Studies on extracts of Elaeocarpus sphaericus fruits on in vitro rat mast cells.

Elaeocarpus sphaericus (Syn: E. ganitrus), in Ayurvedic Medicine commonly known as Rudraksha is known to have wide range of pharmacological activities. We reported previously the protective action of E. sphaericus in experimental bronchial asthma. The present study on rat mesenteric mast cell was undertaken to investigate the effect of E. sphaericus fruits on autacoid release. The petroleum ether (PE), benzene (BE), chloroform (CE), acetone (AE) and ethanol (EE) extracts of E. sphaericus fruits were found to have mast-cell stabilizing activity, substantiating the efficacy of E. sphaericus against bronchial asthma.     

Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A

Molecular probes are useful for both studying and controlling the functions of enzymes and other proteins. The most useful probes have high affinity for their target, along with small size and resistance to degradation. Here, we report on new surrogates for nucleic acids that fulfill these criteria. Isosteres in which phosphoryl [R-O-P(O(2)(-))-O-R'] groups are replaced with N-acylsulfonamidyl [R-C(O)-N(-)-S(O(2))-R'] or sulfonimidyl [R-S(O(2))-N(-)-S(O(2))-R'] groups increase the number of nonbridging oxygens from two (phosphoryl) to three (N-acylsulfonamidyl) or four (sulfonimidyl). Six such isosteres were found to be more potent inhibitors of catalysis by bovine pancreatic RNase A than are parent compounds containing phosphoryl groups. The atomic structures of two RNase A·N-acylsulfonamide complexes were determined at high resolution by X-ray crystallography. The N-acylsulfonamidyl groups were observed to form more hydrogen bonds with active site residues than did the phosphoryl groups in analogous complexes. These data encourage the further development and use of N-acylsulfonamides and sulfonimides as antagonists of nucleic acid-binding proteins.     

Animal Welfare in Nepal

The small densely populated country of Nepal rises from just above sea level to more than 8800 m and encompasses many agro-ecological zones. Rich in both nature and culture, livestock are integral to household and national economies. Most Nepalese consider the cow sacred, and slaughter is forbidden. Other nonhuman animals are less esteemed, slaughter is not proscribed, and many are sacrificed during religious festivals. Limited financial and material resources, inadequate feed supplies, poor access to veterinary services, absence of appropriate legislation, and ignoring the needs of livestock as sentient beings can compromise welfare and lead to inhumane treatment. This article reviews the welfare status of various groups (food-producing stock, work animals, street dogs, experimental animals, and elephants in captivity). Several animal welfare charities are represented. Suggestions for improved welfare are discussed, but the prognosis is not encouraging especially in view of the widespread struggle and people’s own struggle for survival.     

Prediction of structural consequences for disease causing variants in C21orf2 protein using computational approaches

Amyotrophic lateral sclerosis (ALS), a progressive motor-neurone disease, affects individuals usually aged between 50 and 70 years. C21orf2, recently identified as the new ALS susceptibility gene, harbours rare missense mutations that cause this fatal disease. We used bioinformatics and molecular modelling approaches to study specific ALS-associated mutations in C21orf2. Both native and mutant structures of the protein obtained from homology modelling were analysed in detail to gain insights into the potential impact of these mutations on the protein structure and its function. Our analyses reveal that more than 75% of the mutations are likely to be deleterious. These effects seem to carry through to mouse C21orf2 as well, indicating that mouse would make a viable animal model to study this ALS gene in detail.     

Trophoblasts regulate the placental hematopoietic niche through PDGF-B signaling

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRβ, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.     

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.