The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.     

Possible role of NO modulators in protective effect of trazodone and citalopram (antidepressants) in acute immobilization stress in mice

Stress is an aversive stimulus which disturbs physiological homeostasis and is reflected on a variety of biological systems. The present study was designed to investigate the nitric oxide mechanism in neuroprotective effect of trazodone and citalopram against acute immobilization-induced behavioral and biochemical alteration in mice. Mice were immobilized for a 6 h. Acute immobilization stress caused anxiety, hyperalgesia, impaired locomotor activity and oxidative damage. Pretreatment with trazodone and citalopram significantly reversed immobilized stress-induced behavioral and biochemical alterations. L-arginine, pretreatment with trazodone or citalopram significantly reversed their protective effects. However, L-NAME or methylene blue pretreatment with trazodone or citalopram significantly potentiated their protective effects alone. Results suggest the involvement of nitric oxide pathways in the protective effect of trazodone and citalopram against immobilization stress induced behavioral and biochemical alterations.     

Differential protein expression in human gliomas and molecular insights

Gliomas are the most common of the primary intracranial tumors with astrocytomas constituting about 40%. Using clinically and histologically assessed astrocytomas, we have studied their protein profiles using a two-dimensional gel electrophoresis-mass spectrometry approach and identified differentially expressed proteins which may be useful molecular indicators to understand these tumors. Examination of the protein profiles of 27 astrocytoma samples of different grades revealed 72 distinct, differentially expressed proteins belonging to various functional groups such as cytoskeleton and intermediate filament proteins, heat shock proteins (HSPs), enzymes and regulatory proteins. Based on the consistency of their differential expression, 29 distinct proteins could be short-listed and may have a role in the pathology of astrocytomas. Some were found to be differentially expressed in both Grade III and IV astrocytomas while others were associated with a particular grade. A notable observation was underexpression of Prohibitin, a potential tumor suppressor protein, Rho-GDP dissociation inhibitor, Rho-GDI, a regulator of Rho GTPases and HSPs as well as destabilization of glial fibrillary acidic protein, GFAP, major protein of the glial filaments, in Grade III malignant tumors. We attempt to explain glioma malignancy and progression in terms of their combined role.     

Signaling through Disabled 1 requires phosphoinositide binding

The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled 1 (Dab1) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1.     

The discovery of a selective, high affinity A(2B) adenosine receptor antagonist for the potential treatment of asthma

Adenosine has been suggested to play a role in asthma, possibly via activation of A(2B) adenosine receptors on mast cells and other pulmonary cells. We describe our initial efforts to discover a xanthine based selective A(2B) AdoR antagonist that resulted in the discovery of CVT-5440, a high affinity A(2B) AdoR antagonist with good selectivity (A(2B) AdoR K(i)=50 nM, selectivity A(1)>200: A(2A)>200: A(3)>167).     

Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR,and their antifungal activity using NMR based metabolic profiling

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer’s structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time ¹H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.     

Modeling Curvature-Dependent Subcellular Localization of the Small Sporulation Protein SpoVM in Bacillus subtilis

Recent in vivo experiments suggest that in the bacterium, Bacillus subtilis, the cue for the localization of the small sporulation protein, SpoVM, an essential factor in spore coat formation, is curvature of the bacterial plasma membrane. In vitro measurements of SpoVM adsorption to vesicles of varying sizes also find high sensitivity of adsorption to vesicle radius. This curvature-dependent adsorption is puzzling given the orders of magnitude difference in length scale between an individual protein and the radius of curvature of the cell or vesicle, suggesting protein clustering on the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane adsorption and clustering of SpoVM. Based on our analysis, we hypothesize that the radius dependence of SpoVM adsorption observed in vitro is governed primarily by membrane tension, while for in-vivo localization of SpoVM, we propose a highly sensitive mechanism for curvature sensing based on the formation of macroscopic protein clusters on the membrane.