Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.     

A finite element based method to determine the properties of planar soft tissue.

A finite element based method to determine the incremental elastic material properties of planar membranes was developed and evaluated. The method is applicable to tissues that exhibit inhomogeneity, geometric and material nonlinearity, and anisotropy. Markers are placed on the tissue to form a four-node quadrilateral element. The specimen is loaded to an initial reference state, then three incremental loading sets are applied and the nodal displacements recorded. One of these loadings must include shear. These data are used to solve an over-determined system of equations for the tangent stiffness matrix. The method was first verified using analytical data. Next, data obtained from a latex rubber sheet were used to evaluate experimental procedures. Finally, experiments conducted on preconditioned rat skin revealed nonlinear orthotropic behavior. The vector norm comparing the applied and calculated nodal force vectors was used to evaluate the accuracy of the solutions.     

Metabolic pathways involved in the oxidation of isopropanol into acetone by the intact rat

Isopropanol administered in a large (6 g/kg, orally) as well as in a lower dose (1 g/kg, I.P.) is slowly oxidized into acetone by the intact rat. Using two inhibitors, 3 amino-1,2,4-triazole and pyrazole, investigations on the hepatic enzymatic system involved in the oxidation of isopropanol show that catalase does not play an important part in this pathway, contrary to alcohol dehydrogenase which is the major enzyme responsible for this oxidation. Although isopropanol oxidation is mainly catalysed in the liver through alcohol dehydrogenase, no alteration of the hepatic extramitochondrial redox state occurs after the administration of a large as well as of a lower dose of isopropanol. From these experiments it may be concluded that alterations of the liver NAD⁺/NADH ratio, which seem to play an important part in the ethanol induced fatty liver, are not involved in the isopropanol induced one.     

SSU1 encodes a plasma membrane protein with a central role in a network of proteins conferring sulfite tolerance in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SSU1 gene was isolated based on its ability to complement a mutation causing sensitivity to sulfite, a methionine intermediate. SSU1 encodes a deduced protein of 458 amino acids containing 9 or 10 membrane-spanning domains but has no significant similarity to other proteins in public databases. An Ssu1p-GEP fusion protein was localized to the plasma membrane. Multicopy suppression analysis, undertaken to explore relationships among genes previously implicated in sulfite metabolism, suggests a regulatory pathway in which SSU1 acts downstream of FZF1 and SSU3, which in turn act downstream of GRR1.     

Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate.

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.     

Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.