Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Recent developments in the activation process of bovine chymotrypsinogen A

A brief account is given of the history, distribution, and activation events of proteins of the bovine chymotrypsinogen family. Recent developments in the investigation of the activation process of bovine chymotrypsinogen A are discussed, and a revised scheme for the overall activation process is presented.     

Daboxin P,a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A₂ activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca⁺² binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A₂ enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.     

Length–weight relationships of six indigenous fish species from the River Cauvery and its estuary,India

The present study estimated length–weight relationships (LWRs) for six indigenous fish species (Barilius gatensis, Salmostoma acinaces, S. boopis, Puntius amphibius, Hemibagrus punctatus and Ambassis miops) based on specimens collected from River Cauvery (including estuary) during July 2017–January 2020. The sampling surveys were carried out in three distinct sampling seasons, viz., the pre-monsoon (March–May), the monsoon (July–October) and the post-monsoon (November–February). Majority of the fish specimens dealt in the study were collected from multi-meshed monofilament gill nets (mesh sizes 18, 30, 45, 60, 90, 110, 120 and 150 mm) operated by local fishers. For those sites situated in the protected areas, sampling was carried out by cast nets with prior permission from the local administration and the collected fishes were released back into river after length–weight measurements. The length measurements were noted as total length (TL) measured to the nearest 0.1 cm by using a digital Vernier caliper. A digital balance was used for weight measurements with an accuracy of 0.01 g. The study recorded a new maximum length of 48 cm for H. punctatus. The LWR data generated from the present study are significant for proper assessment of the stock status and their management, if collected together with other essential biological and physical parameters.     

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

Vitamin D attenuates HMGB1-mediated neointimal hyperplasia after percutaneous coronary intervention in swine

Molecular and Cellular Biochemistry - Intracoronary stenting is a common procedure in patients with coronary artery disease (CAD). Stent deployment stretches and denudes the endothelial layer,...     

Molecular interactions of MnO2@RGO (manganese dioxide-reduced graphene oxide) nanocomposites with bovine serum albumin

Abstract

Graphene based materials have attracted global attention due to their excellent properties. GO-metal oxide nanocomposites have been conjugated with biomolecules for the development of novel materials and potentially used as biomarkers. Herein, a detailed study on the interaction of Bovine serum albumin (BSA) with MnO₂@RGO (manganese dioxide-reduced graphene oxide) nanocomposites (NC) has been carried out. MnO₂@RGO nanocomposites were prepared through a template/surfactant free hydrothermal route at 180?°C for 12?h by varying the graphene oxide (GO) concentration. Different biophysical experiments have been carried out to evaluate molecular interactions between BSA and NCs. Intrinsic fluorescence has been used to quantify the quenching efficiency of NCs and the binding association of BSA-NC complexes. NCs effectively quenched the intrinsic fluorescence of BSA via static and dynamic mechanism. Further, the results indicate that the molecular interactions of NC with BSA are dependent on the GO percentage in NC. Circular dichroism results demonstrate nominal changes in the secondary structure of BSA in presence of NCs. Also, the esterase-like activity of BSA was marginally affected after adsorption upon NCs. In addition, the FESEM micrographs reveal that the protein-NC complexes consist of nanorod and sheet-like morphologies are forming aggregates of different sizes. We hope that this study will provide a basis for the design of novel graphene based and other related nanomaterials for several biological applications.

Communicated by Ramaswamy H. Sarma