Effects of remote mutation on the autolysis of HIV-1 PR: X-ray and NMR investigations

Autolysis rates of the C95M and C95M/C1095A mutants of a HIV-1 protease tethered dimer have been determined by real time NMR and it is observed that the double mutant has approximately two times higher rate. X-ray structure of the C95M/C1095A double mutant has been solved and refined to 2.1 A resolution. Comparison of the double mutant structure with that of C95M single mutant reveals that there is a shift in the position of the catalytic aspartates and the bound catalytic water. The mutation also causes a loss of hydrophobic packing near the dimerization domain of the protein. These observations demonstrate that subtle changes are adequate to cause significant changes in the rate of autolysis of the double mutant. This provides a rationale for the effects of remote mutations on the activity and drug resistance of the enzyme.     

Biosorption of americium using biomasses of various Rhizopus species

Biomasses from eight different Rhizopus species were tested for the sorption of americium from nitric acid medium. Rhizopus arrhizus NCIM 997 showed maximum sorption at pH 2. Laboratory scale experiments were carried out using this biomass in packed columns for the sorption of -activity from an americium spiked low level waste stream of PUREX process. The biomass was found to be an excellent sorbent for remediation of low level waste streams on once through basis.     

E-NTPase/E-NTPDase: a potential regulatory role in E-kinase/PKA-mediated CD36 activation

CD36 is a platelet surface receptor protein that plays a major role in platelet aggregation and accumulation that is mediated by parasitic attachment. The CD36 receptor is constitutively phosphorylated by E-kinase/PKA, resulting in increased affinity for collagen, but preventing spontaneous platelet aggregation. Dephosphorylation of CD36 by protein phosphatase 2A (PP2A) leads to increased affinity for thrombospondin at a different rate than that of collagen-mediated platelet aggregation. Depletion of the E-kinase/PKA substrate [ATP](0)by E-NTPase-mediated hydrolysis, in conjunction with inhibition of PP2A by okadaic acid, could prove to be a valuable tool in inhibiting CD36 activation, thus preventing platelet aggregation and thrombus formation.     

Cardiovascular effects of leptin and orexins

Leptin, the product of the ob gene, is a satiety factor secreted mainly in adipose tissue and is part of a signaling mechanism regulating the content of body fat. It acts on leptin receptors, most of which are located in the hypothalamus, a region of the brain known to control body homeostasis. The fastest and strongest hypothalamic response to leptin in ob/ob mice occurs in the paraventricular nucleus, which is involved in neuroendocrine and autonomic functions. On the other hand, orexins (orexin-A and -B) or hypocretins (hypocretin-1 and -2) were recently discovered in the hypothalamus, in which a number of neuropeptides are known to stimulate or suppress food intake. These substances are considered important for the regulation of appetite and energy homeostasis. Orexins were initially thought to function in the hypothalamic regulation of feeding behavior, but orexin-containing fibers and their receptors are also distributed in parts of the brain closely associated with the regulation of cardiovascular and autonomic functions. Functional studies have shown that these peptides are involved in cardiovascular and sympathetic regulation. The objective of this article is to summarize evidence on the effects of leptin and orexins on cardiovascular function in vivo and in vitro and to discuss the pathophysiological relevance of these peptides and possible interactions.     

Improvement in ductility of chitosan through blending and copolymerization with PEG: FTIR investigation of molecular interactions

Chitosan is an important biomaterial used widely in medical applications. One of the key concerns about its use is the fragile nature of chitosan films. By comparing the component molecular interactions using FTIR, this study attempts to understand how the ductility of chitosan can be improved by blending and copolymerizing with poly(ethylene glycol) (PEG). An improvement in ductility was obtained for all compositions of blend as manifested by a decrease in modulus and an increase in strain at break. For comparable PEG composition (approximately 30%), the properties of the solution-cast blend were better than those of the grafted copolymer. Therefore, blending may be a more efficient way to improve ductility of chitosan. FTIR characterization of the materials revealed subtle decreases in molecular interactions upon annealing the partially miscible blend. These may not be apparent in DSC or X-ray diffraction, yet they play a key role in the mechanical behavior. It appears that in the case of the graft copolymer the improvement in the properties comes from suppression of the crystallinity of each component and not from component interactions. On the other hand, in the blend, the improvement appears to come predominantly from the "well-dispersed", "kinetically trapped" phase morphology and from the intermolecular interactions. Therefore, annealing the blend leads to decreased intermolecular interactions, phase coarsening, and deterioration in properties.     

Fibril modelling by sequence and structure conservation analysis combined with protein docking techniques: beta(2)-microglobulin amyloidosis

Obtaining atomic resolution structural models of amyloid fibrils is currently impossible, yet crucial for our understanding of the amyloid mechanism. Different pathways in the transformation of a native globular domain to an amyloid fibril invariably involve domain destabilization. Hence, locating the unstable segments of a domain is important for understanding its amyloidogenic transformation and possibly control it. Since relative conservation is suggested to relate to local stability, we performed an extensive, sequence and structure conservation analysis of the beta(2)-microglobulin (beta(2)-m) domain. Our dataset include 51 high resolution structures belonging to the "C1 set domain" family and 132 clustered PSI-BLAST search results. Segments of the beta(2)-m domain corresponding to strands A (residues 12-18), D (45-55) and G (91-95) were found to be less conserved and stable, while the central strands B (residues 22-28), C (36-41), E (62-70) and F (78-83) were found conserved and stable. Our findings are supported by accumulating observations from various experimental methods, including urea denaturation, limited proteolysis, H/D exchange and structure determination by both NMR and X-ray crystallography. We used our conservation findings together with experimental literature information to suggest a structural model for the polymerized unit of beta(2)-m. Pairwise protein docking and subsequent monomer stacking in the same manner suggest a fibril model consistent with the cross-beta structure.     

Adenoviral mediated anti-sense CD38 attenuates TNF-alpha-induced changes in calcium homeostasis of human airway smooth muscle cells

CD38 is a membrane-bound protein involved in the synthesis and degradation of cyclic-ADP-ribose (cADPR). cADPR mobilizes calcium from intracellular stores in airway smooth muscle cells. To determine the role of CD38/cADPR signaling in calcium regulation in human airway smooth muscle (HASM) cells, we downregulated CD38 expression using a recombinant replication-defective adenovirus with anti-sense human CD38 (Ad-asCD38). CD38 expression was determined by RT-PCR and real-time quantitative PCR, and ADP-ribosyl cyclase (cyclase) activity was determined by competitive binding assay. In HASM cells infected with Ad-asCD38, TNF-alpha-induced, augmented-CD38 expression and cyclase activity were significantly lower than in TNF-alpha-treated cells. The net intracellular calcium responses to 10 nmol/L bradykinin were measured in HASM cells by fluorescence imaging. In cells infected with Ad-asCD38 in the presence of TNF-alpha, the net intracellular Ca2+ responses were significantly lower than in cells treated with TNF-alpha in the presence of the control vector (p < 0.001). These results provide evidence for the feasibility of using adenoviral vectors for gene transfer to down regulate gene expression, and confirm the role of CD38 in calcium homeostatis in ASM cells.     

Central stresscopin modulates cardiovascular function through the adrenal medulla in conscious rats

Stresscopin (SCP or urocortin III), a member of the corticotropin-releasing factor (CRF) neuropeptide family, is a high-affinity ligand for the type 2 CRF receptor (CRF(2)). When administered peripherally, SCP suppresses food intake, delays gastric emptying and decreases heat-induced edema. Central administration of CRF produces marked hypertension and increased plasma catecholamine. However, the effects of SCP on the cardiovascular system are unknown. Thus, the present study compared the effects of intracerebroventricular (i.c.v.) administration of CRF and SCP on cardiovascular function. Central administration of SCP (0.05 or 0.5 nmol) elicited transient increases in mean arterial blood pressure (MABP) and heart rate (HR), and the higher dose of SCP (0.5 nmol) resulted in increased plasma epinephrine. In contrast, central administration of CRF provoked long-lasting increases in MABP, HR and plasma catecholamine levels (norepinephrine and epinephrine). Intravenously administered CRF and SCP (0.5 nmol) did not elicit significant changes in MABP and HR. Therefore, these data suggest that centrally administered SCP modulates cardiovascular function, likely through the sympatho-adrenal-medullary (SAM) system.