Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

Binding of [3H]befunolol to beta-adrenoceptors in cardiac muscles of fetal and neonatal rat

Characterization of beta-adrenoceptors was studied in heart muscles of rat fetus and neonate. The results of binding assay with [3H]befunolol, a beta-adrenergic partial agonist, to membrane fractions from rat heart muscles indicate that beta-adrenoceptors contain two different affinity sites. In the presence of 5'-guanylylimidodiphosphate, the low affinity site was reduced, while the high affinity site was not affected. The dissociation constants for both sites did not change during pre- and post-natal development. But the maximum binding sites for both sites decreased slightly but significantly (p less than 0.05) during development. A 10-fold decrease in norepinephrine sensitivity and isoprenaline sensitivity during pre- and post-natal development was not explained by the slight decrease in the maximum binding sites.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).     

Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.     

Anti-inflammatory action of gentamycin through inhibitory effect on neutrophil NADPH oxidase activity

The effects of gentamycin on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Gentamycin was found to inhibit, concentration-dependently, the superoxide generation of neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of the drug required for 50% inhibition of the oxidase (IC₅₀) were 150 μM in the whole-cell system and 10 μM in the cell-free system. In addition, in the cell-free system, the drug did not change the K_m value for NADPH of the oxidase. However, gentamycin did not the superoxide generation of NADPH oxidase after its activation in the cell-free system, suggesting that the drug do not have superoxide-scavenger action. These results suggest that gentamycin, an aminoglycoside antibiotic, may exhibit an anti-inflammatory action due to inhibition of neutrophil NADPH oxidase activation.     

Chronic ethanol administration decreases fatty acyl-CoA desaturase activities in rat liver microsomes

The Δ⁹-desaturase system in liver microsome from rats treated chronically with ethanol was studied. Stearoyl-CoA desaturase activity decreased by 80% and palmitoyl-CoA desaturase activity was not detectable in microsomes from ethanol-fed rats, while activities of electron transport components such as NADH-cytochrome c and NADH-ferricyanide reductases remained unchanged. However, chronic ethanol administration resulted in an adaptive induction of the activity of NADPH-cytochrome c reductase and the contents of cytochrome b₅ and P-450. The activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system was greatly depressed by ethanol treatment. The NADH/NAD ratio in microsomes of ethanol-fed rats increased over 2-fold. These results suggest that, during chronic ethanol ingestion, decreased activities of Δ⁹-desaturases are due mainly to a decreased content of the terminal component of the desaturase system.