RESUMEN
We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065-1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5' restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT-PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22 alpha [Pearlstone et al., 1987: J. Biol. Chem. 262:5985-5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518-530], and human WS3-10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1-7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins alpha and beta [Takahashi and Nadal-Ginard, 1991: J. Biol. Chem. 266:13284-13288], and Drosophila mp20 [Ayme-Southgate et al., 1989: J. Cell Biol. 108:521-531] suggest that all of these proteins may be classified as members of a new transgelin multigene family.
Asunto(s)
ADN Complementario/genética , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Línea Celular Transformada , Clonación Molecular , ADN Complementario/análisis , Electroforesis en Gel de Poliacrilamida , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/fisiología , Datos de Secuencia Molecular , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Reacción en Cadena de la Polimerasa , Ratas , Virus 40 de los SimiosRESUMEN
In-vitro reaction mixtures were set up containing bacteria, human serum, human neutrophils and ampicillin or cefotaxime at the MIC, MIC/2 or MIC/4. Serum was untreated, heat-inactivated, absorbed or both. Five indices measuring intra- and extracellular killing were calculated and the effects of heat-labile and absorbable serum factors and of the various concentrations of antibiotics on these indices were calculated. Both heat-labile and absorbable factors enhanced intra- and extracellular killing; antibiotics had a similar effect except that phagocytosis was reduced. Antibiotics at MIC/4 exerted as great an effect as did higher concentrations and this was especially so for cefotaxime. Both heat-labile and absorbable serum factors had a greater effect on all five indices the lower the concentration of antibiotic with advantage for cefotaxime. In the presence of immunological serum factors the overall effect against Salmonella enteritidis is maintained as the concentration of antibiotic falls below the MIC and this is especially seen in the presence of cefotaxime.