Comparative characterisation of Russell's viper (Daboia/Vipera russelli) venoms from different regions of the Indian peninsula.

Russell's viper (Daboia/Vipera russelli) venom from different regions of India was subjected to chromatographic, electrophoretic, biochemical and immunological analysis. The elution profiles from ion-exchange chromatography and protein banding pattern from SDS-PAGE showed a significant variation in the constituents of venoms. The acidic proteins are found to be predominant in the venoms of eastern and western regions while basic proteins are the major contributors of the northern and southern regional venoms. The major variation of phospholipases A(2) in the venom samples of India may be described as: southern regional venom is rich in basic, toxic PLA(2) while this activity showed a dramatic decrease as one moves towards west, north and eastern regions of India. In addition, the caseinolytic, TAME-hydrolytic, anticoagulant, oedema-inducing and haemorrhagic activities of the venoms have also varied from one region to another. The muscle specimens of mice injected with venoms of different regions showed variable change in the muscle fibre damage and cell morphology. The eastern regional venom is most lethal among all the venoms. The lethal potencies for four regional venoms vary as: eastern>western>southern>northern. The polyclonal antibodies prepared against the venom of southern region showed cross-reaction with the venoms of other regions, but the extent of cross-reaction and diffusion patterns are different. However, the polyclonal antibodies prepared against southern regional venom showed no protection against lethal toxicity of other regional venoms.     

Use of short-lived green fluorescent protein for the detection of proteasome inhibition

Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the constitutive levels of d2EGFP expression was observed. Based on flow cytometry, proteasome inhibitors induced a 5- to 10-fold increase in the fluorescent intensity of d2EGFP in HEK293 cell clones. However, in the presence of proteasome inhibitors, HEK293 clones showed a 4- to 6.5-fold increase in d2EGFP concentration as determined by western blot analysis. Our data suggest that d2EGFP is a useful indicator of proteasome inhibition. Therefore, stable expression of d2EGFP in mammalian cells is potentially useful for high-throughput screening of cDNAs or pharmaceutical drugs that repress proteasome functions in vivo.     

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.     

Role of phospholipid head groups in ethanol tolerance of Saccharomyces cerevisiae

Pre-incubation of cells of Saccharomyces cerevisiae with 2 M-ethanol led to decreased rates of L-alanine uptake, H+ efflux and fermentation rate. However, these responses were modified in yeast cells with altered phospholipid composition. Using L-alanine transport and H+ efflux as indices of ethanol tolerance, it was observed that cells enriched with phosphatidylserine had greater tolerance to ethanol. This resulted from altered charge of membrane phospholipids rather than changes in membrane fluidity. It is suggested that the anion:zwitterion ratio of phospholipids may be one of the important determinants of ethanol tolerance in S. cerevisiae.     

The gp120 Protein Is a Second Determinant of Decreased Neurovirulence of Indian HIV-1C Isolates Compared to Southern African HIV-1C Isolates

Regional differences in neurovirulence have been documented among subtype/clade-C HIV-1 isolates in India and Southern Africa. We previously demonstrated that a C31S substitution in Clade-C Tat dicysteine motif reduces monocyte recruitment, cytokine induction and direct neurotoxicity. Therefore, this polymorphism is considered to be a causative factor for these differences in neurovirulence. We previously reported on the genotypic differences in Tat protein between clade-C and rest of the clades showing that approximately 90% of clade-C HIV-1 Tat sequences worldwide contained this C31S polymorphism, while 99% of non-clade C isolates lacked this Tat polymorphism at C31 residue (Ranga et al. (2004) J Virol 78∶2586–2590). Subsequently, we documented intra-clade-C differences in the frequency of Tat dicysteine variants between India and Southern Africa, as the basis for differential disease severity and showed the importance of the Tat dicysteine motif for neuropathogenesis using small animal models. We have now examined if determinants of neurovirulence besides Tat are different between the clade-C HIV-1 isolates from Southern Africa and India. Envelope glycoprotein gp120 is a well-documented contributor to neurotoxicity. We found that gp120 sequences of HIV-1 isolates from these two regions are genetically distinct. In order to delineate the contribution of gp120 to neurovirulence, we compared direct in vitro neurotoxicity of HIV-infected supernatants of a representative neurovirulent US clade-B isolate with two isolates each from Southern Africa and India using primary human neurons and SH-SY5Y neuroblastoma cells. Immunodepletion of gp120 of both US clade B and the Southern African clade C isolates revealed robust decreases in neurotoxicity, while that of the Indian isolates showed minimal effect on neurotoxicity. The gp120 as a cause of differential neurotoxicity was further confirmed using purified recombinant gp120 from HIV isolates from these regions. We conclude that gp120 is one of the key factors responsible for the decreased neurovirulence of Indian clade C HIV-1 isolates when compared to South African clade C HIV-1.     

Functional characterization of purified zinc transporter from renal brush border membrane of rat

Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase