Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.     

Beta(2)-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G(s) and G(i)

Cardiac G protein-coupled receptors that couple to Galpha(s) and stimulate cAMP formation (eg, beta-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the beta(2)-adrenergic receptor (AR), also couples to the inhibitory G protein Galpha(i) (activation of which inhibits cAMP formation). If beta(2)ARs could be shown to couple to Galpha(i) in the human heart, it would have important ramifications, because levels of Galpha(i) increase with age and in failing human heart. Therefore, we investigated whether beta(2)ARs in the human heart activate Galpha(i). By photoaffinity labeling human atrial membranes with [(32)P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Galpha(i), we found that Galpha(i) is activated by stimulation of beta(2)ARs but not of beta(1)ARs. In addition, we found that other Galpha(s)-coupled receptors also couple to Galpha(i), including histamine, serotonin, and glucagon. When coupling of these receptors to Galpha(i) is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that beta(2)AR and many other Galpha(s)-coupled receptors in human atrium also couple to Galpha(i) and that abolishing the coupling of these receptors to Galpha(i) increases the receptor-mediated adenylyl cyclase activity.     

Interaction of Polymorphonuclear Neutrophils with Escherichia Coli: EFFECT OF ENTEROTOXIN ON PHAGOCYTOSIS, KILLING, CHEMOTAXIS, AND CYCLIC AMP

Enterotoxigenic Escherichia coli are associated with noninflammatory diarrhea and stimulate adenylate cyclase activity of mammalian cells, thereby increasing intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP). Increased concentrations of cyclic AMP in polymorphonuclear neutrophils (PMN) inhibit phagocytosis, candidacidal activity, granule discharge, and chemotactic responsiveness. We examined the effect of enterotoxin on the interaction of human PMN with E. coli. Enterotoxigenic and nonenterotoxigenic strains, including serotypes of E. coli identical except for the presence or absence of the plasmid coding for enterotoxin production, were utilized. Enterotoxigenic and nonenterotoxigenic E. coli, tumbled with PMN, were phagocytized and killed (>97%) equally well, and these strains stimulated PMN hexose monophosphate shunt activity equivalently.However, a chemotaxis assay under agarose demonstrated that filtrates of 10 enterotoxigenic strains were less chemotactic for PMN by 15+/-2% total migration or 46+/-1% directed migration, when compared with 6 non-enterotoxigenic strains (P < 0.001). Inactivation of the enterotoxin by heat (65 degrees C for 30 min) or antibodies formed to E. coli enterotoxin eliminated the inhibitory effect of the enterotoxic filtrates for PMN chemotaxis. Addition of purified E. coli enterotoxin directly to the PMN decreased chemotaxis to E. coli filtrates by 32+/-2% (P < 0.001). These data suggest that the effect was due to the heat-labile enterotoxin. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.1 mM), which potentiates effects due to an increase in intracellular cyclic AMP, further decreased total PMN migration (random plus directed) toward enterotoxic filtrates to 46% of that to nonenterotoxic filtrates (P < 0.001). Addition of cholera toxin (1 mug/ml), which is similar to E. coli enterotoxin, to the PMN inhibited total migration toward nonenterotoxic filtrates by 16+/-2% (P < 0.001). Exogenous dibutyryl cyclic AMP (2 mM) inhibited total PMN migration toward E. coli filtrates by 32% (P < 0.001). PMN intracellular cyclic AMP levels increased by 220% after 2 h of incubation with purified E. coli enterotoxin. The decreased chemotactic attractiveness of enterotoxic E. coli filtrates appears to be related to the ability of enterotoxin to increase cyclic AMP in PMN. Enterotoxin production by E. coli may be advantageous to the microbe by decreasing its chemotactic appeal for PMN.     

Efficacy of UK-49,858 (fluconazole) against Candida albicans experimental infections in mice.

UK-49,858 (fluconazole), a new, orally absorbed bis-triazole derivative, has been evaluated against systemic infections with Candida albicans in normal and immunosuppressed mice and against an intestinal infection with C. albicans in immunosuppressed mice. Orally administered ketoconazole was used as a comparison agent throughout, and orally administered amphotericin B was included for comparative in the experimental intestinal infection. In a 10-day dosage regimen, UK-49,858 was far more active than ketoconazole against systemic infections with C. albicans in normal and immunosuppressed mice. In normal mice, extension of UK-49,858 dosing to 30 days resulted in prolongation of survival to over 90 days, and up to 60% of treated animals had no detectable C. albicans in their kidneys. In addition, over 90% of mice with intestinal candidiasis had culture-negative feces after a 3-day treatment with UK-49,858, but only 62 and 23% of mice gave this response after amphotericin B and ketoconazole therapy, respectively. These data suggest that UK-49,858 may be of value in the treatment of systemic and gastrointestinal infections due to C. albicans in humans.     

Clinical alarm systems testing--a program assessment model

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ONE HEALTH CARE SYSTEM developed a clinical alarm systems testing program in an effort to meet the Joint Commission on Accreditation of Healthcare Organizations' national patient safety goal related to improving the effectiveness of clinical alarm systems.

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IT IS IMPERATIVE that all staff members are aware of the importance of clinical alarms and are prepared to deal with an alarm that is sounding.

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THIS ARTICLE DEFINES clinical alarms and clinical alarm systems, provides recommendations for developing and designing a clinical alarm systems testing program, and presents a format for a clinical alarm systems testing program. AORN J 80 (August 2004) 280-288.

     

Randomised controlled trial and cost consequences study comparing initial physiotherapy assessment and management with routine practice for selected patients in an accident and emergency department of an acute hospital

Objective: The Department of Health is reviewing the effectiveness of accident and emergency (A&E) departments. This study aimed to compare health and economic effects of physiotherapy initial assessment and management with routine practice in an A&E department.

Methods: Randomised controlled trial and cost and consequences study. Patients presenting at A&E were eligible if suspected at triage to have soft tissue injury without fracture. The efficacy end point was "days to return to usual activities". Secondary end points included patient satisfaction with their care and further health outcomes and cost data.

Results: 766 of 844 (915) patients were randomised. The median days before return to usual activities (available for 73% of those randomised) was greater in the physiotherapist group (41 days compared with 28.5 days; hazard ratio 0.85 p = 0.071). The physiotherapy group expressed greater satisfaction with their A&E care (on a scale of 1 to 5, median was 4.2 compared with 4.0, p<0.001), were more likely to be given advice and reassurance, and more likely to be provided with aids and appliances. Costs were the same between the two arms.

Conclusion: There is evidence that physiotherapy leads to a prolonged time before patients return to usual activities. This study shows no clear danger from physiotherapy intervention and long term outcomes may be different but given these findings, a best estimate is that introducing physiotherapist assessment will increase costs to the health service and society. Routine care should continue be provided unless there is some reason why it is not feasible to do so and an alternative must be found.

     

The Effects of Insulin/IGF-I on Glucose and Leucine Metabolism in the Redclaw Crayfish (Cherax quadricarinatus)

In recent years, invertebrate peptides have been identified which share substantial homologies with vertebrate insulin and insulin-like growth factors (IGFs), indicating a high degree of conservation of insulin/IGF systems through animal evolution. In a previous study, we provided evidence for the presence of IGF-I-like peptides in the redclaw (Cherax quadricarinatus), a species of freshwater crayfish endemic to northern Australia river systems which has attained support as a culture species. The general aim of the current study was to elucidate the functional significance of IGF-I-like peptides in this species by examining the effects of mammalian IGF-I on glucose and leucine metabolism. Juvenile redclaw were injected with a single dose of purified human insulin, recombinant human (rh) IGF-I, or Des-1-3-IGF-I. Glucose levels in redclaw tissues were then determined over an 8-hr period using enzymatic approaches. It was shown that injection of rhIGF-I induced an acute increase in free glucose content in hepatopancreas while Des-1-3-IGF-I and insulin raised free glucose levels in abdominal muscle. Radiolabel tracer approaches also demonstrated that injection of rhIGF-I increased glycogen synthesis in abdominal muscle and elevated the incorporation of leucine into protein in both abdominal muscle and hepatopancreas. Taken together, the findings of this study suggest that IGF-I-like peptides are biologically active in this species and may be of significance to metabolic and growth-related processes.