Biomedical subjects
Publications and source records attributed to J A Talbot.
Two cases of invasive Staphylococcus aureus are reported in which human to human transmission resulted in primary bacteremia and endocarditis. The identity of the organism was confirmed by phage typing, antibiograms, coagulase gene polymorphisms and ribotyping. This is the first documented case of such transmission not involving an intravenous drug abuser.
Toxin A produced by Clostridium difficile, the causative agent of pseudomembranous colitis and antibiotic-associated diarrhea, was shown to bind to synthetic oligosaccharide sequences attached to an inert support (SYNSORB). The oligosaccharide sequences that bind to toxin A were related to sequences previously identified as potential receptors for the toxin. Various SYNSORBs containing a variety of oligosaccharides were examined for their potential to neutralize toxin A activity from toxin-containing solutions as well as clinical stool samples from patients with either pseudomembranous colitis or antibiotic-associated diarrhea. The results from neutralization experiments suggest SYNSORB can effectively neutralize toxin A activity from stool samples and thus could serve as a potential therapy for C. difficile-associated diarrhea.
Since 1985 there have been worldwide reports of increases in severe invasive Group A streptococcal (IGAS) infections. We reviewed the charts of all children with IGAS infections (defined as isolation of Group A streptococcus from a normally sterile site) presenting to our institution over a 7-year period (January, 1985, to December, 1991) and the literature. Streptococcal toxic shock syndrome required hypotension and multisystem organ involvement. Twenty-four patients (mean age, 4.96 +/- 4.4 years) were identified with IGAS infection. One patient (presenting in 1989) met the criteria for probable streptococcal toxic shock syndrome and none died. Eight of 19 Group A streptococcal isolates tested were streptococcal pyrogenic exotoxin (SPE) A producers, most (90%) had the speC gene and all had the speB gene and produced the toxin. No M or T type predominated. The low rates of streptococcal toxic shock syndrome and fatalities among children with IGAS infection are consistent with other pediatric but not with adult series. The apparent differences in outcome of IGAS between children and adults were not explained by the virulence factors we examined and may warrant further investigation.
Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, alpha 2-macroglobulin, but not alpha 1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host-parasite interactions.
We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/l) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091 +/- 322 to 1480 +/- 84 pmol/l (p less than 0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360 +/- 45 to 1242 +/- 133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 mumol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 mumol/l) and A23187 (2 mumol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by greater than 80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 mumol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 mumol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.
We have examined the effect of inhibition of protein kinase C activity by staurosporine on estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Staurosporine lead to a dose-related increase in estradiol secretion independent of FSH, such that with 100 nmol/l staurosporine basal estradiol levels increased 10-fold. The maximal response seen with staurosporine alone (100 nmol/l) or in combination with FSH (0.4-8 IU/l) was similar to that seen with a saturating dose of FSH (8 IU/l). There was no evidence of synergy between FSH and staurosporine. Activation of protein kinase C by phorbol 12,13 dibutyrate (10(-7) mol/l) resulted in a 53-74% inhibition of estradiol production provoked by FSH (8 IU/l), staurosporine (5-100 nmol/l) or staurosporine in combination with FSH. Staurosporine (5-100 nmol/l), in the absence or presence of FSH, was unable to overcome inhibition of estradiol secretion by phorbol ester, indicating the presence of at least two independent binding sites on protein kinase C for these molecules. Forskolin (1 mumol/l)- and dibutyryl cAMP (1 mmol/l)-stimulated estradiol secretion was inhibited by 31 +/- 5% and 64 +/- 5% respectively, by phorbol 12,13 dibutyrate (10(-7) mol/l). We conclude that FSH-induced estradiol secretion in immature rat Sertoli cells is affected by protein kinase C activity.
Bioactive-LH (B-LH) was measured in plasma by in-vitro bioassay and immunoactive-LH (I-LH) by immunoassay at 10 min intervals for 6 h in five men after standard chemotherapy for Hodgkin's disease. Eleven normal men acted as controls. Follicle-stimulating hormone (FSH) was markedly raised in the treated patients (mean +/- SEM; 12.8 +/- 2.8 vs. 2.7 +/- 0.4 IU l-1, P less than 0.006) reflecting damage to the germinal epithelium. Bioactive (27.4 +/- 2.8 vs. 12.9 +/- 1.3 IU l-1) and I-LH (9.6 +/- 2.0 vs. 4.9 +/- 0.4 IU l-1) were elevated (P less than 0.006) in the patient group whilst testosterone levels (24.0 +/- 3.8 vs. 19.6 +/- 2.4 nmol l-1) were normal. The testosterone I-LH ratio, a putative index of Leydig cell dysfunction, was negatively correlated with FSH levels (r = -0.85, P less than 0.02). Bioactive and I-LH pulse peak amplitude were elevated, as were pulse maxima (P less than 0.05). In contrast, B-LH pulse frequency was similar between the patients (2 pulses per 6 h) and controls (median 2, range 1-3 pulses per 6 h) as was the I-LH pulse frequency (median 2, 1-2 pulses per 6 h in both groups). The mean B:I LH ratios were similar (2.94 +/- 0.09 vs. 2.63 +/- 0.14) in both groups, although the inter-pulse B:I ratio was increased (P less than 0.007) in the patient group.(ABSTRACT TRUNCATED AT 250 WORDS)
We have measured plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (To) by radioimmunoassay (RIA) and bioactive LH (B-LH) by in vitro bioassay at 10-min intervals over 6 h in men treated by haemodialysis for renal failure and in men after renal transplantation. Eleven normal male volunteers acted as controls. Immunoreactive LH (I-LH) and FSH levels were elevated (p less than 0.03) in uraemia (mean +/- SE; 10.0 +/- 1.0 and 4.6 +/- 0.7 IU/l for LH and FSH, respectively) and following renal transplantation (8.1 +/- 1.2 and 5.3 +/- 0.5 IU/l) compared to controls (C) 4.9 +/- 0.5 and 2.7 +/- 0.4 IU/l) whereas B-LH [17.3 +/- 2.5, 14.8 +/- 1.8 and 12.9 +/- 1.3 IU/l in dialysis (D), transplant (T) and C groups, respectively] levels were normal. Prolactin levels were elevated (p less than 0.03) in the D group (median 348, range 162-1,780 mU/l) compared to the T group (161, 91-206 mU/l) and controls (163, 124-312 mU/l) whereas total To levels (mean +/- SE; 17.3 +/- 4.8, 15.5 +/- 1.3 and 19.6 +/- 2.1 nmol/l in the D, T and C groups, respectively) were similar as was the free To index. B-LH (median frequency, 2 and range 1-3 pulses/6 h) and I-LH (median frequency, 2 and range 1-2 pulses/6 h) was pulsatile in all the C group but B- and I-LH pulses were absent in 2 of the 5 subjects treated by dialysis. Following renal transplantation B-LH pulses were detected in all subjects, whilst I-LH pulses were absent in 1 subject.(ABSTRACT TRUNCATED AT 250 WORDS)
We have studied bioactive and immunoreactive LH pulsatility in 11 normal men. The temporal relationship of plasma LH, testosterone, and FSH were also investigated. Blood samples were taken at 10-minute intervals for 6 h and bioactive LH levels were determined using an in vitro mouse Leydig cell bioassay. Testosterone, LH and FSH were determined by standard radioimmunoassay. Twenty-two bioactive LH pulses were detected (amplitude 8.5 +/- 4.9 IU/l, mean +/- SD) with a frequency of 2 +/- 0.8/6 h compared with only 18 immunoreactive LH pulses (amplitude of 3.6 +/- 1.8 IU/l) and a frequency of 1.6 +/- 0.5/6 h. Bioactive:immunoreactive LH ratios increased (p less than 0.01) from the preceding pulse nadirs (2.26, range 1.66-4.28) to the pulse peaks (2.71, range 1.99-4.67). Twenty FSH pulses (seen in all but one subject) of low amplitude (0.7 +/- 0.6, median 0.5 IU/l) were also present. There was a close temporal relationship between testosterone and FSH secretion with bioactive and immunoreactive LH pulses with lags of 30-60 and 0 min, respectively. We conclude that immunoreactive LH pulses are discordant from bioactive LH pulses in 18% of occasions. Further, the mean amplitude of bioactive episodes were approximately 2.6 times greater than that of immunoreactive episodes, whereas interpulse period and pulse duration were similar. The increase in bioactive:immunoreactive ratio at pulse peaks may indicate that in normal men LH pulses are enriched with a more biopotent form of the molecule.
Lamb-specific odor is used by ewes to discriminate between their own and alien offspring. The objective of the present study was to investigate the feasibility of transferring own-lamb odor to alien young to facilitate fostering. Shortly after birth, cloth stockinettes (body suits) were placed on the offspring of 37 multiparous ewes. At 48 to 72 h after parturition, a lamb from each ewe was replaced by an alien lamb. In 31 cases the odor-impregnated stockinette of the ewe's own lamb was placed on the alien lamb; in the remaining six cases the alien lamb retained its own stockinette (control condition). Lamb acceptance tests were conducted every 12 h for 2 d after fostering and once daily for the following 2 d. Twenty-six of the 31 experimental ewes (84%) adopted alien lambs using the odor-transfer technique. All of the control lambs were rejected. Of the 26 ewes that adopted foster young, 10 (38%) showed immediate acceptance and 22 (85%) were exhibiting acceptance behaviors by 36 h after fostering. There were no significant differences in the fostering success of ewes bearing single lambs and those with twins. The presence of own-lamb odor rather than the absence of alien lamb odor appears to be the salient cue for lamb acceptance. Lamb-specific odor transfer represents a relatively inexpensive and rapid technique to achieve maternal acceptance of alien lambs by postpartum ewes.
Five peptide analogs of the actomyosin ATPase inhibitory region of troponin I (Tn-I) have been synthesized by the solid-phase method. One analog was constructed with a sequence identical to that found in species of Tn-I isolated from chicken fast, rabbit fast and rabbit slow skeletal muscle. A second peptide was made identical to the sequence of the homologous inhibitory region of Tn-I from rabbit cardiac muscle. The remaining three analogs were hybrids of the two sequences. We have verified that rabbit skeletal fast Tn-I was a better inhibitor than rabbit cardiac Tn-I in a rabbit skeletal actomyosin assay system and extended these studies to show that the same results were found in an assay system which used rabbit cardiac actomyosin. Our skeletal fast muscle peptide analog was also a better inhibitor in both assay systems than the cardiac Tn-I peptide analog. The hybrid peptides indicated that the changing of proline 110 to a threonine residue had no effect and suggested that position 110 was not essential to inhibition. The other amino acid change, the replacement of arginine 113 by a leucine residue, was shown to be the crucial difference and resulted in lower activity. Thus, th differences in relative inhibitory activity of rabbit skeletal fast and cardiac Tn-I can be at least partially and possibly solely explained by the single amino acid substitution at position 113. Short active sequences of proteins, like the Tn-I inhibitory region are potentially of enormous value as probes of structure-function relationships.
Twelve peptide analogs of the actomyosin ATPase inhibitory region of rabbit skeletal troponin I (Tn-I) have been synthesized by the solid phase method and tested for biological activity in both actomyosin and acto-S1(A1) systems. Acto-S1(A1) is a cleaner and more facile system and we found no important discrepancies in the results for the two systems. These studies indicate that the sequence 105 to 114 is necessary for inhibition and that the inhibition is on the order of that reported for the 21-residue cyanogen bromide fragment of Tn-I (residues 96 to 116). We have shown the importance of lysine 105 and that of a bulky side chain at position 114 to this inhibition. Both the high activity of peptides containing the sequence 105 to 114 compared to Tn-I (45% on a molar basis) and the previously demonstrated tropomyosin specificity of this activity indicate that the relative inhibitory activity of these peptides is applicable to the discussion of the inhibitory activity of Tn-I. We have proven that actin concentration is a significant factor determining the extent of inhibition of both Tn-I and the peptides. We have also established that the charges on alpha-amino and alpha-carboxyl groups of a peptide, which do not appear in the parent protein, can modify the activity of the peptides. This must be taken into account when extrapolating from the activity of a peptide to the activity of the protein.
A 20-residue peptide analog of the actomyosin ATPase inhibitory region of rabbit skeletal troponin I (Tn-I) has been synthesized by the solid phase method. The analog exhibited biological activity similar to both Tn-I and a 21-residue cyanogen bromide fragment of Tn-I. At ionic strengths where the inhibition of the actomyosin ATPase due to tropomyosin alone is low, the synthetic peptide in the presence of tropomyosin inhibits 90% of the original ATPase activity. In the absence of tropomyosin, the inhibition due to the peptide is much reduced. In contrast, salmine, a basic protein also known to inhibit the actomyosin ATPase, shows less inhibition in the presence of tropomyosin than it does in its absence. Gel electrophoresis data showed that the enhancement of the analog's inhibition by tropomyosin may be related to the analog's promotion of tropomyosin binding to F-actin similar to that reported for Tn-I and that the reduction of salmine inhibition by tropomyosin may be due to the binding of salmine by tropomyosin. At ionic strengths where binding and inhibition of tropomyosin is significant, the analog enhanced inhibition in a manner similar to that reported for whole Tn-I.
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