Infusing Sodium Bicarbonate Suppresses Hydrogen Peroxide Accumulation and Superoxide Dismutase Activity in Hypoxic-Reoxygenated Newborn Piglets

The effectiveness of sodium bicarbonate (SB) has recently been questioned although it is often used to correct metabolic acidosis of neonates. The aim of the present study was to examine its effect on hemodynamic changes and hydrogen peroxide (H(2)O(2)) generation in the resuscitation of hypoxic newborn animals with severe acidosis.Newborn piglets were block-randomized into a sham-operated control group without hypoxia (n = 6) and two hypoxia-reoxygenation groups (2 h normocapnic alveolar hypoxia followed by 4 h room-air reoxygenation, n = 8/group). At 10 min after reoxygenation, piglets were given either i.v. SB (2 mEq/kg), or saline (hypoxia-reoxygenation controls) in a blinded, randomized fashion. Hemodynamic data and blood gas were collected at specific time points and cerebral cortical H(2)O(2) production was continuously monitored throughout experimental period. Plasma superoxide dismutase and catalase and brain tissue glutathione, superoxide dismutase, catalase, nitrotyrosine and lactate levels were assayed.Two hours of normocapnic alveolar hypoxia caused cardiogenic shock with metabolic acidosis (PH: 6.99 ± 0.07, HCO(3)(-): 8.5 ± 1.6 mmol/L). Upon resuscitation, systemic hemodynamics immediately recovered and then gradually deteriorated with normalization of acid-base imbalance over 4 h of reoxygenation. SB administration significantly enhanced the recovery of both pH and HCO(3-) recovery within the first hour of reoxygenation but did not cause any significant effect in the acid-base at 4 h of reoxygenation and the temporal hemodynamic changes. SB administration significantly suppressed the increase in H(2)O(2) accumulation in the brain with inhibition of superoxide dismutase, but not catalase, activity during hypoxia-reoxygenation as compared to those of saline-treated controls.Despite enhancing the normalization of acid-base imbalance, SB administration during resuscitation did not provide any beneficial effects on hemodynamic recovery in asphyxiated newborn piglets. SB treatment also reduced the H(2)O(2) accumulation in the cerebral cortex without significant effects on oxidative stress markers presumably by suppressing superoxide dismutase but not catalase activity

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus

The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of Mr 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a m 1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via m 1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters

The role of arginine-rich motif and beta-annulus in the assembly andstability of sesbania mosaic virus capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein,protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the \beta-annulus structure. Here,mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T = 3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less. stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assemblyis entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of \beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T = 3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of \beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T = 3 capsids is not the-annulus. The ordered \beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize inter subunit interactions

A Molecular Switch in the Capsid Protein Controls the Particle Polymorphism in an Icosahedral Virus

The recombinant coat protein (CP) of Sesbania mosaic virus (SeMV; genus Sobemovirus) was found to self-assemble into capsids encapsidating 23S rRNA and CP mRNA in Escherichia coli. The CP lacking 22 amino acids from the N-terminus assembled into stable T = 3 capsids that appeared similar to SeMV, indicating that the N-terminal 22 amino acid residues are dispensable for T = 3 assembly. Two distinct capsids, T = 1 and pseudo T = 2, were observed when the N-terminal 36 amino acids encompassing the arginine-rich motif (N-ARM) were removed. Only T = 1 particles were observed upon deletion of 65 amino acids from the N-terminus, which also included the sequence element for the β-annulus. These results reveal that N-ARM acts as a molecular switch in regulating T = 3 assembly. Formation of stable pseudo T = 2 particles shows that pentamers of AB dimers could nucleate assembly at icosahedral-5-folds. Capsids assembled from the N-terminally truncated proteins also encapsidated 23S rRNA and CP mRNA, suggesting the presence of sites outside the N-terminal 65 residues that may be involved in RNA–protein interactions

Role of Metal Ion-mediated Interactions in the Assembly and Stability of Sesbania Mosaic Virus T=3 and T=1 Capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA–protein, protein–protein and calcium-mediated protein–protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS).The CBS environment is found to be identical even in the recombinant CP-N$\Delta$65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146ND149N mutation in CP-N$\Delta$65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the TZ3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the $\beta$-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM–RNA interactions that lead to the formation of the $\beta$-annulus, which is essential for T=3 capsid assembly

T=1 Capsid Structures of Sesbania Mosaic Virus Coat Protein Mutants: Determinants of T=3 and T=1 Capsid Assembly

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29 kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-N \Delta 36) and 65 (CP-N \Delta 65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20 nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-N \Delta 65 lacks the \beta-annulus believed to be responsible for the error-free assembly of T=3 particles. Though the CP-N \Delta 36 mutant has the b-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CPND36 and CP-N \Delta 65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-ND65, were mutated to asparagine (CP-N \Delta 65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the \beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles

Determination of the structure of the recombinant T = 1 capsid of Sesbania mosaic virus

The recombinant coat protein (CP) of Sesbania mosaic virus lacking segments of different lengths from the N-terminus expressed in E. coli was shown to selfassemble into a variety of distinct capsids encapsidating 23S rRNA from the host and CP mRNA in vivo.Particles with 60 copies (T = 1) of protein subunits were observed when protein lacking 65 amino acids from the N-terminus was expressed. This recombinant protein possesses the sequence corresponding to the S-domain of the native, T = 3 icosahedral particles but lacks the $\beta$-annulus, the $\beta$A strand (residues 67–70) and the arginine-rich ARM motif (residues 28–36). Purified T = 1 particles crystallized in the monoclinic space group $P2_1$ with cell parameters of a = 188.4 $\AA$, b = 194.6 $\AA$, c = 272.1 $\AA$ and $\beta$ = $92.6^°$. The structure of the T = 1 particles was determined by X-ray diffraction at 3.0 $\AA$ resolution. As expected, the poly-peptide fold of the subunit closely resembles that of the S-domain of the native virus. The recombinant particles bind calcium ions in a manner indistinguishable from that of the native capsids. The structure reveals the major differences in the quaternary organization responsible for the formation of T = 1 against T = 3 particles