Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model

The present study was conducted to investigate the role of iron-regulated outer membrane proteins (IROMP) of Pasteurella multocida B:2 in mice as potential immunogens. Outer membrane proteins extracted from P. multocida B:2 grown under normal (OMP) and iron-deficient (IROMP) conditions were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 85.1 to 16.7 kDa from OMP preparations and two additional polypeptides of MW 95.4 and 89.1 kDa from IROMP preparations were observed with bands of MW 37.2 and 34.7 kDa as major proteins. Mice were immunized twice with OMP, IROMP-enriched fractions and whole cell lysate (WCL) via subcutaneous route at day 0 and 21. Antibody titers were determined from sera collected at weekly interval and protection was studied against challenge using 10(2) cfu of P. multocida two weeks after secondary immunization via intranasal and subcutaneous routes. IROMP and OMP immunized mice provoked significant antibody responses and IROMP induced higher antibody responses. IROMP and OMP immunized mice showed protection (100%) upon intranasal challenge and a protection (84%) following subcutaneous challenge as compared to high mortality (84%) in control mice. These results indicate that OMP enriched with IROMP fractions can be superior means of immunization.     

The Membrane Subunit NuoL(ND5) Is Involved in the Indirect Proton Pumping Mechanism of Escherichia coli Complex I

Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H⁺ translocation site(s), because of a high sequence similarity to multi-subunit Na⁺/H⁺ antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na⁺/H⁺ antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K₃Fe(CN)₆ reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H⁺/e⁻), however, was decreased in most NuoL mutants by 30–50%, while the IC₅₀ values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H⁺/e⁻ stoichiometry has changed from 4H⁺/2e⁻ to 3H⁺ or 2H⁺/2e⁻ without affecting the direct coupling site. Furthermore, 50 μm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na⁺/H⁺ antiporters, caused a 38 ± 5% decrease in the initial H⁺ pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H⁺ pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.     

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.     

Synthesis and characterization of pH-sensitive hydrogel composed of carboxymethyl chitosan for colon targeted delivery of ornidazole

In the present study, carboxymethyl chitosan was prepared from chitosan, crosslinked with glutaraldehyde and evaluated in vitro as a potential carrier for colon targeted drug delivery of ornidazole. Ornidazole was incorporated at the time of crosslinking of carboxymethyl chitosan. The chitosan was evaluated for its degree of deacetylation (DD) and average molecular weight; which were found to be 84.6% and 3.5×10(4) Da, respectively. The degree of substitution on prepared carboxymethyl chitosan was found to be 0.68. All hydrogel formulations showed more than 85% and 74% yield and drug loading, respectively. The swelling behaviour of prepared hydrogels checked in different pH values, 1.2, 6.8 and 7.4, indicated pH responsive swelling characteristic with very less swelling at pH 1.2 and quick swelling at pH 6.8 followed by linear swelling at pH 7.4 with slight increase. In vitro release profile was carried out at the same conditions as in swelling and drug release was found to be dependant on swelling of hydrogels and showed biphasic release pattern with non-fickian diffusion kinetics at higher pH. The carboxymethylation of chitosan, entrapment of drug and its interaction in prepared hydrogels were checked by FTIR, (1)H NMR, DSC and p-XRD studies, which confirmed formation of carboxymethyl chitosan from chitosan and absence of any significant chemical change in ornidazole after being entrapped in crosslinked hydrogel formulations. The surface morphology of formulation S6 checked before and after dissolution, revealed open channel like pores formation after dissolution.     

The magnitude of dengue virus NS1 protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model

There are conflicting data on the relationship between the level of secreted NS1 (sNS1), viremia, and disease severity upon dengue virus (DENV) infection in the clinical setting, and therefore, we examined this relationship in the widely accepted AG129 mouse model. Because of the failure of a routinely used NS1 detection kit to detect sNS1 of the mouse-adapted DENV2 strain, we screened 15 previously undescribed NS1 monoclonal antibodies and developed a robust capture enzyme-linked immunosorbent assay (ELISA) with detection sensitivity at the low nanogram level (0.2 ng/ml) using recombinant baculovirus-expressed sNS1 as well as sNS1 that was immunoaffinity purified from the various DENV2 strains employed in this study. Using this test, we demonstrated that increased viremia paralleled severe pathologies; however, sNS1 level did not correlate with viremia or severity. Furthermore, among the DENV2 strains that were tested, the level of NS1 secretion did not correspond to virus replication rate in vitro, at the cellular level. Together, our data indicate that the magnitude of NS1 secretion appears to be strain dependent and does not correlate with viral virulence in the AG129 mouse model.     

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the β-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.     

Synthesis and characterization of pyruvate-isoniazid analogs and their copper complexes as potential ICL inhibitors

Currently used anti-tubercular drugs target actively growing Mycobacterium tuberculosis (Mtb) but there are no current therapies targeting persistent mycobacteria. Isocitrate lyase (ICL) is an important enzyme of the glyoxylate shunt pathway used by Mtb for sustaining intracellular infection in inflammatory macrophages under conditions of stress such as nutrient depletion and anaerobic metabolism. Since the humans do not possess this enzyme it constitutes an attractive target for selective drug design. Present work describes synthesis and structural characterization of pyruvate-isoniazid conjugates and their copper complexes with potent anti-tubercular activities against M. tuberculosis H37Rv.