Year round patchiness of Vibrio vulnificus within a temperate Texas bay.

AIMS: To investigate with high geographical resolution the small-scale spatial and temporal distribution of the pathogen Vibrio vulnificus throughout the water column in a temperate Texas bay where numerous V. vulnificus infections had been reported by the regional media the previous summer. METHODS AND RESULTS: Surface and bottom water samples were collected from 19 sites between April 2005 and October 2006 from Matagorda Bay, TX. Physicochemical parameters were measured and V. vulnificus were analysed using quantitative polymerase chain reaction (Q-PCR) as a means of overcoming constraints of traditional culturing techniques. V. vulnificus was detected through out the year, although its temporal and spatial distribution was patchy. V. vulnificus abundances at individual sites ranged from <10 to >1·1×10(3)cellsml(-1) . No statistically reliable predictive model related to the physicochemical parameters could be developed for this pathogen. CONCLUSIONS: This study demonstrates that year round detection of V. vulnificus while likely in the viable but nonculturable (VBNC) state during the winter months and emphasizes why physicochemical factors are insufficient metrics for robust regression modelling of this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an effective new tool, Q-PCR, to study environmental distribution of V. vulnificus and that in the light of the patchy distribution observed, new reliable approaches and a mechanistic understanding of pathogen ecology need to be considered to effectively model the aquatic distribution of V. vulnificus.

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.

Digital plethysmography for assessing erythrityl tetranitrate bioavailability.

The bioavailability or oral, sublingual, and chewable tablets or erythrityl tetranitrate (ETN) was evaluated in 15 normal men. In a randomized, complete crossover investigation with nitroglycerin and placebo controls, drug-induced changes in the diastolic amplitude response intensity were measured with a digital plethysmogram. Values for area under the response intensity curve (AUC), maximum response intensity (Imax), and time lapse from dosing to peak response (tmax) were obtained by computer processing and converted to intensity values and AUC segments for specific time intervals. Sublingual nitroglycerin induced a response (p less than 0.05) from placebo within the first hour. Although somewhat slower in reaching peak intensity, all forms of ETN induced significant responses over placebo (p less than 0.05) for 2 hr, with oral (swallowed) ETN different 6 hr. Our results indicate that all the ETN dosage forms were bioavailable, with the longest duration of effect by the oral form.

Pharmacokinetics and pharmacodynamics of a new cardiotonic vasodilator agent, 349U85, in normal subjects.

OBJECTIVE: To assess the pharmacodynamics and pharmacokinetics of single oral doses of a new vasodilator-cardiotonic agent, 349U85 hydrochloride [6-piperidino-2(1H)-quinolinone hydrochloride], in healthy male subjects. METHODS: This randomized, parallel, double-blind, placebo-controlled, dose escalation trial was conducted at a university-based clinical research center among 27 healthy male subjects. Data measurements used in the study included cardiac index, supine and standing blood pressure, 24-hour ambulatory electrocardiography, and 12-lead electrocardiography. RESULTS: Doses from 2 mg to 250 mg were well tolerated. Cardiac index, supine heart rate, and orthostatic hypotension, indicators of inotropic, chronotropic, and vasodilator effects, respectively, correlated to plasma concentrations of 349U85 and of its metabolite, 661U88. Results suggest that 349U85 may be more responsible for inotropic effects, whereas 661U88 may be more responsible for vasodilatory and chronotropic effects. These results are consistent with the preclinical pharmacologic profile for these two compounds. Headache, orthostatic dizziness, and hypotension tended to occur more frequently at higher doses and were temporally related to drug administration. Pharmacokinetic analyses indicate nonlinearity of 349U85 and 661U88, suggestive of saturation of metabolism and large interindividual variability in maximum plasma drug concentration and area under the plasma concentration-time curve. The source of the variability is not known. The time to maximum distribution was approximately 0.7 hours for both 349U85 and 661U88; the terminal elimination half-life was 1 hour for 349U85 and 3 hours for 661U88. Holter monitoring revealed asymptomatic increases in ventricular and supraventricular ectopic activity in some volunteers; ectopy appeared to be related to the dose of 349U85 and generally occurred at higher doses.

Synthesis and antimicrobial evaluation of substituted 5,6-dihydro-5-nitrouracils.

Reaction of 5-nitrouracil derivatives with sodium borohydride in methanol-water, followed by neutralization of the product with acid, has produced 5,6-dihydro-5-nitrouracil (5) 5,6-dihydro-6-dihydro-6-methyl-5-nitrouracil (7), 5,6-dihydro-5-nitro-1-(4-nitrophenyl)uracil (10), and 5,6-dihydro-5-nitro-1(beta-D-ribofuranuronic acid ethyl ester)uracil (12). In assays for antimicrobial activity using strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Trichophyton mentagrophytes, significant inhibition of growth was not found.

Synthesis and antimicrobial activity of certain 6H-1,2,4-oxadiazin-3(2H)-ones.

Treatment of 6H-1,2,4-oxadiazin-3(2H)-one-5(4H)-thione (2) with hydroxylamine, hydrazine, methylamine, or benzylamine afforded the corresponding N5-substituted 5-amino-6H-1,2,4-oxadiazin-3(2H)-ones 3c-f. Refluxing a dioxane solution of 6H-1,2,4-oxiazine-3,5(2H,4H)-dione (1) with benzylamine or aminodiphenylmethane and hexamethyldisilazane in the presence of ammonium sulfate gave 5-benzylamino-6H-1,2,4-oxadiazin-3(2H)-one (3f) and the corresponding 5-diphenylmethylamino derivative 3g. Reaction of 1 with methyl iodide, benzyl chloride, dihydropyran, dihydrofuran, or benzyloxycarbonyl chloride afforded the corresponding 2-substituted 6H-1,2,4-oxadiazine-3,5(2H,4H)-diones 6a-e. Reaction of 2-methyl-6H-1,2,4-oxadiazine-3,5(2H,4H)-dione (6a) or the corresponding 2-benzyl derivative 6b with phosphorus pentasulfide in dioxane gave 2-methyl-6H-1,2,4-oxadiazin-3(2H)-one-5(4H)-thione (8a) and the corresponding 2-benzyl derivative 8b, respectively. Reaction of 8a with ammonia in dioxane afforded 2-methyl-5-amino-6H-1,2,4-oxadiazin-3(2H)-one (9). The degree of in vitro activity and the presence of antibacterial activity in the urine of animals given 5-amino-6H-1,2,4-oxadiazin-3(2H)-one (3a) by oral route of administration prompted selection of this compound for further study.

Synthesis and antitumor and antiviral activities of 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-iminopyrimidine and its derivatives.

1-beta-D-Arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine (10), 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine 3'-phosphate (9), and 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-chloropyrimidine (11) have been synthesized from 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (5), 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine 3'-phosphate (4), and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-chlorocytosine (6), respectively. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine 3'-phosphate (7), 1-beta-D-arabinofuranosyl-2-amino-1,4-(2H)-iminopyrimidine (13), 1-beta-D-arabinofuranosyl-2-amino-1,3(2H)-iminopyrimidine 3'-phosphate (12), and compounds 4, 5, and 9 showed significant in vitro activity against a number of DNA viruses. Compounds 7 and 12 were also effective in vivo against type 1 herpes simplex virus. Compounds 7, 12, and 13 were extremely effective in the treatment of mice bearing leukemia L1210.

A steady-state evaluation of the effects of propantheline bromide and cholestyramine on the bioavailability of digoxin when administered as tablets or capsules.

Drug interactions can profoundly alter the absorption of digoxin in tablet form. This study evaluated whether digoxin solution in capsules, a new dosage form with 90% to 100% bioavailability, would reduce such alterations, specifically those caused by cholestyramine and propantheline bromide. The investigation used a six-treatment, steady-state, balanced, incomplete block design with 18 healthy adults studied for four continuous two-week treatment periods. Treatments were either two 0.25 mg digoxin tablets or two 0.20 mg digoxin capsules administered alone, with propantheline, 15 mg qid, or with cholestyramine, 8 g qd. Bioavailability was determined from steady-state, 24-hour area under the serum concentration-time curve (AUC, ng X h/mL) and from 0- and 24-hour trough serum digoxin concentrations (ng/mL). The AUCs for tablets alone, with cholestyramine, and with propantheline were 32.8 +/- 13.3 (+/- SD), 22.4 +/- 12.1, and 40.6 +/- 13.9, respectively, while corresponding values for capsules were 31.7 +/- 9.3, 24.7 +/- 7.9, and 35.9 +/- 12.8. The trough concentrations for tablets alone, with cholestyramine, and with propantheline were 0.88 +/- 0.47, 0.61 +/- 0.38, and 1.09 +/- 0.35, respectively; trough concentrations for capsules were 0.77 +/- 0.28, 0.74 +/- 0.28, and 0.96 +/- 0.48, respectively. The only significant differences in AUC were seen when comparing tablets alone versus tablets with cholestyramine (P less than .0005) and tablets with propantheline (P less than .01). A significant finding was also observed when comparing trough concentrations for tablets alone versus tablets with cholestyramine (P less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Bioavailability of organic nitrates: a comparison of methods for evaluating plethysmographic responses.

Erythrityl tetranitrate, a long-acting organic nitrate, was compared with isosorbide dinitrate in a double-blind, placebo-controlled complete crossover study in 15 healthy male volunteers. A digital plethysmogram and ear densitogram were used to assess the physiologic response to these two sublingual nitrates, with the intensity and duration of drug effect calculated by differences in diastolic amplitude intensity before and after drug administration. Both 5 mg sublingual erythrityl tetranitrate and 5 mg isosorbide dinitrate produced significant increases in diastolic amplitude intensity for up to 3 hours. The erythrityl tetranitrate peak effect was less than that of the isosorbide dinitrate, but the incidence of headaches was also less. The ear densitogram was found to be an effective means of assessing the diastolic amplitude intensity changes.

Erythrityl tetranitrate compared with isosorbide dinitrate. Effects on systolic time intervals and nitrate modification of effects of food.

The pharmacologic effects of erythrityl tetranitrate (ETN) and isosorbide dinitrate (ISDN) were compared to placebo using systolic time intervals (STI) in a randomized, double-blind study in 15 fasted male volunteers. Sublingual doses of ETN 5 mg, ISDN 5 mg, and placebo were administered to each volunteer at weekly intervals, and measurements of heart rate and STI [pre-ejection period (PEP), left ventricular ejection time (LVET), and PEP/LVET ratio] were made serially for up to 6 hours after each dose. STI were determined using ear densitography. Evaluation of the pharmacologic effects of ETN and ISDN were based on placebo-corrected changes from baseline values. Ejection time index (ETI) [LVET corrected for heart rate] was shortened, but the changes were not statistically significant for either drug. However, after ETN and ISDN, statistically significant (p less than 0.05) changes in PEP and PEP/LVET ratio were demonstrated for up to 240 minutes after dosing. Unexpected marked changes in the baseline corrected PEP/LVET ratio were observed following food at 4 hours after dosing. This suggests increased inotropy during the postprandial period.

Antagonistic interactions among marine pelagic bacteria.

Recent studies suggest that bacterial abundance and species diversity in the ocean's water column are variable at the millimeter scale, apparently in response to the small-scale heterogeneity in the distribution of organic matter. We hypothesized that bacterium-bacterium antagonistic interactions may contribute to variations in community structure at the microscale. We examined each of the 86 isolates for their inhibition of growth of the remaining 85 isolates by the Burkholder agar diffusion assay. More than one-half of the isolates expressed antagonistic activity, and this trait was more common with particle-associated bacteria than with free-living bacteria. This was exemplified by members of the alpha subclass of the class Proteobacteria (alpha-proteobacteria), in which production of antagonistic molecules was dominated by attached bacteria. We found that gamma-proteobacteria (members of the orders Alteromonadales and Vibrionales) are the most prolific producers of inhibitory materials and also the most resilient to them, while members of the Bacteriodetes were the organisms that were least productive and most sensitive to antagonistic interactions. Widespread interspecies growth inhibition is consistent with the role of this phenomenon in structuring bacterial communities at the microscale. Furthermore, our results suggest that bacteria from pelagic marine particles may be an underutilized source of novel antibiotics.

Physiological alterations secondary to perfusion and revascularization of canine intestine.

Our studies suggest that the perfusion rates of canine intestinal segments during extracorporeal perfusion are directly related to the quality of preservation; namely, better preserved specimens exhibit higher perfusion rates and vice versa. Production of intraluminal fluid (ILF) during preservation and following revascularization is related to the quality of preservation. Poorly preserved intestine seems generally to produce more fluid than well preserved specimens. Conversely, continuous pulsatile perfusion techniques resulted in more ILF production than specimens perfused with low-flow gravity-dependent systems. Ex vivo intestinal perfusion resulted in organ oedema inversely proportional to the quality of preservation. Poor preservation is also associated with washout of potassium, lactic dehydrogenase, and tissue acidosis both during preservation and revascularization. Perfusion with a high concentration of potassium ions results in poor preservation presumably owing to vasoconstriction. Mucosal malperfusion as demonstrated by the Microfil technique is the circulatory abnormality most closely associated with inadequate preservation. Smooth-muscle function, as measured by electrical activity recordings, is well preserved by pulsatile flow methods, whereas gravity perfusion results in disorganized and spastic muscular activity. The best preservation appeared to be obtained by the use of pulsatile flow with cryoprecipitated plasma as the perfusate.

Energy requirements for the transport of methylthio-beta-D-galactoside by Escherichia coli: measurement by microcalorimetry and by rates of oxygen consumption and carbon dioxide production.

The energy cost for maintenance of gradients of methylthio-beta-d-galactoside in Escherichia coli was evaluated. Information was also obtained concerning the energy flow associated with gradient establishment under some circumstances. Energy flow was evaluated from transport-induced changes in the rate of heat evolution, oxygen consumption, and carbon dioxide production in metabolically active cells. Heats were measured with an isothermal calorimeter. Energy expenditure behavior was characterized by a transition that depended on the level of accumulation. The data for steady-state maintenance could be rationalized in terms of the Mitchell hypothesis, two models for influx and efflux, and a transition between them. At low levels of uptake, steady-state proton-methylthio-beta-d-galactoside (TMG) symport for influx and efflux occurred via a nonenergy-requiring exchange process. The only energy requirement was that necessary to pump back in any TMG exiting via a leakage pathway (model I). Above the transition, all influx occurred with proton symport, but all exit, leak and carrier mediated, occurred without proton symport (model II). The H(+)/TMG stoichiometric ratio computed for the region of model II applicability (carbon source present, high level of uptake) approached 1. This value agreed with that of other workers for downhill beta-galactoside flow, suggesting that the energy cost for both downhill and uphill flow was approximately the same. For low levels of uptake, initial establishment of the gradient was followed by a burst of metabolism that was much larger than that expected on the basis of the chemiosmotic hypothesis. In the absence of carbon source, the stimulation in respiration was sufficient to produce 13 times more protons than are apparently necessary to establish the gradient. The results indicate also that the nature of the biochemical process stimulated by TMG depends on its level of uptake. Insight into several aspects of the nature of these processes was provided through analysis of the heat, oxygen, and CO(2) data. The key factor controlling the transition in energy flow behavior is suggested to be rate of flux. The present data suggest that it occurs at a flux of approximately 120 nmol/min per mg of protein.

Purification and characterization of avian dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (EC 1.14.17.1) has been purified from the chromaffin granules of avian adrenals. The enzyme has a molecular mass of approximately 320K daltons and consists of four apparently identical subunits joined in pairs by disulfide bonds. Analysis of the products formed from dopamine tritiated in the beta position indicated that 1.72 times as much tritium was retained in norepinephrine as was released as water. Ferrocyanide could serve as a reductant, but ascorbate at equal concentrations afforded higher rates. The enzyme had a pH optimum of 5-6 and was activated by either fumarate or acetate, with fumarate being far more effective. Kinetic experiments varying the concentrations of the substrates ascorbate and dopamine and those of the products dehydroascorbate and norepinephrine suggested that the mechanism was un-uni bi-uni ping pong. By this mechanism, the enzyme released dehydroascorbate after being irreversibly reduced by ascorbate and then sequentially bound oxygen and dopamine and released the product norepinephrine. The enzyme was inhibited by high but probably physiological concentrations of the substrate ascorbate and was activated by low concentrations of the product dehydroascorbate. Ascorbate inhibition was noncompetitive with dopamine, and dehydroascorbate activation was due to an increase in the enzyme's affinity for ascorbate with little or no change in its Vmax. Substrate inhibition by ascorbate and product activation by dehydroascorbate might together ensure that the rate of norepinephrine synthesis in vivo remains relatively unaffected by changes in the ratio of ascorbate to dehydroascorbate within chromaffin granules.

Existence of phospholipid bilayer structure in the inner membrane of mitochondria.

The presence of ordered phospholipid lamellar structure in inner membranes of mitochondria was detected with the use of a spin-labeled 2,4-dinitrophenol. A phospholipid bilayer may be an important structural and functional component of the inner mitochondrial membrane.

A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis.

Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.