Detection and localization of two constitutive NOS isoforms in bull spermatozoa.

Bull spermatozoa were examined for the presence and localization of constitutive Nitric Oxide Synthase (NOS), as nitric oxide (NO) is involved in calcium-dependent capacitation. In bull spermatozoa, NO generation is enhanced by l-arginine (3 microm) and abolished by the NOS-inhibitor N-nitro-l-arginine methyl ester (l-NAME). In addition, presence of NOS in bull spermatozoa was verified by immunohistochemistry, revealing the existence of both neuronal NOS (nNOS) and endothelial NOS (eNOS) immunoreaction. These findings were confirmed by Western blot technique, showing immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS). Confocal laser microscopy localized nNOS related immunofluorescence at the acrosome cap of sperms and their flagellum-mainpart. This technique also identified eNOS staining spread over the spermatozoan head. In conclusion, immunohistochemistry, Western blot technique, and NO generation suggest the presence of n- and eNOS in bull spermatozoa.

Atypical time course of clinical signs in a dog poisoned by strychnine.

The general health of a German shepherd dog had deteriorated slightly when it was found after being loose for one hour. After 10 hours of observation, the dog showed signs of pain for the first time and signs of poisoning, such as tenseness of muscles, slight opisthotonus, regurgitation, salivation, mydriasis, dyspnoea and cyanosis, were observed; it died 15 minutes after showing the first clinical signs but it had no seizures or tetanic spasms at any time. A postmortem examination did not reveal any pathological changes. A screening test for alkaloids was positive for strychnine (strychnidin-10-one). The presence of strychnine was confirmed and its concentration was determined by gas chromatography/mass spectrometry in urine (728.5 ng/ml) and in the stomach contents (44.6m microg/g). No strychnine was detected in the dog's serum, but traces of brucine (2,3-dimethoxystrychnidin-10-one), the dimethoxy derivative of strychnine, were detected. This case was compared with other strychnine poisonings recorded in the authors' laboratory over the previous six years, taking into account the species, type of samples, the clinical signs and their duration, the postmortem findings, and the concentrations of strychnine. This was the only case to show such an atypical time course of clinical signs.

Lipopolysaccharide receptor CD14 polymorphism and risk of stroke in a South-German population.

BACKGROUND: The monocyte receptor for bacterial lipopolysaccharide CD14 is an important mediator of the inflammatory response. Recently, a polymorphism in the promotor of the CD14 gene, C (-260) T, was detected as a risk factor for coronary heart disease. OBJECTIVE: We tested the hypotheses that this polymorphism is a risk factor 1. for cerebral ischemia in general and 2. for cerebral ischemia due to large artery atherosclerosis or microangiopathy in particular. PATIENTS AND METHODS: We performed a case control study including 151 consecutive patients with acute cerebral ischemia treated at our university hospital and 149 control subjects. All control subjects were randomly selected from the general population of the same region in South-West Germany. Genotype frequencies of the C(-260) T polymorphism in the promotor of the CD14 gene were examined by restriction length analysis. RESULTS: The TT-genotype was not associated with cerebral ischemia in general either in univariate or in multivariate analysis together with classical vascular risk factors (odds ratio in multivariate analysis: 1.11; 95 %CI, 0.63 to 1.95). In 70 patients with cerebral ischemia due to atherosclerosis of large arteries or microangiopathy, there was a significantly higher prevalence of the TT-genotype than into control subjects (38.6 % vs. 23.5 %; odds ratio in multivariate analysis 2.26; 95 %CI, 1.13 to 4.54). CONCLUSIONS: We demonstrated that the TT-genotype of the CD14 C(-260) T polymorphism in a South-German population is not associated with an increased risk of cerebral ischemia in general. However, we found that the TT-genotype is associated with a risk of atherosclerotic or microangiopathic stroke. This finding requires confirmation by future studies in larger populations.

Increased cytokine release by leucocytes in survivors of stroke at young age.

BACKGROUND: Enhanced stimulus-induced release of pro-inflammatory cytokines by leucocytes may contribute to the pathogenesis of ischaemic stroke. DESIGN: We investigated the lipopolysaccharide-induced release of interleukin-1beta (IL-1beta), IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) in whole blood from 20 patients with a history of ischaemic stroke under the age of 50, 20 patients with a history of cervical artery dissection (CAD) and 21 age- and sex-matched healthy control subjects. RESULTS: Release of IL-8 was higher (P = 0.006) and release of TNF-alpha and IL-6 tended to be higher (P < 0.1) in young stroke patients than in control subjects. No increased release existed in CAD patients. Vascular risk factors or history of infection before stroke did not modify IL-8 production. A common T(250) --> A polymorphism in the IL-8 gene promotor was newly identified but did not correlate with the variability of IL-8 release. The C(260) --> T polymorphism in the gene of the monocytic LPS-receptor CD14--a risk factor for myocardial infarction--was not associated with increased cytokine release. CONCLUSIONS: We conclude that high inducible release of IL-8--and possibly of TNF-alpha and IL-6--may contribute to the odds of ischaemic stroke in young adults.

Elimination of doxepin isomers from the horse following intravenous application.

The tricyclic antidepressant doxepin, representing a 5:1 mixture of trans- and cis-isomers, owns tranquilizing properties. This compound has been associated with illicit medication of racing horses, and therefore should be considered in doping control. Because analysis of doxepin in equine body fluids has not been documented in the literature, a highly sensitive analytical method was developed to individually monitor the doxepin isomers in blood and urine of horses by the use of gas chromatography/mass spectrometry. Following a dose of 1 mg doxepin-HCl/kg intravenously (i.v.), both the isomers were quantified for up to 24 h in serum of horses (n=4). The beta-half-lives of the trans- and cis-isomers were 3.5 and 3.1 h, respectively. The ratio of the trans/cis-isomers was found to be constant (4.7:1) during drug elimination and thus corresponded to the original composition of the antidepressant. Up to 12 h following administration low trans-isomer concentrations in an average range of 2-6 ng/mL were detected in urine of each of the horses, while the cis-isomer was only present in two of four horses for up to 8 and 12 h, respectively. In serum, mean trans-isomer concentrations exceeded urine levels maximally 120-fold after 3 h and at least sixfold after 12 h. As serum exhibits considerably higher concentrations of the doxepin isomers as compared with urine, blood of horses is the recommended body fluid when screening for the antidepressant.

[Detection of levamisole and ivermectin in organ samples from a dead collie]. / Nachweis von Levamisol und Ivermectin in Organproben eines verendeten Collies.

A collie, known for its breed-dependent adverse reaction to ivermectin, was without any clinical signs. The dog was prophylactically treated with 3 mg/kg KG (s.c.) of levamisole. Within 15 minutes, the dog showed convulsions, vomitus, and dyspnea, and perished 2.5 hours after injection of the drugs. The pathological findings were not informative as to the cause of death, and with regard to the adverse reactions, additional application of ivermectin was not excluded. Therefore, organ samples were submitted for toxicological analysis of both levamisole and ivermectin. For detection of levamisole and ivermectin, modified GC/MS and HPLC procedures were developed. Concentrations up to 535 micrograms levamisole and up to 26 ng ivermectin were found per g tissue. Both analytical methods are sensitive enough to detect these drugs after application of low doses. This study elucidates that combination of low-dosed ivermectin and levamisole is no recommendable means against adverse effects of ivermectin, with respect to collies. Moreover, the synergistic effects of ivermectin and levamisole suggests the same drug incompatibility in other dog breeds and animal species.

Cotinine effects on bile flow and biliary NNK elimination.

Nicotine and its major metabolite cotinine inhibit alpha-hydroxylation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) suggesting that an alternative pathway of NNK metabolism and elimination, biliary excretion of the O-glucuronide of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL-Gluc) may be enhanced. To verify the possible role of cotinine on biliary elimination of NNK and its metabolites, bile duct cannulated rats were administered a single i.p. dose of 50 mg/kg [56sup;-3 H]-NNK with or without i.p. co-administration of 5 mg/kg cotinine or nicotine. Cotinine significantly reduced cumulative bile flow and biliary elimination of NNK-derived radioactivity within six hours to 42 and 27 percent, respectively. The pattern of NNK metabolites in bile was unchanged. Nicotine had a similar inhibitory effect on bile flow. This result constitutes the first experimental evidence that cotinine inhibits bile flow. In rats, biliary elimination of NNK is reduced accordingly which may lead to an increased carcinogen burden in the body. In humans, inhibition of bile flow by tobacco alkaloids may contribute to the appetite suppressing effect of tobacco products.

Development of a method for determination of cyanide concentrations in serum and rumen fluid of cattle.

OBJECTIVES: To develop a reliable method for measurement of cyanide concentrations in cattle, using gas chromatography-mass spectrometry (GC-MS), and establish reference ranges of cyanide concentrations in cattle. ANIMALS: 52 Fleckvieh cattle. PROCEDURE: Cattle were allocated to 3 groups; 12 were fed leguminous grass and hay, 36 were fed whole-maize and corn-cob silages, and 4 were fed other feedstuffs. Samples of blood, rumen fluid, and liver were collected at time of slaughter. Serum, rumen fluid, and liver homogenate were assayed for cyanide content, using a derivatization procedure. A technique for analysis by GC-MS that used selected ion monitoring was developed. RESULTS: Compared with a spectrophotometric method, detection of cyanide in serum and rumen fluid by use of GC-MS was selective and sensitive, with a limit of detection of 0.7 microM. Spectrophotometric analysis yielded false-negative and false-positive results. Thus, the GC-MS method was used for subsequent analysis. In all cattle except 1, cyanide concentration ranged from < 0.7 to 35 microM in serum and from < 0.7 to 28 microM in rumen fluid; cyanide concentration in that 1 animal was 206 microM. Cattle fed clover, grass, grass hay, and clover hay had 8.3- to 8.6-fold higher mean cyanide concentrations in rumen fluid and serum than cattle fed whole-maize and corn-cob silages. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study suggest a reference range that should be useful for aiding in the diagnosis of cyanide poisoning. Also, cattle can apparently accommodate a serum cyanide concentration of 206 microM without adverse effects.

[Pyrogallol concentrations in rumen content, liver and kidney of cows at pasture]. / Pyrogallolkonzentrationen in Panseninhalt, Leber und Niere von Weidekühen.

Pyrogallol (1, 2, 3-trihydroxybenzene), the decomposition product of hydrolysable tannins in oak bark, leaves and acorns, is suspected to be poisonous to animals. The aim of our investigations was to correlate clinical signs and pathological findings with pyrogallol concentrations in organs of poisoned and healthy animals. In a field study, pyrogallol concentrations were determined in liver, kidney, and rumen from seven cattle. In a herd of twelve cows, five animals suffered from hemorrhagic diarrhea, anorexia, weakness, rumen stasis, dyspnoea, and colic symptoms. Death was observed in five cows within five weeks after repeated intake of green acorns and oak leaves. Toxicological analyses of rumen content, liver, and kidney specimens of one cattle confirmed the suspicion of pyrogallol contamination. In this animal, values ranged from 6 to 13 ng pyrogallol per gram specimen. In control cattle, concentrations were clearly lower than in perished cattle. Under antioxidative work-up conditions, detection limit was 0.6 ng/g in rumen content and 1.0 ng/g in liver and kidney, respectively.

Pyrogallol poisoning of pigeons caused by acorns.

Green acorns are known to contain high concentrations of pyrogallol. Here, we describe an extended case report of two pigeons found dead with a filled muscular stomach of acorns. The following pathologic findings were observed: irritation of mucosal membranes in the gastrointestinal tract, blackish discolored chyme, hyperemic organs, and general edemas. The muscular stomach (ventriculus) was filled with pieces of acorns, and the abdominal cavity contained bloody aqueous fluid. In order to uncover the cause of death, we determined pyrogallol in liver and kidney of one dead pigeon and in ventriculus contents of both pigeons by gas chromatography/mass spectrometry. A further aim of our study was to compare pathologic findings and pyrogallol concentrations in kidney, liver, and ventriculus of poisoned pigeons with those of healthy pigeons. The pyrogallol concentrations in samples of dead pigeons were 16-1200-fold higher than in control animals fed grass and maize-corn. Altogether, the acorn-filled ventriculus, the pathologic findings, the well nourished state, and the high pyrogallol concentrations in the dead pigeons suggest an acute pyrogallol poisoning by acorn. With respect to controls, we conclude that pyrogallol concentrations of 6 ng/g of kidney, 8 ng/g of liver, and 2 ng/g of gastric content do not affect the health of pigeons.

Methadone screening of racehorses.

The misuse of opiates in racehorses relates to their effect of increasing locomotor activity. Because methadone, a narcotic analgesic, has been suspected of use as a doping compound in the past, it was added to the list of banned drugs and should be considered in doping control. Because the literature fails to provide information on detection of methadone in blood or urine of horses, an enzyme-linked immunosorbent assay was developed to monitor this narcotic in equine body fluids. Combined with high-performance liquid chromatography, the immunoassay also served to confirm positives indicated by screening. Following intravenous administration of methadone (0.1 mg/kg), the drug was found for up to 24 h in blood but was never identified in urine (10-pg/mL detection limit). Thus, urine is dismissed as a source of methadone control, and the use of blood to screen racehorses for this narcotic analgesic is suggested.

[Zinc intoxication dogs with simultaneous zinc deficiency--an expanded case study]. / Zinkintoxikation eines Hundes bei gleichzeitigem Kupfermangel--eine erweiterte Fallstudie.

A dachshund suffered from vomitus, anorexia, apathy, and anemia. A metal button consisting of brass (alloy of copper and zinc) was diagnosed in the stomach and was removed. Blood analysis revealed an elevated level of zinc (10.7 micrograms/ml) associated with a low copper concentration (0.02 microgram/ml). This apparently paradoxical finding of copper deficiency despite of copper exposition (brass button) caused us to determine the diaminoxidase activity in serum, representing an indicator for the copper accumulating ceruloplasmin. The enzyme activity was found to be decreased. The case study brings to mind the mutual interaction between zinc and copper. Apparently, a copper deficiency is not necessarily due to a reduced supply of copper. Moreover, as shown by further investigations, an attenuated diaminoxidase activity represents an important parameter to diagnose a copper deficient state.

[Psychotropic drugs and glaucoma (author's transl)]. / Psychopharmaka und Glaukom

The i.o. pressure and tonography were studied on 51 patients with endogenous depressions. The patients were treated 10-43 days with psycho-pharmacological drugs (Indoline, Imipramine, Amitriptyline, Noxiptiline and Phenothiazine). In no case does the therapy with the drugs induce an increase of i.o. pressure or-in normal eyes-of tonography. In latent of manifest glaucoma (7 patients) Clomipramine increased the tonography in 1 case, whereas Amitriptyline and Noxiptiline reduced it in 4 cases. The study demonstrated that the commonly used psycho-pharmaca are no contraindication to wide-angle glaucoma.