achieved by the use of water in oil emulsions (w/o) with dimensions in the range of 30,60 ,m. In particular, cell-free EGFP (enhanced green fluorescent protein) synthesis takes place in Tween 80/Span 80 w/o emulsions, and the extent of the reaction can be monitored directly by fluorescence. The medium is mineral oil, containing 0.5,% v/v aqueous solution. Different premixing configurations of the components (plasmid, amino acids, E. Coli extract) are used and compared. The in vitro synthesis of EGFP in emulsion droplets proceeds for 1 h, and the yield is 7.5 ng,,L,1protein. EGFP synthesis in aqueous solution takes place for at least 5 h. The yield is 10.5 ng,,L,1protein after 1 h and 15.8 ng,,L,1protein after 5 h.The results with the w/o emulsions show that solubilisate exchange takes place among the different water droplets, but it is not possible to demonstrate clearly that a true fusion takes place. [source] | |||||||||||||