PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum.

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.

Comparison between the characteristics of follicular fluid and the developmental competence of bovine oocytes.

There are great differences in the developmental competence of oocytes collected from individual cows. Oocytes grow and mature in the follicular fluid (FF). In the present study, characteristics of the FF of each ovary and the developmental competence of enclosed oocytes were investigated, and these data were then compared. A total of 37 pairs of ovaries were collected from beef heifers. The concentration of magnesium (Mg), aspirate aminotransferase (AST), nonesterified fatty acids (NEFA), and lactate dehydrogenase (LDH) in the FF were great compared with serum standard. Several significant correlations among these characteristics were detected. Forty-eight hours after fertilization, the stage of embryo development at an advanced developmental stage (>6 cell stage) is related to the rate of blastulation 8 days after fertilization. In addition, a significantly positive or negative correlation was observed between the developmental competence (the rate of cleavage in the embryo and blastulation) and the concentration of the icterus index (ICT) or blood urea nitrogen (BUN) in the FF. In conclusion, the quality of oocytes is affected by the environment in the follicle, and BUN or ICT is a predictable index of the developmental competence of oocytes.

Effects of seirogan (wood creosote) on propulsive colonic motility and stool characteristics in ambulatory mini-pigs.

Our goal was to determine whether Seirogan, an herbal medicine used as an antidiarrheal agent, modifies colonic function, including motility. Experiments were performed on four female Yucatan mini-pigs with established permanent cecal fistulas providing direct access to the colon. Long-term recordings of proximal colonic motility were accomplished by a solid-state probe (six pressure ports 10 cm apart), and a motility index was calculated. Stool viscosity was also measured. The laxative bisacodyl (15 mg/kg) was used to induce colonic motility (increase in motility index) and stool softening, prior to investigating the effect of Seirogan (2-15 mg/kg per os twice a day) or a vehicle control. Seirogan (15 mg/kg), but not the placebo, reversed the bisacodyl-induced stool softening and restored the motility index to normal values by reducing the number of propagating contractions. Taken together the results suggest that inhibition of proximal colonic motility by Seirogan may contribute to its antidiarrheal action.

No significant differences were observed in the amounts of DNA strand breaks produced by copper between male and female liver cells of long-evans cinnamon (LEC) strain rats.

The amounts of DNA single strand breaks that are oxidative damage produced by copper were examined by comet assay in the liver cells of an inbred strain of Long-Evans Cinnamon (LEC) rats that spontaneously develops fulminant hepatitis. At 4 weeks of age, copper contents in the liver of LEC rats were approximately 30-fold higher than those of WKAH rats that are control rats used in the present study. Copper accumulated in the liver of LEC rats in an age-dependent manner and no significant differences were observed between copper contents in the livers of males and females at each week of age from 4 to 15 weeks. No significant amounts of DNA strand breaks were found in the liver cells of both male and female WKAH rats from 4 to 15 weeks of age. DNA strand breaks were produced in the substantial population of LEC rat liver cells at 10 weeks of age and induced in an age-dependent manner from 10 to 15 weeks of age. The amounts of DNA strand breaks produced by copper accumulation in the liver cells of female LEC rats are not more abundant than those in the cells of male rats, although it has been reported that hepatitis in female rats is more serious than that in male rats.

In vitro effects of wood creosote on enterotoxin-induced secretion measured electrophysiologically in the rat jejunum and colon.

Secretory diarrhea occurs when the balance between intestinal absorption and secretion is disturbed by excessive secretion caused by enterotoxins produced by the pathogen. Wood creosote has long been used as a traditional antidiarrheal remedy. The goal of our study was to extend our knowledge about the antisecretory action of wood creosote against Escherichia coli enterotoxin-induced secretion in the small intestine and colon. Experiments were performed in mucosal sheets of rat jejunum and colon which were stripped of the external muscle layers to eliminate interactions with smooth muscle activity and local blood flow. Mucosal sheets were placed in modified Ussing chambers and hypersecretory conditions were induced by heat-labile (LT) or heat-stable (STa) E. coli enterotoxins added cumulatively (0.01-10 microg/ml) to the mucosal bathing solution. Intestinal secretion was monitored electrophysiologically as transmucosal short circuit current (Isc). LT induced a concentration-dependent increase in Isc in the rat jejunum, with no effect in the colon. In contrast, STa induced a significant increase in colonic Isc, without causing any change in Isc across the jejunum. In separate experiments the effects of increasing concentrations of wood creosote (0.1-50 microg/ml), added to the mucosal or serosal bathing solution, were examined against the secretory responses induced by LT or STa. In the small intestine the antisecretory activity of wood creosote against LT-induced secretion was more potent following serosal application, whereas in the colon wood creosote inhibited STa-induced secretion with equal potency following either serosal or mucosal addition. In summary, our findings demonstrate that wood creosote possesses antidiarrheal activity suppressing E. coli enterotoxin-induced secretion in both the small intestine and colon.

Lack of oncogenicity of wood creosote, the principal active ingredient of Seirogan, an herbal antidiarrheal medication, in Sprague-Dawley rats.

Seirogan, an herbal medicine containing wood creosote (tablets, 10.0% w/w), has been developed and marketed for almost a century in various countries for the control of acute diarrhea and treatment of associated symptoms, such as abdominal cramping. Wood creosote (CAS no. 8021-39-4) is a mixture of simple phenolic compounds, including guaiacol and creosol and related compounds, and is chemically distinct from, and should not be confused with, coal tar creosote, a known carcinogen. In the current study, the oncogenic potential of wood creosote was assessed in a 96/103-week oral gavage study in Sprague-Dawley rats. Groups of 60 rats/sex received wood creosote at dose levels of 20, 50, or 200 mg/kg body weight [bw]/day. An additional group of rats received the vehicle, 0.5% carboxymethylcellulose in deionized, distilled water, at the same dose volume as the treatment groups (10 ml/kg) and served as the controls. Treatment-related decreases in survival, body weight, and food consumption, as well as increased incidences of clinical signs that included rales, decreased activity, and salivation, were noted at 200 mg/kg bw/day when compared with the control group. There was an increased incidence of reddened and edematous lungs in rats from the 200 mg/kg bw/day group that died during the study. The lung findings were suggestive of test article aspiration during dose administration or agonal aspiration preceding and possibly resulting in death, especially because these observations were not seen in animals that survived to scheduled sacrifice. Additionally, phenols are generally recognized as having corrosive properties. There were no changes in clinical pathology and no increases in neoplastic or non-neoplastic lesions, excluding the lung findings, related to treatment with wood creosote at any dose level. Although the results of this study indicate that the maximum tolerated dose of wood creosote was met or exceeded at 200 mg/kg bw/day, there was no evidence of oncogenicity at any dose level. The lack of any evidence of oncogenicity supports the safety profile of the active ingredient in Seirogan, wood creosote.

Hepatic copper accumulation induces DNA strand breaks in the liver cells of Long-Evans Cinnamon strain rats.

Effects of accumulation of copper and iron on the production of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) strain rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-copper food prevented the accumulation of copper in the liver, but did not prevent accumulation of iron. When the amounts of DNA single strand breaks were estimated by comet assay, the number of DNA strand breaks in the liver cells of rats fed standard food increased with age from 4 to 15 weeks. The number of DNA strand breaks in the liver cells from rats fed low-copper food were the same as those of rats at 4 weeks of age. Thus, the copper accumulation in the liver of LEC rats induced DNA strand breaks, but accumulation of iron did not.

Comparison of the antidiarrheal effects of wood creosote and loperamide in the rat jejunum and colon in vitro.

Wood creosote, a mixture of guaiacol, creosol and related compounds, has long been used as an antidiarrheal agent. The goal of our study was to investigate the antisecretory effect of wood creosote and to compare it to the effect of loperamide, a synthetic opioid widely used in the treatment of diarrhea. Experiments were performed in rat jejunal and colonic mucosal sheets, mounted in modified Ussing chambers. Active electrogenic transport was monitored electrically as short circuit current (Isc) and hypersecretory responses were induced by acetylcholine (ACh). Neither loperamide nor wood creosote had any significant effect on basal lsc, when added to the serosal bathing solution at concentrations of 0.1-50microg/ml. In contrast, under hypersecretory conditions, both agents showed concentration-dependent (0.1--100microg/ml) antisecretory effects inhibiting ACh-induced responses in the jejunum and colon. However, the effects suggest regional differences, with loperamide being most potent in the jejunum, while wood creosote showed equal potency in both jejunum and colon. Based upon these in vitro findings, we conclude that like loperamide, the antidiarrheal action of wood creosote is due, at least in part, to its antisecretory activity.

Anti-diarrhoeal effects of seirogan in the rat small intestine and colon examined in vitro.

BACKGROUND: Seirogan is a beechwood extract composed of guaiacol, creosol and other related phenolic compounds which is widely used as an anti-diarrhoeal agent in Asia. Abnormalities in water and electrolyte transport are often the cause of diarrhoea, but the mechanism of action of seirogan on small intestinal and colonic mucosal ion transport is unknown. AIM: To examine the effect of seirogan on electrogenic ion transport in vitro. METHODS: Sheets of rat jejunum and colon were mounted in Ussing chambers, and transmural potential difference (PD) was used as an electrical marker of changes in mucosal ion transport. Hypersecretory conditions were induced by acetylcholine (ACh). RESULTS: Serosal or mucosal application of seirogan (0.1-100 microg/mL) decreased basal jejunal transmural PD. Pre-treatment of the tissue with the neurotoxin, tetrodotoxin, did not inhibit the seirogan-induced changes in basal electrical activity. Seirogan had no effect on basal transmural PD in the ileum and colon. Under ACh-induced hypersecretory conditions in the small intestine and colon, addition of serosal or mucosal seirogan produced antisecretory effects determined indirectly by measurement of transmural PD. CONCLUSION: The ability of seirogan to decrease basal transmural PD in the jejunum, and inhibit the ACh-induced electrical responses, may contribute to its anti-diarrhoeal action.

Suppression of enterotoxin-induced intestinal fluid secretion by wood creosote.

Wood creosote suppresses intestinal fluid secretion induced by heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). When rabbit jejunum is ligated into a 5-cm segment and LT is administered locally, it actively induces intestinal fluid secretion in a dose-dependent manner. Local administration of wood creosote together with a fixed dose of LT suppressed the LT-induced fluid secretion in a dose-dependent manner. At a 50-ng/segment dose of LT, 7.4 +/- 1.1 ml (n = 5) of fluid is secreted into an intestinal segment; coadministration of wood creosote (150 micrograms/segment) significantly (p < 0.01) suppressed the fluid secretion to 2.4 +/- 2.3 ml. Based on these results, we conclude that the antidiarrheal activity of wood creosote is attributable to its antisecretory or proabsorptive effect (or both) on the intestine.

Effect of ethanol on the development and maturation of synapses in the rat hippocampus: a quantitative electron-microscopic study.

The effects of chronic ethanol administration on the development and maturation of synapses in the strata radiatum and lacunosum-moleculare of CA1 in the hippocampus were quantitatively examined in rats exposed to ethanol for the entire period of fetal life as well as the whole period of postnatal life. Synapse densities in the strata radiatum and lacunosum-moleculare of the ethanol-treated group were significantly lower than those of the control group at 2, 14, 21, and 70 days of age. However, the rates of density reduction did not change between either of the strata that receive different groups of afferent fibers. The ratio of axospinous to axoshaftic synapses also did not change between control and ethanol-treated groups. These data suggest that chronic administration of ethanol reduces the density of synapses in this area and that this effect is not specific to neither the type of afferent fibers nor the type of synapses.

[Effect of ethanol on the development and maturation of synapses in the rat hippocampus].

Rats of both sexes were exposed to ethanol during the entire period of the fetal life as well as the whole period of postnatal ages, and their brains were investigated at 2, 7, 14, 21, and 70 days postnatally. Control animals were examined at each age. Densities of synapses in the strata radiatum and lacunosum-moleculare of CA1 in the hippocampus were analyzed by quantitative ultrastructural techniques. Densities of all synapses in the strata radiatum and lacunosum-moleculare in ethanol-treated group were significantly lower than those of control group on 2, 14, 21 and 70 days. However, there were no significant differences in the ratio of axo-spinous to axo-shaftic synapses between control and ethanol-treated group. In addition, in regard to the rate of reduction in densities of all synapses, no change was detectable between both strata that receive different groups of afferent fibers. These data suggest that chronic administration of ethanol during the fetal and postnatal ages reduces the synaptic density in this area, and that this effect is neither specific to types of synapses nor to sorts of afferent fibers.

[The study of Chinese herbal medicinal prescription with enzyme inhibitory activity. VI. The study of makyo-kanseki-to with adenosine 3',5'-cyclic monophosphate phosphodiesterase].

A Chinese herbal medicinal prescription, Makyo-kanseki-to, was studied for the inhibitory activity of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase. The effect for the inhibitory activity of cAMP phosphodiesterase by combination with constituent crude drugs of the prescription was studied. Gypsum acted as a mitigatory component for Ephedra herb and Glycyrrhiza in cAMP phosphodiesterase inhibition test.

Thermally induced conformational transition of double-stranded xanthan in aqueous salt solutions.

The thermally induced conformational transition of double-stranded xanthans (degree of pyruvate substitution, DSp = 0.45) having Mw = 3.1, 5.7, and 20.3 x 10(5) has been studied in aqueous salt solutions by high-sensitivity differential scanning calorimetry (DSC). The double strandedness of these samples in the ordered conformation was ascertained by the value of mass per unit length, ML = 2090 +/- 270 g mol-1 nm-1, which was determined from the contour length obtained by electron microscopic observations and the molecular weight by light scattering measurements. The temperature at half completion of the transition T 1/2 for these samples increased linearly with the logarithm of the cation (Na+, K+) concentration. The plot of 1/T1/2 vs the natural logarithm of cation (Na+) concentration in mM for the sample with Mw = 5.7 x 10(5) (15-SX) yielded the equation 10(3)/T1/2 = 3.45-0.159 ln [Na+]. The specific enthalpy delta hcal for 15-SX, essentially independent of salt concentration above 20 mM, was 8.31 +/- 0.39 J/g (SD, n = 6). No systematic dependence of molecular weight on the transition temperature and the enthalpy was observed. Application of the Manning polyelectrolyte theory to the system using the DSC data suggested that the separation of the double strand of xanthan into two single chains was not completed at the temperature where the endothermic peak was finished. This suggestion is consistent with recent findings by light scattering measurements as a function of temperature. Our DSC study was extended to include four other samples from various sources. It was found that T1/2 and delta hcal depend on the pyruvate contents of the samples. For example, the t1/2 (t1/2/degrees C = T1/2/K - 237.15) values for samples with high pyruvate content (DSp = 0.9) and depyruvated (DSp = 0.14) in 20 mM aqueous NaCl were 48.8 and 85.3 degrees C, respectively. Two other samples showed relatively broad DSC curves having shoulders, which were resolved into two independent components. Thermodynamic parameters for each component were examined as a function of salt concentration, and the results obtained were interpreted in terms of the heterogeneity of the pyruvate content of the samples.

Inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase by flavonoids from licorice roots and 4-arylcoumarins.

Isoliquiritigenin, glabridin, licoarylcoumarin and licoricidin were identified as strong inhibitors of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase in waste materials which were obtained during the industrial extraction of glycyrrhizin from licorice roots. The structure-activity relationships of 12 flavonoids from licorice roots and 34 4-arylcoumarins were studied. In 4-arylcoumarins, 5,7-dihydroxy derivatives were generally highly inhibitory towards cAMP phosphodiesterase.

[The study of Chinese herbal medicinal prescription with enzyme inhibitory activity. IV. The study of the prescription containing mineral drug with adenosine 3',5'-cyclic monophosphate phosphodiesterase].

Twenty five Chinese herbal medicinal prescriptions containing gypsum, kaolin, longgu, oyster shell and sodium sulfate were studied for the inhibitory activity of adenosine 3',5'-cyclic monophosphate phosphodiesterase. The inhibitory activity of 15 prescriptions without mineral drug was higher than that of each original prescription. On the contrary, four were lower and six were not recognized to be different. All 11 prescriptions containing gypsum with an exception increased the inhibitory activity by removing gypsum. The half prescriptions containing kaolin or sodium sulfate also increased the inhibitory activity by removing the drug.

[The study on Chinese herbal medicinal prescription with enzyme inhibitory activity. II. The study of kanbaku-taiso-to with adenosine 3',5'-cyclic monophosphate phosphodiesterase].

A Chinese herbal medicinal prescription, Kanbaku-taiso-to, was studied for the inhibitory activity of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase. The effect for the inhibitory activity of cAMP phosphodiesterase in combination with constituent crude drugs and difference in kind of crude drugs of the prescription were studied. The inhibitory activity of the prescriptions which were combined with low protein content wheat were higher than that with high protein content wheat.