In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.
In small RNA viruses, arm-like segments located at the N or C termini have been suggested as mediators in the assembly of the capsid proteins. In many cases the arms of several subunits converge at a common point (the symmetry axis). Recent advances in studies of the hepatitis B virus (HBV) core protein attest the convergence of the segments preceding the protamine region, around the symmetry axis, where five or six HBc protein subunits converge. We report a mutation study of the region that we have suggested forms an armlike structure, which reveals that a single mutation, Pro-138 Gly, prevents the full-length HBV core protein self-assembling into particles.In many viruses, extended conformations frequently found at the N terminus of the capsid protein are supposed to play an important role in the assembly of the viral particles (Harrison, 1992). In simian virus 40, for example, the capsid contains 72 pentamers of the VP1 protein. The pentamers are linked by the interaction of the C-terminal arms of the VP1 protein with five or six neighbouring pentamers (Liddington et al., 1991). These interactions supplant the absence of complementary interfaces between pentamers.The human hepatitis B virus (HBV) has been shown to form particles of two different sizes, one major population of capsids containing 240 copies of HBc protein (T l 4 capsids, where T is the triangulation number) and a minor population of capsids containing 180 copies (T l 3 capsids) (Crowthers et al., 1994 ;Kenney et al., 1995 ;Zlotnick et al., 1996). In both cases, the capsids reveal tightly clustered dimers composed of a shell domain and a protruding spike. Two recent papers (Bo$ ttcher et al., 1997 ;Conway et al., 1997) have made an important contribution to the knowledge of the structure of the HBV capsid. It has been revealed that the previously reported
DNA-based vaccination is a potent technique to prime cellular (T-cell mediated) immune responses (reviewed in 1). Many details of the priming of T-cell precursors by antigen translated from injected expression plasmid DNA are unknown. The relevant cell that is transfected in situ after DNA vaccination and that can process and present the protein in an immunogenic form has not yet been identified. Alternatively, the transfected cell may initiate 'cross-priming' in vivo by transferring processed antigen to a professional antigen-presenting cell (APC).
Background Hypoxic pulmonary vasoconstriction (HPV) is a reaction of the pulmonary vasculature upon hypoxia, diverting blood flow into ventilated areas to preserve oxygenation. It is impaired in endotoxemia or ARDS. Voltage gated potassium channels have been shown to play a key role in the regulation of HPV. The aim of the study was to identify a voltage gated potassium channel involved in dysregulated HPV during endotoxemia. Methods Lungs of male C57BL/6 mice with and without endotoxemia (n = 6 ea. group) were analyzed for Kv3.4 gene and protein expression. HPV was examined in isolated perfused lungs of mice with and without endotoxemia and with and without selective Kv3.4 blocker BDS-I (n = 7 ea. group). Pulmonary artery pressure (PAP) and pressure-flow curves were measured during normoxic (FiO2 0.21) and hypoxic (FiO2 0.01) ventilation. HPV was quantified as the increase in perfusion pressure in response to hypoxia in percent of baseline perfusion pressure (ΔPAP) in the presence and absence of BDS-I. Results Kv3.4 gene (3.2 ± 0.5-fold, p < 0.05) and protein (1.5 ± 0.1-fold p < 0.05) expression levels were increased in endotoxemic mouse lungs. Endotoxemia reduced HPV (∆PAP control: 121.2 ± 8.7% vs. LPS 19.5 ± 8.0%, means ± SEM) while inhibition of Kv3.4 with 50 nM BDS-I augmented HPV in endotoxemic but not in control lungs (∆PAP control BDS-I: 116.6 ± 16.0% vs. LPS BDS-I 84.4 ± 18.2%, means ± SEM). Conclusions Kv3.4 gene and protein expressions are increased in endotoxemic mouse lungs. Selective inhibition of Kv3.4 augments HPV in lungs of endotoxemic mice, but not in lungs of control mice.
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