Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.     

Infection of the Cat Flea, Ctenocephalides felis (Bouché) by Neoaplectana carpocapsae Weiser

Infection of cat flea, Ctenocephalides felis, larvae by the entomophilic nematode Neoaplectana carpocapsae was accomplished in the laboratory. The Breton strain of N. carpocapsae provided higher larval mortality at lower dosages than did the DD-136 strain. Adult nematodes were evident in the insect hemocoel after 48 h; however, no infective third-stage larvae were produced. Larval flea infection increased with an increase in the moisture content of sand from 2% to 7% and of sandy clay from 7% to 12%. Larval flea infection was also obtained on turf containing dauer larvae. Nematode penetration of cocoons with invasion of prepupal and pupal fleas was apparent.     

Interaction of phlorizin and sodium with the renal brush-border membraned-glucose transporter: Stoichiometry and order of binding

Summary The order and stoichiometry of the binding of phlorizin and sodium to the renal brush-border membraned-glucose transporter are studied. The experimental results are consistent with a random-binding sites is one-to-one. When the kinetics of phlorizin binding are measured as a function of increasing sodium concentration no significant variation is found in the apparent number of binding sites; however, the apparent binding constant for phlorizin decreases rapidly from approximately 16 m at [Na]=0 to 0.1 m at [Na]=100mm and approaches 0.05 m as [Na]. The experimental data are fit to a random carrier-type model of the coupled transport of sodium andd-glucose. A complete parameterization of the phlorizin binding properties of this model under sodium equilibrium conditions is given.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.     

Sex dependence of clearance rates of aldosterone and its metabolites from plasma of intact rats.

Following I.V. injection of 3H-aldosterone, the rates of clearance of plasma 3H-radioactivity was demonstrated to be sex-dependent in intact rats. Even though the percentages of CH2Cl2-extractable plasma radioactivity are greater in female than in male rats, the quantities of CH2Cl2-extractable label are similar until 60 min post-injection. However, the quantities of non-extractable, polar metabolites of aldosterone (NEPD) are markedly greater in the plasma of males and rapidly reach peak levels 10 min post-injection of aldosterone. In females, these polar metabolites (NEPD) are rapidly cleared from the blood. After bile-duct cannulation, the rate of excretion of aldosterone radiometabolites was demonstrated to be rapid and sex-dependent. Within 1 hr., female rats excreted via the bile 82% of the injected dose of 3H-aldosterone, compared to 49% in male rats. In both sexes, greater than 95% of the total radioactivity excreted in the bile are non-extractable polar metabolites of aldosterone (NEPD). The sex hormones appear to influence not only the nature of metabolism of aldosterone in the liver, but also the rates of clearance of aldosterone and its metabolites from the plasma into the bile.     

Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

A natural outbreak of transmissible murine colonic hyperplasia in A/J mice.

Transmissible murine colonic hyperplasia was diagnosed in 6-month-old A/J mice kept under standard laboratory conditions. Bacterial cultures revealed the presence of Citrobacter freundii (4280). Clinical signs included rough coats, feces adhering to the anus, slight dehydration and rectal prolapses. A nonclotting sanquinous intestinal fluid and gross colonic thickening were frequently seen at necropsy. Morbidity was approximately 50%; mortality approximately 25%. Tetracycline appeared to be effective in controlling the disease.