Activated G␣13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, G␣12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from G␣12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with G␣12 and G␣13. RGS domain of LARG demonstrated G␣12-and G␣13-specific GAP activity. LARG synergistically stimulated SRF activation by G␣12 and G␣13 in HeLa cells, and the SRF activation by G␣12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by G␣13 but not G␣12. However, when LARG was phosphorylated by Tec, G␣12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through G␣12 and that the regulation of RhoGEFs by heterotrimeric G proteins G12͞13 is further modulated by tyrosine phosphorylation.

Cyclin-dependent kinase (Cdk) enzymes are activated for entry into the S phase of the cell cycle. Elimination of Cdk inhibitor protein p27 Kip1 during the G 1 to S phase is required for the activation process. An inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase prevents its elimination and leads to G 1 arrest. Mevalonate and its metabolite, geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, restore the inhibitory effect of pravastatin on the degradation of p27 and allow Cdk2 activation. By the addition of geranylgeranyl pyrophosphate, Rho small GTPase(s) are geranylgeranylated and translocated to membranes during G 1 /S progression. The restoring effect of geranylgeranyl pyrophosphate is abolished with botulinum C3 exoenzyme, which specifically inactivates Rho. These results indicate (i) among mevalonate metabolites, geranylgeranyl pyrophosphate is absolutely required for the elimination of p27 followed by Cdk2 activation; (ii) geranylgeranylated Rho small GTPase(s) promote the degradation of p27 during G 1 /S transition in FRTL-5 cells.

We investigated the role of the intrinsic mevalonate cascade in the neuronal cell death (NCD) induced by the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in rat primary cortical neurons cultured from the brains of 17-d-old fetal SD rats. HMG-CoA reductase inhibitors induced NCD [HMG-CoA reductase inhibitor-induced NCD (H-NCD)] in time- and dose-dependent manners. The apoptotic characteristics were revealed by the formation of the DNA ladder and by the electron microscopical observation. During the progression of H-NCD, p53 was induced followed by the expression of Bax. Although the mevalonate completely inhibited H-NCD, the cholesterol did not. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, prohibited H-NCD with inhibition of the induction of p53 and Bax. The inhibition of HMG-CoA reductase decreased the amount of membrane-associated Rho small GTPase families, but not Ras small GTPase, and GGPP restored the blockage by HMG-CoA reductase inhibitor in the translocation or redistribution of Rho small GTPase families to membrane. These data indicated that (1) the inhibition of the intrinsic mevalonate cascade induces the apoptotic NCD with the induction of p53 followed by that of Bax, (2) the inhibition of HMG-CoA reductase concomitantly causes blockage of the translocation or redistribution of Rho small GTPase families, not Ras small GTPase, to membrane, and (3) GGPP, not FPP, is one of the essential metabolites in the mevalonate cascade for protecting neurons from H-NCD.

We have applied laser-induced breakdown spectroscopy to quantitative analysis of colloidal and particulate iron in water. A coaxial sample flow apparatus developed in our previous work, which allowed us to control the atmosphere of laser-induced plasma, was used. Using sequential laser pulses from two Q-switched Nd:YAG lasers as excitation sources, the FeO(OH) concentration in the tens of ppb range was determined with an optimum interval between two laser pulses and an optimum delay time of a detector gate from the second pulse. The detection limit of Fe decreased substantially using two sequential laser pulse excitations:  the 0.6 ppm limit of single pulse excitation to 16 ppb with sequential pulse excitation. The effects of the second laser pulse on the plasma emission were studied. The concentration of iron in fine particles in boiler water sampled from a commercially operated thermal power plant has been determined successfully by this method. The results show the capability of laser-induced breakdown spectroscopy in determining suspended colloidal and particulate impurities in a simple and quick way.

No abstract

Ras and Rho are involved in the regulation of signal transduction events governing cell growth and cell proliferation. Protein prenylation is essential for the activation and/or the translocation of these small GTPases; however, protein geranylgeranylation rather than farnesylation is required for G 1 /S transition. We studied prenylation and translocation of Ras and Rho A during

No abstract

Neoplasms may be one of the systemic complications to which we attribute high mortality in acromegaly. The present study was designed to investigate the incidence of malignant tumors in patients with acromegaly in the Japanese population. In this report, 44 patients (25 men and 19 women) with biochemically proven acromegaly were studied retrospectively and had a total 670 patient years of the duration of acromegaly. We investigated the incidence of malignant tumors. There were 5 patients with malignant tumors (5 in men) in this study (11%). Male patients with acromegaly had nearly a 3.5 times higher ratio of malignancy than expected and this increased cancer incidence was considered significant (P=0.01). There was no significant increase in cancer incidence of either the total patient population or female patients. The malignant tumors were two thyroid cancers and one colon, one gastric and one bladder cancer. It is of note that the colon cancer of one patient was diagnosed 2 years after transsphenoidal surgery even though the levels of serum GH and insulin-like growth factor (IGF-1) were reduced to normal after operation. This preliminary study has suggested that male patients with acromegaly might have a high risk of malignancy and that careful screening for tumors is needed both before and after surgical and medical treatment, even in patients with normalized serum GH and IGF-1 levels.