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Although the delta ribozymes have been stud ied for more than ten years the most im- por tant in for ma tion con cern ing their struc ture and mech a nism of ca tal y sis were only ob tained very re cently. The crys tal struc ture of the genomic delta ribozyme turns out to be an ex cel lent ex am ple of the ex traor di nary prop er ties of RNA mol e cules to fold into uniquely com pact struc tures. De tails of the X-ray struc ture have greatly stim u- lated fur ther stud ies on the fold ing of the ribozymes into func tion ally ac tive mol e cules as well as on the mech a nism of RNA ca tal y sis. The abil ity of the delta ribozymes to carry out gen eral acid-base ca tal y sis by nu cle o tide side chains has been as sumed in two pro posed mech a nisms of self-cleavage. Re cently, con sid er able prog ress has been also made in char ac ter iz ing the cat a lytic prop er ties of trans-act ing ribozyme vari ants that are po ten tially at trac tive tools in the strat egy of di rected RNA degradation.
The molecular determination of viral load in the serum represents the most valuable prognostic marker of HBV infection. In this paper, a new molecular assay for the quantitative measurement of HBV presence is described. It is based on PCR performing with a HBV-specific competitor DNA template. For the construction of the DNA template, a HBV DNA-originated 436 bp DNA fragment was modified by introducing a 110 bp deletion and cloned into pUC19. The resulting vector serves as the competitor DNA template in the competitive PCR. Post-PCR, the competitor DNA generates an amplified fragment of 306 bp; it could be easily distinguished from the product generated from the viral-originated DNA product (416 bp) when the same primers are used. The quantitative ratio between the two products enables the quantitative determination of viral load. The range of the HB-PCR assay is from 3 x 104 to 6 x 1010 particles/ml. A serum HBV load determination performed by HB-PCR assay indicated a close correlation with the results of the Quantiplex HBV DNA assay (bDNA). The HB-PCR assay is cheap, reliable and easy to use in any laboratory working with PCR methods.
Serum response factor (SRF) is a transcription factor, which binds to a serum response element (SRE) associated with a variety of genes including immediate early genes such as c-fos, fosB, junB, egr-1 and –2, neuronal genes such as nurr1 and nur77 and muscle genes such as actins and myosins. By regulating expression of these genes, SRF controls cell growth and differentiation, neuronal transmission as well as muscle development and function. SRF can be activated by a variety of agents, including serum, lysophosphatidic acid (LPA), lipopolysaccharide (LPS), 12-O-tetradecanoylphorbol- 13-acetate (TPA), cytokines, tumor necrosis factor-. (TNF.... ), agents that increase intracellular Ca 2+, T-cell virus1 activator protein, hepatitis B virus activator proteins pX, activated oncogenes and protooncogenes as well as extracellular stimuli such as antioxidant and UV light. SRF itself is regulated by both cellular signal transduction pathways and interaction with other transcription factors e.g. Sp1, ATF6 and myogenic regulatory factors. Its biological function is best eluci-dated for myocardium. Specific cardiac SRF transgenesis demonstrated that overex-pression of SRF caused hypertrophic cardiomyopathy in mouse and the mouse died of heart failure within 6 months after birth. Other transgenic data suggested that suf-ficient SRF was needed for embryogenesis and early development. Since SRF is important regulator of numerous genes involved in cell growth and differentiation, including muscle and neural components, SRF may also play a crucial role in tissue injury and ulcer healing, e.g. healing of gastrointestinal ulcers.
The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis В Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 µg per g of fresh tissue.
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