Changes in the signalling status of the small GTP-binding proteins Rac and Rho do not influence insulin-stimulated hexose transport

Post-receptor signalling molecules that convey the signal from the activated insulin receptor to the actual process of Glut4 translocation and hexose uptake are poorly understood. Various studies have suggested a requirement of the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) in this process. PI3kinase regulates the activation status of the small GTP-binding protein Rac which, in turn, is able to activate another G-protein Rho. Rac and Rho are known to regulate the structure of the membrane- and cytoplasmic actin-cytoskeleton. We have examined whether Rac and Rho transfer the signals generated by PI3kinase towards insulin-stimulated hexose uptake. For that purpose, we expressed in 3T3-L1 adipocytes the dominant-negative mutant of RacN17 using vaccinia virus-mediated gene transfer. The expression levels of the RacN17 protein were monitored by Western blotting. The abrogation of endogenous Rac signalling by expression of RacN17 was inferred from the observed loss of arachidonic acid release in response to insulin. Basal and insulin-stimulated hexose transport were not affected by expression of the RacN17 mutant. A possible contribution of Rho.GTP to stimulation of hexose uptake was examined by pre-incubation of adipocytes with lysophosphatidic acid (LPA). We observed a profound effect of LPA on the structure of the cytoskeleton and on the phosphorylation of Focal Adhesion Kinase (p125FAK), indicating that 3T3-L1 adipocytes respond to LPA and that Rho was activated by LPA. However, no effect was detected on the basal or on the insulin-stimulated hexose transport. We conclude that Rac and Rho are unlikely to be involved in insulin-stimulated hexose transport, suggesting a possible contribution of other signalling pathways, downstream of PI3kinase to this process.     

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.     

Prediction of residual retroperitoneal mass histology after chemotherapy for metastatic nonseminomatous germ cell tumor: multivariate analysis of individual patient data from six study groups

PURPOSE: To develop a statistical model that predicts the histology (necrosis, mature teratoma, or cancer) after chemotherapy for metastatic nonseminomatous germ cell tumor (NSGCT). PATIENTS AND METHODS: An international data set was collected comprising individual patient data from six study groups. Logistic regression analysis was used to estimate the probability of necrosis and the ratio of cancer and mature teratoma. RESULTS: Of 556 patients, 250 (45%) had necrosis at resection, 236 (42%) had mature teratoma, and 70 (13%) had cancer. Predictors of necrosis were the absence of teratoma elements in the primary tumor, prechemotherapy normal alfa-fetoprotein (AFP), normal human chorionic gonadotropin (HCG), and elevated lactate dehydrogenase (LDH) levels, a small prechemotherapy or postchemotherapy mass, and a large shrinkage of the mass during chemotherapy. Multivariate combination of predictors yielded reliable models (goodness-of-fit tests, P > .20), which discriminated necrosis well from other histologies (area under the receiver operating characteristic (ROC) curve, .84), but which discriminated cancer only reasonably from mature teratoma (area, .66). Internal and external validation confirmed these findings. CONCLUSION: The validated models estimate with high accuracy the histology at resection, especially necrosis, based on well-known and readily available predictors. The predicted probabilities may help to choose between immediate resection of a residual mass or follow-up, taking into account the expected benefits and risks of resection, feasibility of frequent follow-up, the financial costs, and the patient's individual preferences.     

Decompression sickness presenting as forearm swelling and peripheral neuropathy: a case report

Bleeding stomal varices is a rare complication of portal hypertension. We report the case of a cirrhotic patient, with a history of colonic adenocarcinoma, who had recurrent bleeding stomal varices. Treatment with transjugular intrahepatic portosystemic shunt and stomal varice embolization was performed because failure of medical treatment of portal hypertension and sclerotherapy. Twenty six months later only one stomal hemorrhage was noted. This suggests that transjugular intrahepatic portosystemic shunt and stomal varice embolization is effective in case of recurrent bleeding of stomal varices.     

Quantitative proton magnetic resonance spectroscopy of childhood adrenoleukodystrophy

Cerebral metabolic disturbances in patients with childhood adrenoleukodystrophy (ALD) were assessed by quantitative localized proton MRS. Patient monitoring by follow-up MRS studies served to identify putative markers for disease onset and progression. Whereas normal-appearing white matter of neurologically asymptomatic patients is characterized by slightly elevated concentrations of choline-containing compounds (Cho), an increase of both Cho and myo-inositol (Ins) seems to indicate the onset of demyelination. Markedly elevated concentrations of Cho, Ins, and glutamine in affected white matter reflect active demyelination and glial proliferation. A simultaneous reduction of the concentrations of N-acetylaspartate and glutamate is consistent with neuronal damage or loss. The observation of elevated lactate is in line with inflammation and/or macrophage infiltration. The more severe metabolic disturbances in cerebral ALD correspond to progressive demyelination, neuroaxonal loss and gliosis leading to clinical deterioration and eventually death. The detection of MRS abnormalities before the onset of neurological symptoms may help in the selection of patients for bone marrow transplantation (BMT). Stabilization and partial reversal of metabolic abnormalities is demonstrated in a patient after BMT.