Biomedical subjects
J C Robbins
Publications and source records attributed to J C Robbins.
Evidence for group A-related M protein genes in human but not animal-associated group G streptococcal pathogens.
Group G streptococci have on their surface antiphagocytic M protein-like antigens. To determine if these organisms have genes similar to the M protein genes of Streptococcus pyogenes (group A), DNA from independent group G isolates of human and animal origin were tested for homology to probes representing sequences encoding the carboxy-terminus and leader peptide of the type 12 M protein (M12) of group A streptococci. All eight human-associated group G strains tested had DNA homologous to the carboxy-terminal probe. Six of these strains also had DNA that hybridized with the leader peptide probe. Using probes representing the group A M12 gene (emm12) and adjacent 5' sequences, we found that one of these strains, known to produce an M12 antigen, had a nearly complete duplication of the group A emm12 gene, differing only in 0.26 kb of sequence at the 5' end. The other human-associated strains did not hybridize with emm12-specific sequences. None of the group G strains had homology to 5' proximal sequences thought to be associated with group A emm12 regulation, but all the human-associated strains had DNA homology to a 1.5 kb DNA segment which mapped 2.5 kb upstream of the emm12 gene in group A streptococci. None of the twelve animal-associated strains tested hybridized with any of the probes used in this study. These results suggest that human but not animal-associated group G isolates have group A-related M protein genes. We propose that expression of these genes are critical for infection of the human host and that group A and G shared upstream sequences could encode additional virulence factors.
Streptococcus pyogenes type 12 M protein gene regulation by upstream sequences.
A partial nucleotide sequence that included 1,693 base pairs of the M12 (emm12) gene of group A streptococci (strain CS24) and adjacent upstream DNA was determined. Type 12 M protein-specific mRNA of strain CS24 is transcribed from two promoters (P1 and P3) separated by 30 bases. The transcription start sites of the emm12 gene were located more than 400 bases downstream of a deletion that causes decreased M-protein gene transcription in strain CS64. Deletion analysis of M protein-expressing plasmids indicated that an upstream region greater than 1 kilobase is required for M-protein gene expression. The M-protein gene transcriptional unit appears to be monocistronic. Analysis of the emm12 DNA sequence revealed three major repeat regions. Two copies of each repeat, A and B, existed within the variable 5' end of the gene; repeat C demarcated the 5' end of the constant region shared by emm12 and emm6.
Platelet activating factor (PAF) involvement in endotoxin-induced hypotension in rats. Studies with PAF-receptor antagonist kadsurenone.
Evidence from three types of experiments indicates that platelet activating factor (PAF)1 is an important mediator of endotoxin-induced hypotension in rats. a) Endotoxin infusion stimulates the time-dependent appearance of PAF in the blood. b) PAF infusion results immediately (less than 30 sec) in hypotension while endotoxin-induced hypotension takes 3-5 min to occur, allowing time for PAF production. c) Infusion of the specific PAF-receptor antagonist kadsurenone (2.2 mumole/kg bolus, 0.9 mumoles/min/kg continuous infusion), which inhibits PAF-induced hypotension by 67%, causes a 67% reversal of endotoxin-elicited hypotension. An additional finding of this study is that rats respond hypotensively to each of a series of low-dose PAF infusions but only to the first low-dose endotoxin infusion. These endotoxin-refractory rats do respond to subsequent PAF infusions.
Cell-surface changes in Ricinus communis toxin (ricin)-resistant variant of a murine lymphoma.
A variant of the murine lymphoma cell line BW5147 that was 250 times more resistant than the parent to Ricinus communis II agglutinin (RCAII, ricin) toxicity (measured in the absence of serum) was selected by repeated exposure of cells to increasing concentrations of the lectin. Quantitative binding of the lectin, however, was decreased by only 30-40% in the variant. In contrast with several reported lectin-resistant variants, most surface glycoproteins on the parental and variant cell surfaces were similar, as judged by electrophoresis after lactoperoxidase-catalyzed iodination and RCAI-affinity chromatography. Surface studies showed that an RCAI- and RCAII-binding protein of about 80,000 daltons on the surfaces of parental cells is altered on the variant cells to a form with a lower apparent molecular weight. We suggested that this protein is important for entry of RCAII molecules in parental cells, but that its altered form on the variant cells no longer mediates efficient RCAII uptake, thus imparting toxin resistance. In addition, a protein of approximately 35,000 daltons, which does not bind RCAI, is weakly lactoperoxidase-iodinated on parental but not variant cells.
Cell interactions in the metastatic process: some cell surface properties associated with successful blood-borne tumor spread.
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Cell surface receptors and their dynamics on toxin-treated malignant cells.
The binding, mobility, and mode of cell entry of the plant toxin ricin (or RCAII) were investigated on susceptible and partially resistant murine cell lines. When susceptible cells (SV40-transformed 3T3 fibroblast cells and BW5147 lymphoma cells) were examined, ricin bound rapidly, induced endocytosis, and entered the cell cytoplasm via broken endocytotic vesicles to inhibit cell protein synthesis, as found previously (1). Addition of lactose within 15 min after initial ricin binding prevented toxicity. After this time lactose addition no longer blocked the inhibition of protein synthesis. In a partially resistant lymphoma (BW5147/RCA3) that shows only a slight reduction in the total number of ricin-binding sites, ricin bound rapidly to the cell surface, but was endocytosed significantly less at low ricin doses compared to its parental line, indicating a possible difference in cell surface behavior. The exposed surface proteins on the BW5147 parental and BW5147/RCA3 resistnat lines were examined by 125I-labeling utilizing lactoperoxidase-catalyzed iodination. The radiolabeled components were solubilized and separated by slab electrophoresis in sodium dodecyl sulfate. Autoradiograms of the slab gels indicated that two surface components of approximately 80,000 and 35,000 mol wt were much less exposed or were missing on the resistant line.
Ricinus communis toxin-mediated inhibition of protein synthesis in cell-free extracts of a toxin-resistant variant mouse lymphoma cell line.
Ricinus communis agglutinin II (RCAII, ricin, toxin) at low concentrations inhibits protein synthesis in cell-free extracts, but not in intact cells, of an RCAII-resistant mouse lymphoma variant cell line. The concentration dependence of the inhibition by RCAII was the same in cell-free extracts of both RCAII-resistant variant and RCAII-sensitive parental cells, while intact parental cells are 250 times more sensitive to RCAII toxicity. The onset of RCAII inhibition of cell-free protein synthesis was extremely rapid in both cases, being complete in a few minutes. Under these conditions RCAII inhibits protein synthesis in intact RCAII-sensitive parental cells, but maximal inhibition requires several hours to occur. These results support our previous electron microscopic observations that the variant cells are defective in the uptake of RCAII by endocytosis at low toxin concentrations.
Cellular interactions in the metastatic process.
Tumor metastasis is a multistep process which is dependent on both host and tumor properties. It is proposed that the interaction of normal host blood and endothelial cells with circulating malignant cells result in tumor cell arrest leading to subsequent metastases at specific secondary sites. In experimental animal models the frequency and location of metastatic foci can be manipulated by repeated in vivo selection for tumor cell variants showing altered preference for organ-specific implantation and survival. These tumor cell variants can be used to determine the cell surface structures and enzymes involved in the various steps of the metastatic process.
Transport properties of merodiploids covering the dagA locus in Escherichia coli K-12.
A membrane componenet of the dag transport system which serves for glycine, D-alanine, and D-serine is coded for by the dagA gene at minute 83 of the Escherichia coli chromosome. Merodiploid strains (dagA+/dagA+) show two to three times the transport activity for only those amino acids that are substrates of the dag transport system. The increased transport activity is a result of a two-to threefold increase in Vmax for amino acid uptake with little or no change in the Km value. The two- to threefold gene dose effect of the merodiploid strains is maintained even during carbon starvation, eliminating the possibility that a greater energy supply for transport activity may account for the effect. Since merodiploids which carry more than one copy of the dagA allele show a gene dose response for transport activity, we conclude that the membrane componenet of the dag transport system which is coded for by the dagA allele is present in limiting amounts.