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Objective To isolate and culture the primary cerebellar granulosa cells(CGNs)of SD rats and evaluate the activity of botulinum neurotoxin A(BoNT/A)based on CGNs.Methods CGNs of 6-to 8-days-old SD rats were isolated and cultured.After 5-7 d,the cells were treated with BoNT/A.The activity of the toxin was evaluated with immunofluo-rescence,and the relationships between the activity and dose of the toxin were analyzed with Western blotting.Results and Conclusion CGNs Of SD rats were successfully cultured,a method for evaluating the activity of BoNT/A was established at the level of primary nerve cells,and the relationships between the activity and dose of toxin were analyzed.This study provides a tool for further detailing the biochemical mechanism of BoNT /A.
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Objective To construct EHEC△z5150 using Red recombination and explore its pathogenicity.Methods Overexpression of the z5150 gene was used to test its toxicity.The kanamycin resistant gene flanked by homologues of target genes was amplified by PCR before being transferred into EHEC containing pKD 46 plasmid,and the target fragments were recombined into genome by Arabia sugar.The kanamycin resistance gene was removed by pFLP 2 plasmid mediated FLP site-specific recombination.Cell adhesion ability and virulence of the mutant were detected by infecting HT 29 cells and BALB/c female mice,respectively.Results and Conclusion The mutant strain EHEC △z5150 was successfully constructed.The absence of z5150 did not affect the normal growth of bacteria,but after excessive expression,it inhibited the growth.Deletion of z5150 showed significant difference in intestinal epithelial cell adhesion and pathogenicity compared with wild strain.This study provides a gene knockout technique for the study of EHEC gene function and of the relationships between RelEz5150 and EHEC pathogenicity.
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<p><b>OBJECTIVE</b>To study the immunohistochemical expression of OCT4, CD117 and CD30 in germ cell tumors and to assess their diagnostic value.</p><p><b>METHODS</b>Immunohistochemical study for OCT4 was performed on formalin-fixed, paraffin-embedded tissues of 63 cases of germ cell tumors, including seminoma (21), dysgerminoma (7), germinoma (8), embryonal carcinoma (8), yolk sac tumor (6), mature teratoma (10) and immature teratoma (3), as well as 25 cases of non-germ cell tumors, including granulosa cell tumor (8), clear cell adenocarcinoma (4), Leydig's cell tumor (5), diffuse large B-cell lymphoma (4) and malignant melanoma (4). Besides, the expression of CD117 and CD30 in all germ cell tumors was studied.</p><p><b>RESULTS</b>All cases of seminoma and germinoma, 6/7 cases of dysgerminoma and 7/8 cases of embryonal carcinoma were positive for OCT4, with strong nuclear staining. All other germ cell tumors and non-germ cell tumors were negative for OCT4, except for 1 case of yolk sac tumor and 1 case of clear cell adenocarcinoma which showed weak staining. Positive membranous expression of CD117 was demonstrated in 19/21(90.5%) seminoma, 5/7 dysgerminoma and 7/8 germinoma. Focal weak membranous staining was also noted in 1 case of yolk sac tumor. The melanocytes in teratoma were also positive for CD117. All cases of embryonal carcinoma were negative. On the other hand, positive membranous expression of CD30 were demonstrated in 6/8 embryonal carcinoma. One case of germinoma and 1 case of yolk sac tumor showed weak cytoplasmic positivity. All cases of seminoma and dysgerminoma, 7/8 germinoma and all cases of teratoma were negative for CD30.</p><p><b>CONCLUSIONS</b>OCT4 is a sensitive and relatively specific marker for diagnosing seminoma, dysgerminoma, germinoma and embryonal carcinoma. CD117 and CD30 immunostains, when used in combination, represent valuable tools for distinguishing embryonal carcinoma and seminoma, dysgerminoma, germinoma.</p>