Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

Chapes, Stephen K.; Beharka, A.A.; Hart, Mark E.; Smeltzer, Mark S.; Iandolo, John J.

doi:10.1002/jlb.55.4.523

Cited by 11 publications

(9 citation statements)

References 42 publications

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“…RNA was isolated from S. aureus grown overnight in BHI, BHI+Methyl Red (0?2 mM), or BHI+ Orange II (0?2 mM), essentially using the RNAzol B procedure (Chapes et al, 1994). The RT-PCR reaction mixture (25 ml) consisted of 50 pmol each primer (Sas1-forward, 59-ggcagtgcacaagtgaattc-39 and Sas1-reverse, 59-ccatgatagtttggcgttcc-39), 4 mg RNA, 0?1 mM (each) deoxynucleoside triphosphate, 1 U Taq DNA polymerase and 10 U AMV reverse transcriptase in PCR buffer with MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

2005

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Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent K m values for NADPH and Methyl Red substrates were 0?074 and 0?057 mM, respectively. The apparent V max was 0?4 mM min "1 (mg protein) "1 . Azo1was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora.

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Section: Methodsmentioning

confidence: 99%

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

2005

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“…These data indicate that SEA and SEB probably also bind to non-MHC II receptors on wild-type macrophages (7,9 Table 2).…”

Section: Methodsmentioning

confidence: 90%

“…Staphylococcal enterotoxins A and B (SEA and SEB, respectively) were obtained from Toxin Technology (Madison, WI) or from the U.S. Army Medical Research and Development command (Ft. Detrick, MD). The exotoxins were free of endotoxin (at <0.001 ng of endotoxin per jug of exotoxin) as determined by the manufacturer or our own analysis (7,9).…”

Section: Methodsmentioning

confidence: 99%

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Staphylococcal enterotoxins bind H-2Db molecules on macrophages.

Beharka

Iandolo²,

Chapes³

1995

Proc. Natl. Acad. Sci. U.S.A.

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We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the a2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the a2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

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“…Balb/c 3T3 (H-2 d) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, CCL 163). The bone-marrow-derived macrophage cell line, B6MP102 (H-2b), derived from C57BId6 mice (8,9,24), was used for comparative analysis in some experiments. B6MP102 was cultured in tissue culture medium consisting of Dulbeeco's modified minimal essentials medium (DMEM, GIBCO, Grand Island.…”

Section: Methodsmentioning

confidence: 99%

Macrophage cell lines derived from major histocompatibility complex II-negative mice

Beharka

Armstrong

Chapes

1998

In Vitro Cell.Dev.Biol.-Animal

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Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

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Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

Cited by 11 publications

References 42 publications

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Staphylococcal enterotoxins bind H-2Db molecules on macrophages.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

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