Mass transfer studies of cell permeabilization and recovery of alkaline phosphatase from Escherichia coli by reverse micellar solutions

Transfer of alkaline phosphatase (AP) directly from Escherichia coli cells into reverse micellar solutions (RMS) was studied by varying the water content, pH, and ionic strength of RMS. Prior to the mass transfer studies, the optimum conditions for the activity of alkaline phosphatase in reverse micellar solutions were determined. The maximum enzyme activity could be detected at higher pH and water content, indicating the ionization of p-nitrophenol to be crucial in the enzyme activity. The transfer of AP from E. coli followed a two-step process with most of the recovery being achieved in the first 3-10 min beyond which it slowed. A model suggesting separate locations for the enzyme in the cell wall has been proposed.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking. Copyright 2001 Academic Press.     

Mitotic regulation: the fine tuning of separase activity

Mitotic progression requires the dissolution of cohesion between sister chromatids. Cohesion is dissolved by an essential protease known as separase. Separase is highly conserved throughout evolution and is subjected to multiple levels of regulation. Here we discuss recent studies that unravel several key mechanisms for regulating separase activity.     

No influence of scopolamine hydrobromide on odor detection performance of rats

Despite speculation that the muscarinic cholinergic antagonist, scopolamine, may influence the olfactory sensitivity of rats, there have been no definitive studies on this point to date. In this study, we examined the influence of a range of doses of scopolamine hydrobromine (namely, 0.10, 0.125, 0.15 and 0.20 mg/kg i.p.) on the odor detection performance of 15 adult male Long-Evans rats to ethyl acetate. Air-dilution olfactometry and a go/no-go operant signal detection task were employed. The drug conditions and a saline control were administered to each animal in an order counterbalanced by Latin squares, with 2 day intervals interspersed between tests. Scopolamine had no significant influence on odor detection performance per se, as measured by percent correct S+ and S- responses and a non-parametric signal detection measure of sensitivity. This is in contrast to the relatively large effects previously observed in the same test paradigm for such drugs as the D-1 agonist SKF 38393 and the D-2 agonist quinpirole. These data suggest that scopolamine has no meaningful influence on a well-practiced odor detection task.     

Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh

Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.     

Identification,purification and characterization of a receptor for dengue virus-induced macrophage cytotoxin (CF2) from murine T cells

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mphi) to produce a biologically active cytotoxic cytokine, the Mphi cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mphi. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22000 receptors per cell). The binding of [125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [125I]CF2. The purified CF2-R is a 90-95-kDa protein as characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [125I]CF2 and its receptor complex showed a product of 100-110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease.     

Monomeric APOBEC3G is catalytically active and has antiviral activity

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif-defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.