Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure:     

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O₂ concentrations and mitochondrial O₂ consumption by high-resolution respirometry over a range of O₂ concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC₅₀ on [O₂] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O₂ and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O₂-dependent oxidation. It also explains the non-linear dependence of IC₅₀ on O₂ concentration, and the hyperbolic increase of c₅₀ as a function of NO concentration.     

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Circulating TNF-alpha and its soluble receptors during experimental acute pancreatitis

Clinical and experimental studies have shown increased concentrations of TNF-alpha and its soluble receptors in serum of patients with acute pancreatitis. In this work, we have investigated the time-course of TNF-alpha and its soluble receptors during taurocholate-induced acute pancreatitis. In addition, since TNF-alpha itself could mediate the shedding of its receptors, we have assessed the effect of inhibiting TNF-alpha production on the release of soluble TNF-alpha receptors in experimental acute pancreatitis. Our results indicate that soluble receptors are released in the early stages of the disease and this increase is concomitant with the release of TNF-alpha, which is mainly bound to specific proteins. The increased concentrations of its receptors strongly suggest that they could be these binding proteins. Inhibition of TNF-alpha generation with pentoxifylline abrogated the shedding of sTNF-alphaR1, but had no effect on sTNF-alphaR2. This finding suggests that the shedding of sTNF-alphaR1 is induced by TNF-alpha itself, but in the case of sTNF-alphaR2, the shedding appears to be induced by another mechanism.     

Effect of ice melting on bacterial carbon fluxes channelled by viruses and protists in the Arctic Ocean

During the last few years, extensive sea ice melting in the Arctic due to climate change has been detected, which could potentially modify the organic carbon fluxes in these waters. In this study, the effect of sea ice melting on bacterial carbon channelling by phages and protists has been evaluated in the northern Greenland Sea and Arctic Ocean. Grazing on bacteria by protists was evaluated using the FLB disappearance method. Lysis of bacteria due to viral infections was measured using the virus reduction approach. Losses of bacterial production caused by protists (PMM_BP) dominated losses caused by viruses (VMM_BP) throughout the study. Lysogenic viral production was detected in 7 out of 21 measurements and constituted from 33.9 to 100.0% of the total viral production. Significantly higher PMM_BP and lower VMM_BP were detected in waters affected by ice melting compared with unaffected waters. Consequently, significantly more bacterial carbon was channelled to the higher trophic levels in affected waters (13.05 ± 5.98 μgC l⁻¹ day⁻¹) than in unaffected waters (8.91 ± 8.33 μgC l⁻¹ day⁻¹). Viruses channelled 2.63 ± 2.45 μgC l⁻¹ day⁻¹ in affected waters and 4.27 ± 5.54 μgC l⁻¹ day⁻¹ in unaffected waters. We conclude that sea ice melting in the Arctic could modify the carbon flow through the microbial food web. This process may be especially important in the case of massive sea ice melting due to climate change.     

Properties of a Novel PBP2A Protein Homolog from Staphylococcus aureus Strain LGA251 and Its Contribution to the ��-Lactam-resistant Phenotype

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A_LGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A_LGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A_LGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.