The roles of growth hormone and somatomedin in stimulating muscle cell proliferation were investigated in a series of experiments on myoblast growth in culture. At levels 100 times normal circulating concentrations, neither growth hormone nor insulin stimulated growth of rat muscle cells in serum-free medium. In contrast, Temin's Multiplication Stimulating Activity (MSA), a close biological and chemical analog which served as a somatomedin surrogate in this study, was active at concentrations corresponding to the reported circulating levels of the somatomedins; MSA gave consistent stimulation of myoblast proliferation, protein accumulation, and [3H]thymidine incorporation by rat myoblasts and by Yaffe's L6 myogenic cell line. (The unreliability of [3H]thymidine in corporation as a quantitative assay for mitogenic activity is illustrated.) Addition of insulin at physiological concentrations and at the very high levels often used to stimulate cell growth did not enhance the effects of MSA on myoblast proliferation; effects were barely additive. We interpret these results to support earlier indications from studies on diaphragm muscle that the somatomedins are physiologically important mediators of the growth-promoting actions of growth hormone in muscle.

The Bordetella bronchiseptica tonB gene was cloned by detection of a chromosomal restriction fragment hybridizing with each of two degenerate oligonucleotides that corresponded to Pro-Glu and Pro-Lys repeats characteristic of known TonB proteins. The tonB Bb gene was situated upstream of exbB and exbD homologues and downstream of a putative Fur-regulated promoter. Hybridization results indicated that the tonB operon and flanking regions were highly conserved between B. bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Disruption of tonB in B. bronchiseptica resulted in inability to grow in iron-limiting media, and inability to utilize alcaligin, enterobactin, ferrichrome, desferroxamine B, haemin and haemoglobin. Although it was not possible to inactivate tonB in a clinical B. pertussis isolate, tonB was disrupted in a laboratory B. pertussis strain previously selected for the ability to grow on Luria-Bertani medium. This B. pertussis tonB mutant shared a similar iron complex utilization deficient phenotype with the B. bronchiseptica tonB mutant. The B. bronchiseptica tonB operon present on a plasmid did not complement an Escherichia coli tonB mutant, but inefficient reconstitution of enterobactin utilization was observed in one fepA mutant harbouring plasmid copies of the B. pertussis fepA homologue and tonB Bb operon.

The etiologic agent of a large 1998 outbreak of poststreptococcal acute glomerulonephritis (PSGN) in Nova Serrana, Brazil, was found likely to be a specific strain of Streptococcus equi subsp.zooepidemicus from contaminated cheese (S. Balter et al., Lancet 355:1776–1780, 2000). In the present study, we used a serologic screen for a known surface-exposed virulence factor to confirm the epidemiologic findings. Using primers flanking a previously characterized M-like protein gene (J. F. Timoney et al., Infect. Immun. 63:1440–1445, 1995), we amplified and sequenced the M-like protein (designated Szp5058) gene and found it to be identical among four independent acute-phase PSGN patient isolates. Convalescent-phase sera from 33 of 44 patients in the PSGN outbreak were found to contain antibodies highly reactive to a purified Szp5058 fusion protein, compared with 1 of 17 control sera (P < 0.0001), suggesting that Szp5058 was expressed during infection and further implicating this strain as the cause of the PSGN outbreak. The predicted signal sequence and cell wall association motif of Szp5058 were highly conserved with the corresponding sequence from S. equi subsp. zooepidemicus SzpW60, while the predicted surface-exposed portions differed markedly between these two proteins. The 5′ end of the szp5058 gene, including its variable region, was identical to the szp gene from another strain associated with a previous PSGN outbreak in England (M. Barham et al., Lancet i:945–948, 1983), and the corresponding szpsequence found from the Lancefield group C type strain isolated from a guinea pig. In addition, the hypervariable (HV) portion ofszp5058 was identical to a previously published HV sequence from a horse isolate (J. A. Walker and J. F. Timoney, Am. J. Vet. Res. 59:1129–1133, 1998). Three other strains ofS. equi subsp. zooepidemicus, including another strain previously associated with a PSGN outbreak, were each found to contain a distinct szp gene. Two of these szpgenes had HV regions identical to szp regions from isolates recovered from different host species.

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