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Application of real-time PCR using Taqman probe was tested for jaagsiekte sheep retrovirus (JSRV) detection. Sensitivity of real-time PCR and hemi-nested PCR methods was compared using plasmid DNA. The methods, along with RT PCR and real-time RT PCR, were tested for the possibility of JSRV genome (LTR region) detection in biological material from experimentally and naturally infected sheep. The experimental group of eight animals was used, including five lambs infected with JSRV by intratracheal inoculation at the age of 2 weeks. The samples collected from the animals ante-mortem included blood and respiratory tract fluid. Lung tissue, mediastinal lymph nodes, spleen, and liver were collected post-mortem. The field studies included blood samples collected from sheep from Polish flocks and lung samples obtained from slaughterhouse. In addition, DNA samples isolated from blood of sheep from the abroad located flocks with history of ovine pulmonary adenomatosis (OPA) were also included. Lung samples were examined histologically for the presence of pulmonary adenocarcinoma. The sensitivity of PCR, hemi-nested PCR, and real-time PCR using Taqman probe was evaluated as 10³, 10², and 10² viral copies, respectively. Both viral RNA and DNA were detected in the lung fluid taken from JSRV infected sheep showing clinical sings of OPA and in all neoplastic tissues. Proviral DNA was found in mediastinal lymph node of one experimental sheep. Five of the 66 DNA samples from the abroad located farms were positive for the presence of JSRV LTR. All blood and lung samples collected from Polish sheep were negative for the presence of JSRV LTR. The characteristic adenocarcinoma lesions were found in all lung sections of experimentally infected sheep. Implementation of the real-time PCR method is a good alternative to traditional PCR and hnPCR in JSRV detection and, apart from histopathological and immunohistochemical examinations, may be used as a confirmatory test in clinically suspected cases, or as a screening method in control or eradication scheme.
Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). In the present study the protein profiles of five neoplastic and three non-neoplastic sheep lung tissues were examined for the identification of proteins overexpressed in ovine pulmonary adenocarcinoma. Lung sections of the experimental group of sheep were collected during necropsies for proteomic and immunohistochemical examination. Two dimensional electrophoresis (2DE) was performed using gel strips with immobilized pH gradient 3-10. As a result of 2DE gel analysis 14 spots characterized by over 2-fold higher expression in tumour proteomes were selected for mass spectrometry. In eleven spots more than one polypeptide was identified indicating overlapping of proteins in gels. In two spots demonstrating over 3-fold higher expression in OPA proteomes, single proteins: cytokerarin 19 (CK19) and aldolase A were identified. Immunohistochemical studies revealed that CK19 and aldolase A were expressed in the cytoplasm of epithelial cells of bronchioles in non-neoplastic lung sections, as well as epithelial cells of bronchioles and neoplastic cells in lung sections of OPA affected sheep. The results indicate that the overexpression of the two proteins reflects the presence of neoplastic cells in the lungs of OPA affected sheep.
Frozen lung tissue sections from 2 healthy and 2 adenocarcinoma affected sheep were lysed in appropriate buffer. The two-dimensional (2D) electrophoresis of the protein lysates was performed. The resulting gels were visualised by silver and Coomassie Blue staining, then scanned and analysed using appropriate software. There was one spot present on the image obtained from the analysis of healthy tissue and no spot was found on cancer 2D gel image. The spot was excised and analysed using mass spectrometry. As a result, cytosolic NADP-isocitrate dehydrogenase was identified. In addition, several other protein spots of different intensity in neoplastic tissues, as compared with healthy ones, were found. The last finding reflects changes in protein expression in neoplastic and healthy tissues. These preliminary results can serve as the basis for more detailed investigations of the neoplastic tissue proteome, e.g.: isoelectric focusing in narrow pH range and analysis of correlation between tissue and serum protein profiles. The analysis of serum proteins from affected sheep can reveal markers of neoplastic process and help in preclinical diagnosis of ovine pulmonary adenocarcinoma.
The possibility of jaagsiekte sheep retrovirus (JSRV) genome detection in peripheral blood leukocytes and respiratory tract fluid of ovine pulmonary adenocarcinoma (OPA) affected sheep was tested. Five of six lambs used in the experiment were infected with JSRV by intratracheal inoculation. The blood samples were taken from all the lambs at 10-d intervals for 3 months and the white blood cells were subjected to DNA isolation followed by PCR amplification for the detection of proviral DNA that gave negative results for all the samples. The respiratory tract fluid was collected from the nostrils of lamb No. 6, which developed clinical sign of OPA 2 months after the inoculation. The fluid was examined using PCR and reverse transcriptase PCR (RT-PCR) for the presence of proviral DNA or viral RNA. The presence of OPA in the lamb was subsequently confirmed by histopathological examination and the detection of proviral DNA in the lung tissue. Results of the standard PCR amplification performed on the DNA isolated from the nasal discharge from lamb No. 6 was negative, while the RT-PCR gave positive results confirming the presence of virions in the lung fluid. Results of the study show the RT-PCR technique may be a useful tool in ante-mortem diagnosis of OPA, especially in distinguishing it from other respiratory diseases.
The importance of magnesium supplements on organ retention of cadmium and allometric parameters after repeated exposure to cadmium chloride were studied in male Wistar rats. Magnesium chloride was given via drinking water (500 mg Mg/L) to rats exposed intragastrically to cadmium chloride (labelled with cadmium 109) at a daily dose corresponding to 25 mg/kg diet for 7, 14, 21, and 28 d. Supplements of magnesium temporarily decreased cadmium retention in the duodenum and liver. No significant differences in cadmium retention were evidenced in the kidneys and testicles. The supplements of magnesium also retain more of the body weight gains and restore the relative liver and testicle weight in rats intoxicated with cadmium. Comparison of the present results with earlier reports suggests a relationship between doses of magnesium and cadmium; higher doses of cadmium need more magnesium to overcome toxic action of the heavy metal.
Samples of morbid bovine tissues obtained from slaughterhouse were submitted to histopathological examination. Neoplastic growth within the peripheral nerves was observed microscopically, with the presence of Antoni A and Antoni B patterns, and sporadically Verocay bodies. Additionally, immunohistochemical examination of the sections revealed positive reaction to vimentine, neuron specific enolase, protein S100, and glial fibrillary acidic protein. The results indicate that the examined neoplasms may belong to the type of benign peripheral nerve sheath tumours called schwannoma.
Histopathological examination of liver, kidney, spleen, pancreas, duodenum, jejunum, skeletal muscle, and bursa of Fabricius samples, collected from broiler chickens, laying hens, fattening pigs, and calves fed genetically modified corn MON 810 and soybean meal MON-40-3-2 (Roundup Ready, RR), was performed. The examination showed no significant differences between the control animals fed diets containing no genetically modified feeds and animals fed genetically modified feeds. In some cases, congestion of parenchyma and focal lymphoid cell infiltrations were observed in all dietary groups, including controls, and therefore, it was assumed that the lesions were not associated with the feeding transgenic feeds.
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