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Biomedical subjects

P A LeBlanc

Publications and source records attributed to P A LeBlanc.

At least 19 recordsLinked to original sources
PubMed1999

Interaction with vesicular stomatitis virus-infected BALB/c3T3 cells inhibits the synthesis of nitric oxide in activated murine bone marrow culture-derived macrophages.

Bone marrow-culture-derived macrophages activated with interferon-gamma and lipopolysaccharide produced less nitric oxide (NO) when cultured with vesicular stomatitis virus (VSV)-infected BALB/c3T3 (3T3-VSV) than macrophages activated in an identical manner and cultured alone, with uninfected BALB/c3T3 (3T3), or with P815. However, all four groups of macrophages produced nearly the same amount of interleukin-6 (IL-6). Addition of VSV to activated macrophages did not change the amount of NO produced. The amount of NO generated by two non-macrophage sources of NO was not affected by the presence of either P815 or 3T3-VSV. Reverse transcriptase-polymerase chain reaction showed a decrease in the amount of inducible nitric oxide synthase (iNOS) but not IL-6 mRNA from macrophages cocultured with 3T3-VSV compared with macrophages cocultured with P815. The reduction in iNOS mRNA was confirmed by ribonuclease protection assay. When RAW 264.7 transfected with an iNOS regulatory construct were activated and incubated with 3T3-VSV there was a decrease in the expression of the reporter luciferase gene and NO production but not IL-6 production compared with cells incubated with either medium alone or with P815.

3T3 Cells↗
PubMed1998

Wall-associated processing of extracellular enzymes of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus produces a staphylolytic glycylglycine endopeptidase (lysostaphin) and a micrococcolytic endo-beta-N-acetylglucosaminidase (hexosaminidase) as proenzymes that are proteolytically processed through multiple intermediates to their mature forms by an extracellular sulfhydryl protease. Analysis of protease production by immunoblots using antiserum prepared against purified protease and by renaturing activity gels using gelatin as the substrate has revealed that the lysostaphin-processing protease also is produced as a proenzyme, which appears to be autocatalytically processed. Very little proprotease could be detected in supernatants from cultures of S. simulans biovar staphylolyticus, which suggested that the protein was being processed before it was released to the culture medium. Analysis of wall-associated proteins revealed that processing of proprotease occurred primarily in the cell wall. Furthermore, processing of prolysostaphin and prohexosaminidase also occurred in the cell wall matrix.

Animals↗
PubMed1997

Mouse natural killer cells express gp49B1, a structural homologue of human killer inhibitory receptors.

NK cells express MHC class I-specific receptors that inhibit tumor killing. In mice, these receptors belong to the highly polymorphic Ly-49 family, which are type II integral membrane proteins homologous to C-type lectins. In contrast, human killer inhibitory receptors (KIR) are type I transmembrane proteins that display minimal allelism and belong to the Ig superfamily. These structural differences suggested that each species evolved distinct receptors to subserve the same function. In this report, however, we show that mouse NK and LAK cells and NK cell clones express full-length transcripts for gp49B1, an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing type I transmembrane protein belonging to the Ig superfamily, and displaying minimal allelism by Southern and sequence analysis. By flow cytometry, gp49B1 is expressed on all NK cells. Therefore, we have established that gp49B1, a structural homologue of human KIR, is expressed on mouse NK cells. This strongly suggests that NK cells express two structurally distinct types of inhibitory receptors and that these receptors may act as coreceptors in mediating inhibition.

Animals↗
PubMed1995

Induction of macrophage binding to virally infected cells by low levels of lipopolysaccharide.

Bone marrow culture-derived macrophages (BMCM) and vesicular stomatitis virus-infected BALB/c-3T3 cells (3T3-VSV) were used to determine whether macrophages could be activated to bind virally infected cells. Although lipopolysaccharide (LPS)-activated BMCM bound some uninfected BALB/c-3T3 cells, the number of targets that were bound increased with increasing times between infection and assay. Furthermore, LPS-activated BMCM bound more 3T3-VSV cells than did control macrophages. As more targets were added, the number of targets bound by the unactivated macrophages remained relatively level. However, the number of targets bound by the activated macrophages increased with increasing concentrations of added targets until they reached a plateau that was eight times greater than that bound by the unactivated BMCM. When BMCM were exposed to LPS for 24 h before assay, they lost both their ability to bind to 3T3-VSV and their cytolytic activity against those targets. However, as when using P815, a standard tumor target, the acquisition of binding of 3T3-VSV could be separated from macrophage cytolytic activity against those targets. The amount of LPS required to activated BMCM for increased binding of 3T3-VSV cells was 10-100 times lower than that needed to induce cytolytic activity for 3T3-VSV cells. Each of these values was approximately 100-fold lower than the amount of LPS required to induce the corresponding activity (binding or cytotoxicity) by using P815 targets.

3T3 Cells↗
PubMed1995

The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus.

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pL150 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr+ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr+ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.

Cell Wall↗
PubMed1994

Plasmid-encoded catalase of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids designated pACK1 through pACK5. Non-denaturing electrophoretic analysis of an extract prepared from wild-type cells revealed three bands of catalase activity, whereas an extract of cells cured of pACK1 produced only two catalase bands. Cloning and Southern hybridization analysis showed that there is a catalase structural gene on pACK1. The plasmid-specified catalase was the major activity produced under both aerobic and anaerobic conditions of growth.

Aerobiosis↗
PubMed1994

Activation of macrophages for cytolysis of virally infected cells by monoclonal antibody to the 73-kDa lipopolysaccharide receptor.

The abilities of lipopolysaccharide (LPS) and mAb5D3 (a monoclonal antibody specific for the 73 kDa LPS receptor) to activate bone marrow culture derived macrophages (BMCM) for cytolysis of vesicular stomatitis virus infected BALB/c3T3 (VSV-3T3) and the tumor target, P815, were compared. Activation for cytolysis of VSV-3T3 cells occurred at a lower level of either LPS or mAb5D3 than activation for cytolysis of P815. With both targets and under several stimulation conditions there was a constant differential between the two activating stimuli. These data indicate that stimulation of the 73 kDa LPS receptor at low levels of either mAb5D3 or LPS can activate BMCM for cytolysis of VSV-3T3 but not P815. Further, the constant relative efficiency of mAb5D3 to LPS in both target systems suggests that there is a common receptor for both LPS mediated effects.

3T3 Cells↗
PubMed1993

Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus secreted two bacteriolytic peptidoglycan hydrolases as proproteins that were activated as they were processed by an extracellular sulphydryl protease. This processing resulted in the production of multiple molecular-mass forms of each enzyme. Cells from early exponential phase cultures were susceptible to lysis by the mature forms of each of the peptidoglycan hydrolases whereas stationary phase cells were resistant. Thus secretion of these bacteriolytic enzymes during early exponential growth as precursors that are activated later by the protease would provide time for the cells to become resistant.

Bacterial Proteins↗
PubMed1991

Characteristics of extracellular protein production by a plasmidless derivative of Staphylococcus simulans biovar staphylolyticus.

A derivative of Staphylococcus simulans biovar staphylolyticus cured of all five plasmids present in the wild-type organism was developed, and the characteristics of extracellular protein production by this plasmidless strain were compared to those of the wild type. Although staphylolytic endopeptidase (lysostaphin) and beta-lactamase are known to be plasmid encoded, analysis of this cured strain revealed that most other extracellular proteins are chromosomally encoded.

Bacterial Proteins↗
PubMed1990

Activated macrophages use different cytolytic mechanisms to lyse a virally infected or a tumor target.

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolites by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.

Animals↗
PubMed1989

Macrophage activation for cytolysis of virally infected target cells.

Bone-marrow-culture-derived macrophages killed virally infected cells but not uninfected cells. This activity could be enhanced by preexposure of the macrophages to lipopolysaccharide (LPS), and/or purified interferons. The ability to kill virally infected targets was not restricted to a single cell type or virus. Comparing the ability of activators to induce activity against virally infected targets or tumor (P815) targets, it was found that much lower levels of LPS or alpha/beta-interferon were able to induce cytolytic activity for virally infected cells than were needed for tumor targets. Further, while the antitumor activity did not change significantly with an increase in the time of exposure to activating stimuli from 4 to 24 h, the activity against virally infected cells decreased dramatically with the longer exposure to stimuli.

Animals↗
PubMed1988

Characteristics of extracellular protein production by Staphylococcus simulans biovar staphylolyticus during aerobic and anaerobic growth.

Aerobic cultures of Staphylococcus simulans biovar staphylolyticus characteristically achieved about 17 times higher bacterial densities and produced about 7 times higher concentrations of exoprotein than did anaerobic cultures. However, total exoprotein secreted per unit of bacterial dry weight typically was 2.3 times greater for anaerobic cultures. As determined by SDS-PAGE, anaerobic cultures also produced a wider variety of exoproteins than did aerobic cultures. Three exoenzymes, a staphylolytic endopeptidase, a micrococcolytic hexosaminidase and a thiol protease, were completely repressed during anaerobic growth, which is further evidence for coordination of their production.

Aerobiosis↗
PubMed1986

Modification of Epstein-Barr virus replication by tunicamycin.

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.

Animals↗
PubMed1986

Antiviral activity of Bordetella pertussis vaccine-elicited peritoneal exudate cells.

Peritoneal exudate cells collected from mice 7 days after treatment with Bordetella pertussis vaccine exhibited significant in vitro antiviral activity against vesicular stomatitis virus (VSV). Vaccine-induced peritoneal exudate cells exhibited both intrinsic and extrinsic antiviral activity in culture with target VSV-infected L cells. Virus replication was poor in the vaccine-induced exudate cells. Coculture of vaccine-induced exudate cells and VSV-infected L cell targets decreased virus yield. The activity appeared specific for infected cells and at least a portion of the antiviral activity was directed against the initial infection cycle. Nonadherent vaccine-induced exudate cells showed an increase in antiviral activity over total vaccine-induced exudate cells.

Animals↗
PubMed1985

Heterogeneity among macrophages cultured from mouse bone marrow. Morphologic, cytochemical and flow cytometric analyses.

The development of macrophages in culture from mouse bone marrow was followed for 14 days by light and electron microscopy, ultrastructural cytochemistry, and flow cytometric analysis. By 10 days greater than 97% of the cells in culture were mononuclear phagocytes, and by 12 days greater than 99% were identifiable as macrophages. Ultrastructurally, three subpopulations of mononuclear phagocytes were distinguished based on the appearance of cytoplasmic structures. Early in culture, cells containing large, membrane-bounded vesicles predominated. With increasing time in culture these cells were replaced to varying degrees first by cells that contained vesicles filled with relatively dense, osmiophilic material and, finally, by macrophages that contained granules of various sizes, shapes and staining densities. Cytochemical (peroxidase and acid phosphatase) and colloidal gold uptake studies at the ultrastructural level suggested that many, if not all, of these cytoplasmic structures arose by pinocytosis and subsequent fusion of pinocytic vesicles with lysosomes. Analysis of DNA content of propidium iodide-stained nuclei by flow cytometry, coupled with the examination of cells treated with colchicine to arrest mitosis in metaphase, suggested that cell cycling was a negligible contributor to heterogeneity within cultured populations. Thus, by waiting until 12-14 days after bone marrow cultures were initiated, with partial replenishment of the culture medium at 7 days, heterogeneity could be greatly reduced in cultured macrophage populations. Taking this fact into consideration could help to reduce the variability seen in functional studies of macrophage populations that are less homogeneous.

Acid Phosphatase↗