The influence of NO-containing ruthenium complexes on mouse hippocampal evoked potentials in vitro.

The influence of different, nitric oxide-containing ruthenium complexes on the evoked potentials recorded from the CA1 region of the mouse hippocampus in vitro has been investigated. Of the compounds tested, only trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 (1-2.5 mM) exerted a strong facilitatory action on the population spike, the EPSP, and the spontaneous activity. Its activity probably depends upon its ability to release NO following reduction. The phosphito ligand is important both in terms of adjusting the reduction potential of the complex to be biologically accessible and in labilizing the coordinated NO. The effects of this compound could not be reversed by perfusion. Scavenging NO in slices preincubated with oxyhemoglobin prior to the addition of this compound eliminated its neurophysiological effects. The control molecules trans-[(P(OEt)3)2(NH3)4Ru](PF6)2, trans-[(H2O)(P(OEt)3) (NH3)4Ru](PF6)3, and [(NO)(NH3)5Ru]Cl3, which are structurally similar, but unable to generate NO, were ineffective. NaNO2 suppressed neuronal firing. Attempts to induce Long-Term Potentiation (LTP) at the time of maximal effect of trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 were unsuccessful, suggesting that the mechanism of amplification induced by trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 and LTP may share common pathways.

A controlled NO-releasing compound: synthesis, molecular structure, spectroscopy, electrochemistry, and chemical reactivity of R,R,S,S-trans-[RuCl(NO)(cyclam)]2+(1,4,8,11-tetraazacyclotetradecane).

The synthesis of trans-[RuCl(NO)(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane) can be accomplished by either the addition of cyclam to K2[RuCl5NO] or by the addition of NO to trans-[RuCl(CF3SO3)(cyclam)](CF3-SO3). Crystals of trans-[RuCl(NO)(cyclam)](ClO4)2 form in the monoclinic space group P2(1)/c, with unit cell parameters of a = 7.66500(2) A, b = 24.7244(1) A, c = 16.2871(2) A, beta = 95.2550(10) degrees, and Z = 4. One of the two independent molecules in the unit cell lies disordered on a center of symmetry. For the ion in the general position, the Ru-N and N-O bond distances and the [Ru-N-O]3+ bond angle are 1.747(4) A, 1.128(5) A, 178.0(4) degrees, respectively. In both ions, cyclam adopts the (R,R,S,S) configuration, which is also consistent with 2D COSY 1H NMR studies in aqueous solution. Reduction (E degree = -0.1 V) results in the rapid loss of Cl- by first-order kinetics with k = 1.5 s-1 and the slower loss of NO (k = 6.10 x 10(-4) s-1, delta H++ = 15.3 kcal mol-1, delta S++ = -21.8 cal mol-1 K-1). The slow release of NO following reduction causes trans-[RuCl(NO)(cyclam)]2+ to be a promising controlled-release NO prodrug for vasodilation and other purposes. Unlike the related complex trans-[Ru(NO)(NH3)4(P(OEt)3)](PF6)2, trans-[RuCl(NO)(cyclam)]Cl2 is inactive in modulating evoked potentials recorded from mice hippocampal slices probably because of the slower dissociation of NO following reduction.

Validation and characterization of a human volunteer challenge model for cholera by using frozen bacteria of the new Vibrio cholerae epidemic serotype, O139.

Until recently, all epidemic strains of Vibrio cholerae were of the O1 serotype. Current epidemics have also been caused by a new serotype, Vibrio cholerae O139. Although the pathogenesis and clinical features of O139 cholera are similar to those of O1 cholera, immunity to serotype O1 does not confer immunity to serotype O139. Therefore, prior to beginning vaccine efficacy studies, we sought to validate the use of a large standardized frozen inoculum of virulent V. cholerae O139 4260B for use in a human volunteer challenge model. Healthy volunteers (n = 25) were recruited for an Internal Review Board-approved inpatient dose-escalation challenge. Our goal was to identify a dose at which the cholera attack rate and the geometric mean purge were sufficient for determining vaccine efficacy against moderate and severe disease. At a dose of 10(5) CFU, 8 of 10 volunteers experienced purging and had a positive stool culture for V. cholerae. However, at this dose, the geometric mean stool volume of 2,175 g was insufficient by study criteria. At a dose of 10(6) CFU, 14 of 15 volunteers experienced purging, with a geometric mean stool volume of 5,621 g. Disease severity was significantly greater in volunteers with blood group O than those with non-O blood types (10,353 g versus 3,555 g, P < 0.001). Following challenge, all volunteers demonstrated a significant rise in antitoxin antibodies but the serum vibriocidal titer was attenuated compared to that seen after challenge with an O1 strain. This model provides a reproducible illness of sufficient severity for testing the efficacies of new O139 or combined O1-O139 vaccines.

Summary of the thirty-first United States-Japan Joint Conference on Cholera and Related Diarrheal Diseases.

The 31st United States-Japan Cholera and Related Diarrheal Diseases Conference was held in conjunction with the United States-Japan Malnutrition Conference at Kiawah Island, South Carolina, 30 November to 3 December 1995. In addition to the overall conference theme of the role of cytokines in the pathogenesis of enteric infections and malnutrition, researchers described substantial advances in cholera epidemiology, detection, molecular mechanisms, and pathophysiology plus new mechanisms for enterotoxigenic, enteroadherent, enterohemorrhagic, and enteroinvasive Escherichia coli. There was also emphasis on new work with and vaccine development with Bacteroides fragilis and Yersinia, Shigella, and Salmonella species.

Growth characteristics of tumors induced by transplantation into athymic mice of BALB/3T3 cells transformed in vitro by residue organics from drinking water.

The growth and histopathological characteristics of tumors produced in nude mice by injection of morphologically transformed or normal BALB/3T3 1-13 cells were studied. Subcutaneous injection of 5 X 10(6) cells transformed in vitro by organic residue mixtures from samples of drinking water resulted in tumor growth in all animals with an average latency period of 25 days. Of the 13 animals receiving non-treated cells, three developed tumors with an average latency period of 127 days. Tumors grew progressively at the site of injection, reaching an average size of 1141 mm2 within 8-37 days. Most of the tumors, primarily those deriving from transformed cells, invaded the tumor bed and extended into the surrounding tissues. In few cases, extension of the tumor into the peritoneal cavity was observed. Microscopically all tumors were characterized as fibrosarcomas.

Malignant transformation of BALB/3T3 cells by residue organic mixtures from drinking water.

Malignant transformation of BALB/3T3 1-13 cells results following treatment with complex mixtures of residue organic chemicals from drinking water samples of five U. S. cities. The U. S. Environmental Protection Agency provided the residues which were obtained by liquid-liquid solvent extraction, and sorption-desorption on XAD-2 resin of samples concentrated by reverse osmosis from drinking water of Philadelphia, PA; New Orleans, LA; Miami, FL; Seattle, WA; and Ottumwa, IA. Transformation was observed using a focus formation assay following 72-hr exposure of cells to residues from one or more samples from each city. Malignancy of several transformed and normal clones was evaluated in athymic (nu/nu) mice. The usefulness of such a transformation assay in detecting potential carcinogenic compounds contained in complex mixtures and the evaluation of possible health effects of residue organics in drinking water are discussed.

Membrane bioenergetic parameters in uncoupler-resistant mutants of Bacillus megaterium.

Mutants of Bacillus megaterium displaying malate-driven ATP synthesis resistant to uncouplers of oxidative posphorylation are further characterized. Both the pH gradient and electrical potential generated across the membrane by malate respiration are equally sensitive to uncouplers in the wild type and uncoupler-resistant mutants. The mutants possess 0 to 10% of the wild type ATPase activity which is not activated by pretreatment with heat or trypsin. Despite this inability to measure ATPase activity, the mutants demonstrate acid-pulse-driven ATPase synthesis which is sensitive to uncouplers as well as malate-driven ATP synthesis which becomes uncoupler sensitive at pH 5.5. N,N' -Dicyclohexylcarbodiimide and valinomycin plus potassium inhibition of ATP synthesis is reversed by uncouplers in the mutants but not in the wild type. The data support the existence of a specific site on the ATPase complex for uncoupler binding which, if altered by mutation, affects uncoupler binding to the complex. The retention of malate-driven ATP synthesis in the absence of a significant pH gradient or electrical potential suggests that an alternative intermediate is involved in coupling oxidation to phosphorylation.

Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.

Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus.

Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2-deoxyglucose and 3-O-methylglucose in cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non-phosphorylatable analog 3-O-methylglucose occurs at the same time as that of 2-deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation-specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non-phosphorylated forms of 2-deoxyglucose in normal cells and in cells transformed by the wild-type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2-deoxyglucose in both normal and transformed chicken embryo cells.

Mutants of Bacillus megaterium resistant to uncouplers of oxidative phosphorylation.

Mutants of Bacillus megaterium displaying malate-stimulated ATP synthesis resistant to uncouplers of oxidative phosphorylation were isolated and partially characterized. ATP synthesis in such mutants was resistant to carbonyl cyanide m-chlorophenyl hydrazone as well as to other uncouplers including 2,4-dinitrophenol, pentachlorophenol, and sodium azide. ATP synthesis in the wild type and in resistant mutants was sensitive to N,N'-dicyclohexylcarbodiimide, tributyltin, valinomycin plus potassium, and potassium cyanide. Active transport of glycine and glutamine which are sensitive to uncouplers in the wild type was also uncoupler-sensitive in the mutants.

Bacillus megaterium mutant deficient in membrane-bound adenosine triphosphatase activity.

An adenosine triphosphatase (ATPase) mutant of Bacillus megaterium was isolated and characterized. This mutant (designated A37) was unable to grow on nonfermentable carbon sources and possessed less than 5% of the wild-type ATPase activity. Oxygen uptake by the mutant was comparable to that in the wild type. Sporulation in the wild type occurred in both glucose- and nitrogen-limiting media; however, A37 sporulated only in the nitrogen-limiting medium. The inability of A37 to sporulate in glucose-limiting medium seemed to be due to insufficient adenosine 5'-triphosphate (ATP) levels during the sporulation stages. Fructose, which can generate ATP via substrate-level phosphorylation, is equally efficient in stimulating ATP synthesis in the wild type and A37. Malate-stimulated ATP synthesis in the wild type was shown to have many characteristics associated with oxidative phosphorylation and was absent in the mutant. These data suggest that the ATPase deficiency results in the loss of oxidative phosphorylation.

Effects of quercetin and F1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles.

Quercetin (3,3',4',5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD+ by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3' and perhaps 3 position are important for the inhibition of ATPase activity.

Development of a membrane-bound resiratory system prior to and during sporulation in Bacillus cereus and its relationship to membrane structure.

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.

Lipid composition of Bacillus cereus during growth and sporulation.

The lipid composition of Bacillus cereus during growth and sporulation was examined. The total lipid extract accounted for 2 to 3% of the dry weight of the cells and consisted of neutral lipids (30 to 40%) and phospholipids (60 to 70%). Phospholipids were separated by thin-layer chromatography into eight components; phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol were the major phospholipids and accounted for over 90% of the total. Also identified was a diglycosyl diglyceride and an alanine ester of phosphatidyl glycerol. Diphosphatidyl glycerol was more difficult to extract than the other components in vegetative and stationary-phase cells, but became increasingly easy to extract during spore maturation, and during sporulation cellular levels increased. Phosphatidyl glycerol had a high turnover rate; it accounted for about 70% of the phospholipid synthesis throughout sporulation but only represented between 30 and 40% of the total phospholipid at any time. Phosphatidyl ethanolamine, on the other hand, accounted for about 20% of the synthesis but was the major phospholipid (50 to 60% of the total).