Effect of tampon composition on production of toxic shock syndrome toxin-1 by Staphylococcus aureus in vitro.

To determine whether tampons composed of cotton or rayon differ in their effects on production of TSST-1 in vitro, two methods of bacterial cultivation, with and without agitation, were used to study the effects of 16 commercially available and experimental tampons on production of TSST-1. Experiments were done in blinded fashion. TSST-1 was produced in medium alone in the absence of a tampon by both methods. Neither cotton nor rayon tampons consistently increased toxin production, nor were there significant differences between them in their effects on TSST-1. In contrast, a tampon composed of carboxymethylcellulose and polyester foam increased production of TSST-1 to a large degree in both culture systems. Thus, neither cotton nor rayon amplifies production of TSST-1 in vitro, and cotton tampons cannot be claimed to be inherently safer on the basis of such data.

N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane.

The exocytotic release of secretory products from fragments of sea urchin egg cortex has been shown to be inhibited by covalent modification of membrane sulfhydryl groups with N-ethylmaleimide (NEM). Exocytotically competent preparations of reconstituted cortex, formed by recombination of purified cortical vesicles (CVs) with fragments of egg plasma membrane (PM) were also inhibited by treatment with NEM. The cellular localization of sulfhydryl-containing constituent(s) responsible for inhibition was investigated by treating CVs and/or PM with NEM prior to reconstitution. Both native cortex and cortex reconstituted with NEM-treated components were challenged with calcium-containing buffers. Exocytosis was monitored by phase-contrast microscopy, and quantitated by light scattering. Evidence for CV-PM fusion was obtained with an immunofluorescence-based assay that permits visualization of the transport of CV content proteins across the PM. Cortex reconstituted by recombination of NEM-treated CVs with untreated PM or by recombination of untreated CVs with NEM-treated PM was exocytotically competent, whereas cortex formed by recombination of NEM-treated CVs with NEM-treated PM was inactive. These results: (1) support the hypothesis that the mechanism of exocytosis in native and reconstituted cortex is the same; (2) provide evidence that both CV and plasma membranes participate in the release of CV contents from reconstituted cortex; and (3) suggest that sulfhydryl-containing protein(s) present on the surface of purified CVs and plasma membrane are involved in exocytosis.

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: assessment of binding specificity.

An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding of S. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4 +/- 3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bind S. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74 +/- 9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.

In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles.

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.

Legionella pneumophila: growth inhibition by human pharyngeal flora.

Several bacterial isolated from human pharyngeal cultures specifically inhibited the growth of Legionella pneumophila. The inhibitory substance from two strains (Streptococcus species 1-3 and Staphylococcus saprophyticus KC) was isolated from a broth supernatant. The inhibitor was active against all strains of L. pneumophilia tested, including five strains of L. pneumophila serogroup 1 and one strain each of serogroups 2, 3, and 4. The substance did not inhibit growth of 18 fresh clinical and laboratory pathogens (12 general). The substance was dialyzable, was resistant to head and proteolysis, and did not precipitate with ammonium sulfate. Nicotinamide adenine dinucleotide, produced by several upper respiratory tract organisms, did not inhibit L. pneumophila, and L. pneumophila could not be isolated when Streptococcus species 1-3, S. saprophyticus KC, and L. pneumophila were cocultivated. These properties may in part explain the difficulty of isolation and may aid in the identification of L. pneumophila.