Round robin investigation of methods for recovering human enteric viruses from sludge

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of low-dose insulin infusions in diabetics after myocardial infarction

Myocardial infarction in diabetics is often accompanied by poor diabetic control. An assessment of a low-dose insulin infusion regimen in 26 diabetic patients after myocardial infarction found this system to be simple, effective, and safe.     

Hormonal regulation of anabolic processes and of protein utilization efficiency in the early postnatal period

The levels of thyroid, pituitary and steroid hormones-thyroxine, triiodothyronine and 11-hydroxycorticosteroids in the blood serum, somatotropin in the pituitary, and processes of protein assimilation were studied in rats in the early postnatal period. The highest endogenous production of thyroxine, triiodothyronine and somatotropin was detected in 15-day-old rats. The highest level of protein utilization was detected in 7 to 15-day-old rats, followed by the lowering of the utilization on changing over to definitive nutrition. Endogenous production of the anabolic hormones thyroxine, triiodothyronine and somatotropin was found to correlate with a high level of protein utilization in rats within the first days of life after birth.     

Decreased virulence in stable,acapsular mutants ofCryptococcus neoformans

Six acapsular strains ofCryptococcus neoformans obtained by chemical mutagenesis failed to produce a capsulein vivo and were avirulent in mice following high dose intramuscular, intraperitoneal or intravenous inoculation. Peritoneal granulomas were observed in all animals inoculated with the acapsular mutants. These granulomas were characterized by a large central mass consisting of intact, degenerating and necrotic yeast cells. This was surrounded by concentric layers of a broad band of histiocytes, a narrow band of fibroblasts, and around the periphery, a mass of lymphocytes and plasma cells. These isolates did not revert to an encapsulated or virulent state after more than a year of subculturing or 18 passages through mice.     

Regulation of adenylate cyclase in E. coli

The intracellular concentrations of cAMP in Escherichia coli are regulated mainly by control of the activity of adenylate cyclase. Withdrawal of the carbon source from the growth medium causes a gradual reduction of cellular energy and a dramatic stimulation of cyclase activity. Manipulations of the proton gradient at the cell membrane of ATP synthase-deficient E. coli (unc-) revealed that this part of the energy compartment is not responsible for the starvation-induced stimulation of cyclase. Neither is the ATP pool involved in regulation of the activity of the cyclase. The intracellular concentrations of ATP were experimentally lowered by purine starvation of auxotrophs, by inhibition of purine synthesis using amethopterin, or by affecting ATP synthesis using arsenate. None of these conditions led to stimulation of cyclase activity. The control of cyclase is exerted not via the energy pools but via uptake systems of energy substrates independent of whether the substrate can be metabolized or not, or how the transport is energized. The stringent coupling between these transport systems and cyclase activity enables the cell to react instantaneously to changes in its environment.     

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.     

Effects of delta 9-tetrahydrocannabinol on testosterone production in vitro: influence of Ca++, Mg++ or glucose

The major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC), influences testicular function. In the present experiments, the addition of THC to incubations of whole decapsulated mouse testes altered testosterone (T) production differentially, depending on the specific gonadotropin used, the dose of THC and/or the amount of divalent cation present in the media. In the presence of luteinizing hormone (LH; 10 ng/ml), and a dose of 25 micrograms THC/ml, T production was significantly decreased, compared to that by testes incubated with LH and vehicle at all Ca++ levels, except at 0.127 or 1.0 mM Ca++. The production of T by these paired testes exposed to either THC or vehicle (ethanol; ETOH), increased as Ca++ concentration approached physiological levels. In contrast, in the presence of follicle-stimulating hormone (FSH; 1 microgram/ml), THC-induced suppression of T production was significant in the absence of Ca++ from the media, and at 12.7 mM Ca++. However, it appeared that the levels of Ca++ did not differentially affect T production in the presence of FSH, whether or not THC was also added. In the presence of human chorionic gonadotropin (hCG; 12.5 mIU/ml), a lower dose of THC (25 ng/ml), stimulated T production at 0.25 to 1 mM Ca++, but had no effect as Ca++ reached 2.5 mM. Without additional Ca++ in the media, this dose of THC significantly reduced T secretion. In contrast, in the presence of hCG, a higher THC dose (25 micrograms/ml), suppressed T accumulation at 0.127, and from 1.0 to 12.7, but had no effect at 0.25 mM, or in the absence of Ca++. In the presence of hCG, the high 25 micrograms/ml dose of THC stimulated T production, in the absence of additional Mg++, and at 0.01 mM Mg++, but THC had no effect at 0.1 mM Mg++, but inhibited T production at 1.1 mM Mg++. In the presence of hCG, 25 micrograms THC/ml produced a consistent suppression of T production across glucose concentrations examined. These findings suggest that the mechanisms by which THC effects testicular steroidogenesis may involve Ca++- and/or Mg++-dependent processes. Differential requirements for these divalent cations by the gonadotropins may explain the interactive effects of THC with LH, hCG or FSH.