Biotransformation of flubendazole and selected model xenobiotics in Haemonchus contortus.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g., sheep and goat). The treatment of haemonchosis is complicated because of frequent resistance of H. contortus to common anthelmintics. The development of resistance can be facilitated by the action of drug metabolizing enzymes of parasites that can deactivate anthelmintics and thus protect parasites against the toxic effect of the drug. The aim of this project was to investigate the Phase I biotransformation of benzimidazole anthelmintic flubendazole in H. contortus and to determine the biotransformation of other model xenobiotics. For this purpose, in vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (live nematodes cultivated in flasks with medium) experiments were used. The results showed that cytosolic NADPH-dependent enzymes of H. contortus metabolize flubendazole via reduction of its carbonyl group. The apparent kinetic parameters of this reaction were determined (V'max=39.8+/-2.1 nM min(-1), K'm=1.5+/-0.3 microM). The reduction of flubendazole in H. contortus is stereospecific, the ratio of (-):(+) enantiomers of reduced flubendazole formed was 90:10. Reduced flubendazole was the only Phase I metabolite found. Effective reduction of other xenobiotics with carbonyl group (metyrapon, daunorubicin, and oracin) was also found. Significant activity of carbonyl-reducing enzymes may be important for H. contortus to survive the attacks of anthelmintics or other xenobiotics with carbonyl group.

Survey of Shiga toxigenic Escherichia coli O157 and drug-resistant coliform bacteria from in-line milk filters on dairy farms in the Czech Republic.

AIMS: To determine the occurrence of Shiga toxin-producing Escherichia coli (STEC) O157 and coliform bacteria isolates resistant to antimicrobial agents in dairy herds by examining milk filters and to analyse the influence of management factors and antibiotic use on antimicrobial resistance. METHODS AND RESULTS: A total of 192 in-line milk filters were sampled on 192 dairy farms in the Czech Republic. Information on feeding, husbandry, production, and antibiotic therapy were obtained by questionnaire. The milk filters were cultured for STEC O157 and coliform bacteria. All recovered isolates were examined for antimicrobial susceptibility and presence of antimicrobial-resistance genes. STEC O157 was detected in four (2%) of the filters. Resistant nonpathogenic E. coli and coliform bacteria isolates with specific genes were detected in 44 (23%) of the filters. CONCLUSIONS: The study demonstrated a high prevalence of resistant coliform bacteria in milk filters obtained on Czech dairy farms. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence of resistant coliform bacteria in milk filters was significantly higher among isolates from farms where antibiotic therapy against mastitis was employed during the dry period (P < 0.05).

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells.

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland.

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4-9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) which reflects PI cycle activation. AVT (10(-5) and 10(-4) M) induced a significant increase in 32P labelling of PI, PIP and PIP2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V1 receptor antagonist. The desmopressin and AVP 4-9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 microg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Stimulatory effect of bombesin on phosphoinositide metabolism in the rat pineal gland.

The pineal gland is under complex peptidergic nervous control originating from hypothalamic nuclei. The daily rhythm of bombesin-like peptide in the hypothalamus suggests that this neuropeptide, similarly as other neuropeptides, might be involved in modulation of the physiological activity of the pineal gland. In our experiments we studied the mechanism of signal transduction of bombesin in the isolated pineal glands of rats. The phosphoinositide signalling system was examined by measuring 32P-labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), which reflects phosphoinositide cycle activation. Bombesin induced a significant increase in 32P-labelling of PI, PIP and PIP2. The antagonist of this neuropeptide, (D-Phe12-Leu14)-bombesin, suppressed the increase in 32P-labelling of all phosphoinositides. Bombesin was without effect on cAMP dependent protein phosphorylation. The data indicate that bombesin activates the PI signalling system via specific receptors.

Arginine vasopressin stimulates 32P labeling of phosphoinositides in rat pineal gland.

The peptidergic innervation of the pineal gland contains arginine vasopressin (AVP) fibers. Since the site and molecular mechanism of AVP action on the pineal signal transduction has not yet been determined, we examined whether the inositol phosphate transduction system is involved. The phosphoinositide signaling system was studied by measuring metabolic turnover of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), which reflects phosphoinositide cycle activation. AVP induced a significant increase in 32P labeling of PI, PIP and PIP2. The V1 receptor site(s) blocker for AVP abolished enhanced 32P labeling of PIP and PIP2. AVP was without effect on cAMP dependent phosphorylation. The data indicate that AVP activates the phosphoinositide signaling system via V1 receptors and is without effect on the cAMP system.

Phosphoinositide metabolism of rat pineal gland in a low ionic strength medium.

Pineal glands were incubated in the presence of 32P orthophosphate. When all NaCl in a conventional incubation medium was replaced by isotonic sucrose, i.e. when the ionic strength of the medium was decreased, there was a marked increase in 32P labelling of phosphatidylinositol (PI) and phosphatidic acid (PA). The 32P labelling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was not affected. No net synthesis of PI was observed. The increased labelling of PI therefore represents an increase in the turnover of PI. The 32P labelling of PI was observed also in media where NaCl was replaced by fructose or mannitol, but not in media, where NaCl was replaced by choline chloride. The effect depends on the concentration of the HEPES buffer and was not found in the medium with a bicarbonate buffer. 32P labelling of PI was not blocked by alpha 1 adrenergic blockers, phentolamine and prazosin, and did not depend on the presence of Ca2+ in the incubation medium. The effect was blocked by a Ca2+ channel blocker, MnCl2. Only 32P labelling of PI and not that of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was increased during prolonged incubation in the sucrose medium. It is suggested that a decrease in the charge distribution across the plasma membrane as a result of the absence of most monovalent cations is responsible for the increased metabolism of phosphatidylinositol.

The effect of chlorpromazine and dibucaine on phospholipid metabolism in the frog sartorius muscle.

The effect of cationic amphiphilic drugs, chlorpromazine and dibucaine, on phospholipid metabolism in the frog sartorius muscle was studied at concentrations affecting excitation-contraction coupling. Labelling patterns of phospholipids using 32P orthophosphate were determined. The drugs at a concentration of 3.10(-4) mol/l inhibited the synthesis of phosphatidylcholine and phosphatidylethanolamine and stimulated the synthesis of phosphatidylinositol and phosphatidylserine. At the 3.10(-3) mol/l concentration the drugs blocked the synthesis of all phospholipids without appreciably affecting their degradation. The effect of cationic amphiphilic drugs was independent of the presence of Ca2+ or K+ ions in the media, however, basal labelling of phospholipids was affected by withdrawal of Ca2+ or K+ ions.

K+ depolarization and phospholipid metabolism in frog sartorius muscle.

K+ depolarization evokes phosphatidylinositol response, i.e. the increased 32P orthophosphate labelling of phosphatidylinositol in frog sartorii muscles. The phosphatidylinositol response seems to be closely related to K+ depolarization and not to the transient Ca2+ release at the beginning of depolarization. It ceases as soon as the muscles depolarized by 90 mmol/l KCl for a short period of time are repolarized, while it continues when the depolarization is maintained. When the muscles are depolarized with 20 mmol/l KCl, the phosphatidylinositol response is also observed. This response is not suppressed by drugs that block Ca2+ mobilization. Other agents like caffeine, azide or EGTA which induce some effects similar to that of K+ depolarization, do not evoke phosphatidylinositol response. Rather, they simply cause a decrease in the labelling of phospholipids, phosphatidylinositol being the least affected. In muscles derived from frogs maintained under healthy conditions Ca2+ release in the early phase of K+ depolarization does not cause significant changes in phospholipid labelling. However, in muscles from frogs starving for many months, a large decrease in the labelling of phospholipids is observed in the early phase of K+ depolarization. It is postulated that the changes in the physicochemical state of the membrane and not Ca2+ gating mechanism or free cell Ca2+ level are crucial in the phosphatidylinositol response in the frog sartorii muscles depolarized by high K+.

Effect of different environmental temperatures on the serotonin concentration and turnover in the brain stem of a hibernator.

The serotonin (5-HT) and 5-hydroxyindoleacttic acid (5-HIAA) levels and 5-HT turnover were studies in the brain stem of warm- (+30 degrees C) and cold- (+6 degrees C) acclimated golden hamsters, exposed for 3 hours to temperatures of +6 degrees C, +30 degrees C and +37 degrees C, respectively. In war-acclimated hamsters kept under conditions the 5-HT level in the brain did not change significantly during the year. The 5-HIAA level was slightly higher in the winter. The 5-HT turnover varied within limits of 0.071 to 0.180 mug/g/hour-1. Three hours' exposure of warm-acclimated golden hamsters to cold (6 degrees C) increased the concentrations of 5-HT and 5-HIAA and the 5-HT turnover in the brain. After long-term adaptation to cold (6 degrees C) the 5-HT level, and the 5-HT turnover returned to the original level. Three hours' exposure of golden hamsters to higher environmental temperatures (warm-acclimated individuals to 37 degrees C and cold-acclimated individuals to 30 degrees C) also increased the 5-HT turnover. The concentrations of 5-HT and 5-HIAA increased in cold-acclimated golden hamsters exposed to 30 degrees C and was not changed in warm-acclimated ones, exposed to 37 degrees C. Although the elevated temperatures induce greater changes in serotonin metabolism than lowered temperatures, the serotonin pathways in the brain do not seem to be affected by short-term temperature changes specifically. The findings are rather indicative that changes in 5-HT turnover may be the primary reaction to stressful conditions.