N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Effect of aminoguanidine on ischemia-reperfusion induced myocardial injury in rats

Myocardial ischemia-reperfusion (MI/R) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, excessive production of NO has been involved in causing myocardial injury. In our in vivo model, we examined the effects of aminoguanidine (AMG), a known iNOS inhibitor, on percentage infarct size in anaesthetized rats. A total of 14 rats were equally divided into two groups (n = 7 in each group). To produce myocardial necrosis, the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion, in anesthetized rats. AMG (200 mg kg⁻¹) was given intravenously 10 min before occlusion. The volume of infarct size and the risk zone were determined by planimentry of each tracing and multiplying by the slice thickness. Infarct size was normalized by expressing it as a percentage of the area at risk. Hemodynamic parameters were measured via the left carotid artery. Compared to MI/R group, whereas AMG administration elevated mean arterial blood pressure, statistically reduced the myocardial infarct size (21± 1 and 14± 4%, respectively) and infract size/risk zone (53± 3 and 37± 5%, respectively) in rat model of ischemia-reperfusion. In conclusion, this study indicates that iNOS inhibitor, AMG, show reduction in NO’s side effect in I/R injury.     

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Testosterone secretion and semen plasma enzyme activity in rams with genital pathology stimulated with GnRH

Testosterone secretion in response to GnRH stimulation and enzymatic activity of semen plasma was evaluated comparatively in rams with or without genital abnormality. Scrota, testes and epididymides of 128 rams between 1.5 and 6 years old from various breeds were examined clinically and ultrasonographically. Bilaterally cryptorchid rams (n = 2), and rams with focal testicular degeneration (n = 3) or unilateral sperm granuloma localized in the caput (n = 3) epididymis or the cauda epididymis (n = 3), diagnosed by either clinical or ultrasonographic examination, were selected for the further investigation of spermatologic parameters, testosterone secretion in response to GnRH stimulation, and enzymatic activity of semen plasma before histopathologic confirmation of lesions. Except for the cryptorchid rams, sperm parameters determined in ejaculates were similar to intact controls (n = 3). GnRH administration increased plasma testosterone levels significantly irrespective of the type of genital pathology (P < 0.01). The testosterone response calculated based on area under the curve following GnRH administration in rams having genital abnormality was not significantly different from the controls. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activity in the semen plasma varied between rams, with the lowest mean values in the bilaterally cryptorchid group (P < 0.05). Spermatic granuloma localized either in the caput or cauda of the epididymis was associated with a significant reduction in the semen plasma AST activity compared to controls (P < 0.01). In conclusion, our results indicated that the ability of testicular tissue to secrete testosterone in response to GnRH stimulation in rams with bilateral cryptorchidism, focal testicular degeneration and unilateral sperm granuloma was similar to that of intact controls, and that reduced semen plasma AST activity may have a diagnostic value in the diagnosis of the epididymal obstruction in rams. Focal testicular degeneration did not influence AST, alkaline phosphatase (ALP) and LDH activity in semen plasma.     

Life history and spatial distribution of Oriental beetle (Coleoptera: Scarabaeidae) in golf courses in Korea

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.     

Gastric H/K-ATPase liberates two moles of Pi from one mole of phosphoenzyme formed from a high-affinity ATP binding site and one mole of enzyme-bound ATP at the low-affinity site during cross-talk between catalytic subunits

The maximum amount of acid-stable phosphoenzyme (E32P)/mol of alpha chain of pig gastric H/K-ATPase from [gamma-32P]ATP (K(1/2) = 0.5 microM) was found to be approximately 0.5, which was half of that formed from 32P(i) (K(1/2) = 0.22 mM). The maximum 32P binding for the enzyme during turnover in the presence of [gamma-32P]ATP or [alpha-32P]ATP was due to 0.5 mol of E32P + 0.5 mol of an acid-labile enzyme-bound [gamma-32P]ATP (EATP) or 0.5 mol of an acid-labile enzyme-bound [alpha-32P]ATP, respectively. The K(1/2) for EATP formation in both cases was 0.12 approximately 0.14 mM. The turnover number of the enzyme (i.e., the H+-ATPase activity/(EP + EATP)) was very close to the apparent rate constants for EP breakdown and P(i) liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of P(i) liberated to that of EP that disappeared increased from 1 to approximately 2 with increasing concentrations of ATP (i.e., equal amounts of EP and EATP exist, both of which release phosphate in the presence of high concentrations of ATP). This represents the first direct evidence, for the case of a P-type ATPase, in which 2 mol of P(i) liberation occurs simultaneously from 1 mol of EP for half of the enzyme molecules and 1 mol of EATP for the other half during ATP hydrolysis. Each catalytic alpha chain is involved in cross-talk, thus maintaining half-site phosphorylation and half-site ATP binding which are induced by high- and low-affinity ATP binding, respectively, in the presence of Mg2+.     

Investigation of the mechanism of nicotine-induced relaxation on the sheep sphincter of Oddi

Possible mechanisms for nicotine-induced relaxation were investigated in the isolated sheep's sphincter of Oddi. Sheep's sphincter of Oddi rings were mounted in tissue bath with modified Krebs-Henseleit solution and aerated with 95% oxygen and 5% carbon dioxide. Tension was measured with isometric force transducers, and muscle relaxation was expressed as percent decrease of precontraction induced by carbachol. Nicotine (1 x 10(-5) to 3 x 10(-3) mol/L) produced concentration-dependent relaxation on sphincter of Oddi precontracted by carbachol (10(-6) mol/L). Nicotine-induced relaxation was 72.8 +/- 4.2% of precontraction with carbachol (10(-6) mol/L) (mean pD2 value, 3.76 +/- 0.05 mol/L). Nicotine-induced relaxation was not affected by N(w)-nitro L-arginine methyl ester (L-NAME) (3 x 10(-5) mol/L), methylene blue (10(-5) mol/L), indomethacin (10(-5) mol/L), hexamethonium (10(-5) mol/L), glibenclamide (10(-5) mol/L), 4-aminopyridine (10(-3) mol/L), tetraethylammonium (3 x 10(-4) mol/L), clotrimazole (10(-6) mol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (10(-6) mol/L), and anthracene-9-carboxylate (9-AC) (10(-6) mol/L), but potentiated by bupivacain (10(-5) mol/L). A calcium-antagonizing effect of nicotine was not observed. The results suggest that nicotine-induced relaxation of the sheep's sphincter of Oddi is not mediated by the release of prostaglandins, nitric oxide (NO), or a related substance; by the activation of potassium channels or chloride channels; or by the stimulation of nicotinic cholinoceptors. Potentiation of the nicotine-induced relaxation by bupivacain indicates that blockade of sodium channels may play a role in this relaxation.     

Evidence for Recombination in the Microcystin Synthetase (<Emphasis Type="Italic">mcy</Emphasis>) Genes ofToxic Cyanobacteria <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Bold">spp</Emphasis>

Recombination has been suggested to be an important factor for the genetic variation of bacterial genes, but few studies have dealt with intragenic recombination between the same or closely related species of cyanobacteria. Here we provide strong evidence for recombination in the microcystin synthetase (mcy) gene cluster of the toxic cyanobacteria Microcystis spp. This gene cluster contains 10 genes (mcyA to J) that encode a mixed polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) complex. mcy gene sequences were determined for four selected regions (within mcyA, D, G, and J) within the mcy gene cluster from 1 Canadian and 10 Asian toxic Microcystis and compared with previously published mcy sequences. Split decomposition analysis indicated a reticulate phylogeny of mcyA, and several potential recombination tracts of mcyA were identified by the RDP analysis and a runs test implemented in GENECONV. In contrast, no recombination was detected in the mcyD, G, and J sequences. However, discrepancies among the four mcy gene genealogies were evident from the results of independent split decomposition analyses, which were further supported by incongruence length difference (ILD) tests. Taken together, these findings suggest that both intragenic and intergenic recombination within the mcy gene cluster contributes to the genetic diversity of the mcy genes of Microcystis spp.This article contains online supplementary material.