G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways

Inactivation of ceramide synthase 6 in mice results in an altered sphingolipid metabolism and behavioral abnormalities

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for beta-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is approximately 35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain

Validation of the diagnostic and prognostic relevance of serum MMP-7 levels in renal cell cancer by using a novel automated fluorescent immunoassay method.

PURPOSE: Despite encouraging results in other cancers, in renal cell cancer, no consensus exists regarding the diagnostic and prognostic relevance of MMP-7. The aim of this study was to assess the diagnostic and prognostic potential of serum MMP-7 levels in renal cell cancer. Furthermore, parallel to the widely used ELISA method, we tested a new, fluid-phase, fluorescent immunoassay (B.R.A.H.M.S KRYPTOR(R)) for the quantitation of MMP-7. METHODS: We analyzed the serum samples of 174 individuals (77 patients and 97 age-matched healthy controls) by a commercially available sandwich ELISA and by a novel, automated, fluid-phase immunofluorescent assay (B.R.A.H.M.S KRYPTOR(R)). Results were correlated with the clinicopathological and follow-up data. RESULTS: MMP-7 concentrations showed a high concordance level (R 2 = 0.979) between the two methods (p < 0.001). Serum MMP-7 concentrations were significantly higher in patients compared to controls. At a cutoff value of 3.15 ng/ml, a specificity and a sensitivity of 70 and 82 % for the detection of RCC was found. Patients with metastasis had significantly higher MMP-7 levels as those without metastasis (p = 0.038 by KRYPTOR, p = 0.011 by ELISA). High MMP-7 levels proved to be independently associated with shorter overall, disease-specific and metastasis-free survival, regardless of the analytical method. CONCLUSIONS: Based on these results, serum MMP-7 levels have both diagnostic and prognostic potential. The KRYPTOR method provided comparable results to the standard ELISA analysis, with a high concordance level and can therefore be considered as a surrogate method. Its flexibility and automated operation make the KRYPTOR MMP-7 assay suitable for routine laboratory use in the daily practice