Mamoru Watanabe

Testosterone uptake by membrane vesicles of Pseudomonas testosteroni

Abstract Uptake of testosterone was demonstrated in membrane vesicles prepared from Pseudomonas testosteroni grown on testosterone. In contrast, membrane vesicles from uninduced cultures revealed no significant transport activity for steroids. The K m of the reaction was 2 · 10 −6 M and the V 28.5 nmoles/min per mg protein. Steroid uptake was maximal within the pH range of 8 to 9 and at incubation temperatures between 30 and 37 °C. Transport of steroid was dependent upon NAD+ and was reduced by NADH, dinitrophenol, and inhibitors of electron transport, such as N 3 − · CN − and amytal. The intravesicular steroid concentration was approx. 800 times the steroid concentration present in the medium at the start of the incubation.

Steroid-receptor in Pseudomonas testosteroni released by osmotic shock.

Abstract An inducible steroid-binding activity, demonstrated in cultures of Pseudomonas testosteroni , was released into the medium after exposure of bacteria to osmotic shock. The protein nature of the binding activity released into the osmotic shock fluid was indicated by its susceptibility to pronase digestion and heat denaturation and resistance to RNase and DNase. The receptor was saturable at low hormone concentrations and had a high affinity for steroids (K d 6.7 × 10 −9 M). It formed a complex with binding steroids which had a sedimentation coefficient of approximately 4 S in sucrose density ultracentrifugation studies.

Membrane bound 3β and 17β-hydroxysteroid dehydrogenase and its role in steroid transport in membrane vesicles of Pseudomonas testosteroni

Abstract Uptake of steroids by membrane vesicles prepared from Pseudomonas testosteroni was specific for testosterone, dihydrotestosterone and dehydroepiandrosterone. The intravesicular steroid was androstenedione when testosterone or dehydroepiandrosterone was transported and androstanedione in the case of dihydrotestosterone. During transport NAD + was reduced to NADH. These results indicated that steroid transport could best be described as a group translocation process and involved 3β and 17β-hydroxysteroid dehydrogenase activity. The steroid itself appeared to function as the physiological electron donor via NADH, generated during transport and oxidation of the steroid.

Periplasmic steroid-binding proteins and steroid transforming enzymes of Pseudomonastestosteroni

Abstract Exposure of induced Pseudomonas testosteroni to osmotic shock resulted in the release of androgen and estrogen-binding proteins, 5-ene-3-ketosteroid isomerase and (3 and 17)β-hydroxysteroid dehydrogenase activities, and variable amounts of 3α-hydroxysteroid dehydrogenase, 1-ene-dehydrogenase and 4-ene-5α-dehydrogenase activities. When binding of C19 and C21 steroids to induced bacteria and to periplasmic proteins was examined, binding correlated with the ability of the steroids to support growth of this organism. Except for estradiol-17β, steroids incapable of supporting growth, such as aldosterone, cortisol, and corticosterone, were not bound. In the case of C19 steroids, the bound form of the steroid was androstanedione and/or androstenedione. On the other hand, progesterone and 17α-hydroxyprogesterone were bound in the form in which they were added. Displacement studies with unlabeled steroids indicated the presence of two steroid receptors, one which bound C19 and C21 steroids, and another which bound estra-diol-17β.

Induction of steroid-binding activity in pseudomonas testosteroni

Abstract Growth of Pseudomonas testosteroni on testosterone was optimal at 30°C. Induction of steroid-binding activity occurred optimally at 25°C to 30°C. Binding activity first appeared after 9 h of growth on testosterone and reached its maximum at 30 h. Dehydrogenation activities, on the other hand, reached a maximum at 12 h. Steroid-binding activity was induced by growth on several C19 and C21 steroids. At incubation temperatures of 37°C or 45°C there was minimal induction of binding activity. Binding activity induced at 30°C was irreversibly destroyed at 45°C and reversibly inhibited at 37°C.

Binding of steroids by a partially purified periplasmic protein from Pseudomonas testosteroni.

Abstract The androgen-binding periplasmic protein of Pseudomonas testosteroni was released by lysozyme-EDTA treatment of induced bacteria and partially purified by ammonium sulfate fractionation. The androgen-binding protein appeared in the 50–80% saturated fraction whereas steroid transforming enzymes and the estradiol-binding protein appeared in the 0-50% saturated fraction. The partial purification, however. did not alter the substrate specificity of the androgen-binding protein. Kinetics of binding at this stage of purification indicated a simple equilibrium binding process with independent binding sites.

Health policy directions for evidence-based decision making in Canada.

The topic, evidence-based decision making, may be seen from many perspectives and levels of policy consideration. Canada has been actively pursuing policy macro-level directions for health information and evidence-based decision making through the National Forum on Health and the Advisory Council on Health Infostructure, together with changes in the federal health ministry, Health Canada, and other important national health organizations such as the Canadian Institute for Health Information. It remains to be determined how these initiatives will fundamentally influence a culture of evidence-based decision making and health information availability and quality for the average Canadian. Canada has, nonetheless, recognized that our health care delivery system has fallen behind in terms of using information management and technology to enable and facilitate the ever-growing information needs of the system. Policy direction provided by the National Forum on Health and the Advisory Council on Health Information offer an agenda for action and strategic direction drawn from two broadly based, highly iterative processes.

Effect of infection with RNA phage R23 on membrane permeability and K+ fluxes in Escherichia coli

Abstract 1. 1. Infection of Escherichia coli by the RNA bacteriophage R23 produced a rapid, transient increase in K + efflux from cells preloaded with 42 K + . R23 also stimulated the influx of 42 K + for the first 5–10 min. The enhanced efflux was terminated by a “sealing reaction” 10–15 min after infection. 2. 2. The phage effect on K + flux, which was multiplicity-dependent, did not appear to require viral RNA penetration or replication but was related to phage adsorption. 3. 3. Analysis of 42 K + influx and efflux indicated little or no net change in intracellular K + content after R23 infection.

Temperature-sensitive RNA synthesis during adaptive growth on testosterone of Pseudomonas testosteroni.

Pseudomonas testosteroni is able to grow on nutrients other than steroids at 37°C, but during adaptive growth on steroids demonstrates temperature sensitivity and grows at 30 but not at 37°C. Similarly, RNA synthesis, which precedes and is necessary for induction of degradative enzymes, steroid-binding proteins, and transport processes occurs only at 30°C and not at 37°C, whereas rate of RNA synthesis during growth on other nutrients is greater at 37°C. The temperature-sensitive step occurs early during the induction period and the critical period appears to be during 2 to 212h of adaptive growth on testosterone. The critical step appears to be the initiation of specific RNA synthesis. Once RNA synthesis is initiated, synthesis continues at the non-permissive temperature. From these data we have postulated that the temperature-sensitive step involves initiation of RNA synthesis and may depend on a temperature-sensitive represser molecule of the steroid operon.

Reversal of inhibition of membrane-bound RNA synthesis in Escherichia coli infected with R23

Abstract 1. Infection by RNA bacteriophage R23 causes a release of ribosomes from a membrane-DNA-RNA polymerase complex isolated from Escherichia coli. This release may be responsible for the reduction in RNA synthetic capacity seen after phage infection. 2. The addition of ribosomes to such a membrane preparation restores the RNA synthetic capacity to uninfected levels. The ribosomes may exert their effect by binding to nascent RNA chains. 3. Exogenous RNA polymerase stimulates RNA synthesis by the membrane complex under conditions which probably prevent the initiation of new RNA chains. The mechanism of its effect may be analogous to that of ribosomes in this system.