Specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase do not change the in vitro release of TRH or its content in rodent brain

Pyroglutamyl diazomethyl ketone and N-benzyloxycarbonyl prolyl prolinal, specific inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase respectively, were used to study the possible role of these enzymes in the regulation of thyrotropin releasing hormone turnover. In vitro thyrotropin releasing hormone release by male rat hypothalamic slices was studied. Combined in vitro treatment with 10(-5)M of both inhibitors totally inhibited both enzymatic activities. The treatment did not affect basal or 56 mM K+ induced thyrotropin releasing hormone release or thyrotropin releasing hormone levels in slices. Repeated combined intraperitoneal injections of the two inhibitors for up to 12 hours produced a 70%-95% reduction in mouse brain pyroglutamyl peptidase I specific activity and a 65%-85% reduction in prolyl endopeptidase specific activity. Thyrotropin releasing hormone levels were unaffected by this treatment in all regions tested. The data suggest that these two enzymes are not involved in the intra- or extracellular control of thyrotropin releasing hormone levels in brain or hypophysis.     

Laser capture microdissection of cervical human papillomavirus infections: Copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation

Research on the pathogenicity of human papillomaviruses (HPVs) during cervical carcinogenesis often relies on the study of homogenized tissue or cultured cells. This approach does not detect molecular heterogeneities within the infected tissue. It is desirable to understand molecular properties in specific histological contexts. We asked whether laser capture microdissection (LCM) of archival cervical tumors in combination with real-time polymerase chain reaction and bisulfite sequencing permits (i) sensitive DNA diagnosis of small clusters of formalin-fixed cells, (ii) quantification of HPV DNA in neoplastic and normal cells, and (iii) analysis of HPV DNA methylation, a marker of tumor progression. We analyzed 26 tumors containing HPV-16 or 18. We prepared DNA from LCM dissected thin sections of 100 to 2000 cells, and analyzed aliquots corresponding to between nine and 70 cells. We detected nine to 630 HPV-16 genome copies and one to 111 HPV-18 genome copies per tumor cell, respectively. In 17 of the 26 samples, HPV DNA existed in histologically normal cells distant from the margins of the tumors, but at much lower concentrations than in the tumor, suggesting that HPVs can infect at low levels without pathogenic changes. Methylation of HPV DNA, a biomarker of integration of the virus into cellular DNA, could be measured only in few samples due to limited sensitivity, and indicated heterogeneous methylation patterns in small clusters of cancerous and normal cells. LCM is powerful to study molecular parameters of cervical HPV infections like copy number, latency and epigenetics.     

Proliferative ischaemic retinopathy in a case of systemic lupus erythematosus and antiphospholipidic syndrome

PURPOSE/METHODS: We report a 34-year-old woman with sytemic lupus erythematosus (S.L.E.) and antiphospholipidic syndrome which presented severe ischaemic retinopathy and neovascular proliferation. RESULTS/CONCLUSIONS: We achieved no progression of the disease and stabilization of visual acuity with panphotocoagulation. We recommend periodic ophtalmologic exams because of the scarce symptomatology.     

Involvement of protein kinase C and Na+/K+-ATPase in the contractile response induced by myricetin in rat isolated aorta

The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.NA:noradrenalineNA+/K+-ATPase pump:sodium-potassium-activated ATPasePKC:protein kinase CPMA:phorbol 12-myristate 13-acetateTXA2:thromboxane A2The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.