Effect of activating and inactivating mutations of GS-and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

Previous investigations have demonstrated that both G_s- and the G_i-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G_sα vs. G_i2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G_sα or the inactivating G226A(H21a)-G_sα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G_i2α wild type or the missense mutations R179E-G_i2α, Q205L-G_i2α, and G204A(H21a)-G_i2α. The activating [R201C]G_sα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G_sα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G_i2α or [Q205L]G_i2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G_i2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G_i2α or [Q205L]G_i2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G_i2α and [Q205L]G_i2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G_i2α and [Q205L]G_i2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G_i2α and [Q205L]G_i2α over differentiated controls. The inactivating [H21a]G_i2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that G_i2 plays a positive and much more important role than G_s in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc.     

Short heparin thrombin clotting time, increased fibrinogen and platelet aggregates count in coronary disease: a probable hypercoagulable state.

Several indices of plasmatic and platelet coagulability (H.T.C.T., AT III, fibrinogen and Wu-Hoak test) were studied in 82 patients with acute and chronic coronary artery disease. The results were compared with those obtained in a control group. The most interesting result is the consistent shortening of H.T.C.T. as compared to the control group, particularly in patients with acute myocardial infarction. H.T.C.T. was always inversely correlated with the fibrinogen level. Those data suggest an important influence of fibrinogen levels on H.T.C.T., but this observation does not rule out the possibility that the heparin neutralizing activity (PF 4) will also influence the test. No positive correlation between H.T.C.T. and AT III could be observed. The elevated levels of AT III in acute myocardial infarction did not confirm the existence of a consumption of AT III due to chronic intravascular coagulation in these patients. The Wu-Hoak test increased only in patients with acute coronary disease, never in the other groups. In conclusion, H.T.C.T. may be assumed to be a valid test for indicating the existence of a possible hypercoagulability state.     

Isolation and characterisation of a Bam HI element in psam 3 a gene of Pisum sativum L. induced during early stages of arbuscular mycorrhiza development

An Alu-like element was identified in the 5′-UTR of psam 3, a Pisum sativum L. gene which shows enhanced expression during early events of AM formation. Two sets of primers specific for the 5′-UTR of the gene psam 3 and for the Alu-like element, respectively, were designed to study psam 3 gene organisation by targeted Alu PCR carried out on pea genomic DNA. PCR products free of Alu-like sequences were produced. A 1.0 kb DNA fragment showing up to 65 percnt; similarity to a Bam HI repeated sequence of Vicia faba was isolated in both wild-type and mycorrhiza-resistant pea. Southern blot analysis revealed the presence of other Bam HI related sequences in pea. The possibility that Bam HI repeated sequences might constitute complex repeating units together with an Alu-like element in the P. sativum genome is discussed.     

Arbuscular mycorrhizal fungi associated with Artemisia umbelliformis Lam, an endangered aromatic species in Southern French Alps, influence plant P and essential oil contents

Root colonization by arbuscular mycorrhizal (AM) fungi of Artemisia umbelliformis, investigated in natural and cultivated sites in the Southern Alps of France, showed typical structures (arbuscules, vesicles, hyphae) as well as spores and mycelia in its rhizosphere. Several native AM fungi belonging to different Glomeromycota genera were identified as colonizers of A. umbelliformis roots, including Glomus tenue, Glomus intraradices, G. claroideum/etunicatum and a new Acaulospora species. The use of the highly mycorrhizal species Trifolium pratense as a companion plant impacted positively on mycorrhizal colonization of A. umbelliformis under greenhouse conditions. The symbiotic performance of an alpine microbial community including native AM fungi used as inoculum on A. umbelliformis was evaluated in greenhouse conditions by comparison with mycorrhizal responses of two other alpine Artemisia species, Artemisia glacialis and Artemisia genipi Weber. Contrary to A. genipi Weber, both A. umbelliformis and A. glacialis showed a significant increase of P concentration in shoots. Volatile components were analyzed by GC–MS in shoots of A. umbelliformis 6 months after inoculation. The alpine microbial inoculum increased significantly the percentage of E-β-ocimene and reduced those of E-2-decenal and (E,E)-2-4-decadienal indicating an influence of alpine microbial inoculum on essential oil production. This work provides practical indications for the use of native AM fungi for A. umbelliformis field culture.     

Taxonomic,functional, and phylogenetic β‐diversity patterns of stream fish assemblages in tropical agroecosystems

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MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability,motility and VEGF response

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets.MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423).Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways.Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.     

Genetic trials improve the transfer of Douglas‐fir distribution models across continents

Climate change is likely to result in novel conditions with no analogy to current climate. Therefore, the application of species distribution models (SDMs) based on the correlation between observed species’ occurrence and their environment is questionable and calls for a better understanding of the traits that determine species occurrence. Here, we compared two intraspecific, trait‐based SDMs with occurrence‐based SDMs, all developed from European data, and analyzed their transferability to the native range of Douglas‐fir in North America. With data from 50 provenance trials of Douglas‐fir in central Europe multivariate universal response functions (URFs) were developed for two functional traits (dominant tree height and basal area) which are good indicators of growth and vitality under given environmental conditions. These trials included 290 North American provenances of Douglas‐fir. The URFs combine genetic effects i.e. the climate of provenance origin and environmental effects, i.e. the climate of planting locations into an integrated model to predict the respective functional trait from climate data. The URFs were applied as SDMs (URF‐SDMs) by converting growth performances into occurrence. For comparison, an ensemble occurrence‐based SDM was developed and block cross validated with the BIOMOD2 modeling platform utilizing the observed occurrence of Douglas‐fir in Europe. The two trait based SDMs and the occurrence‐based SDM, all calibrated with data from Europe, were applied to predict the known distribution of Douglas‐fir in its introduced and native range in Europe and North America. Both models performed well within their calibration range in Europe, but model transfer to its native range in North America was superior in case of the URF‐SDMs showing similar predictive power as SDMs developed in North America itself. The high transferability of the URF‐SDMs is a testimony of their applicability under novel climatic conditions highlighting the role of intraspecific trait variation for adaptation planning in climate change.     

New genera,species and records of Phaneropterinae (Orthoptera,Phaneropteridae) from sub-Saharan Africa

The results of the study of many specimens preserved in different European museums are reported. The tribe Terpnistrini Brunner von Wattenwyl, 1878 is resurrected. The distribution of the following species is enhanced: Pardalota asymmetrica Karsch, 1896, Diogena denticulata Chopard, 1954, Diogena fausta (Burmeister, 1838), Plangiopsis adeps Karsch, 1896, Poreuomena sanghensis Massa, 2013 and Tylopsis continua (Walker, 1869). Further, for their peculiar characteristics, two African representatives of the American genus Symmetropleura Brunner von Wattenwyl, 1878 are included in two new genera: Symmetrokarschia africana (Brunner von Wattenwyl, 1878), comb. n. and Symmetroraggea dirempta (Karsch, 1889), comb. n. A new genus and species from the Democratic Republic of Congo, Angustithorax spiniger gen. n., sp. n., and a new genus and species from Tanzania, Arostratum oblitum gen. n., sp. n. are described. Finally Melidia claudiae sp. n. and Atlasacris brevipennis sp. n. are described and compared with related species.     

Diabetes Mellitus and Prediabetes on Kidney Transplant Waiting List- Prevalence,Metabolic Phenotyping and Risk Stratification Approach

Background

Despite a significant prognostic impact, little is known about disturbances in glucose metabolism among kidney transplant candidates. We assess the prevalence of diabetes mellitus (DM) and prediabetes on kidney transplant waiting list, its underlying pathophysiology and propose an approach for individual risk stratification.

Methods

All patients on active kidney transplant waiting list of a large European university hospital transplant center were metabolically phenotyped.

Results

Of 138 patients, 76 (55%) had disturbances in glucose metabolism. 22% of patients had known DM, 3% were newly diagnosed. 30% were detected to have prediabetes. Insulin sensitivity and-secretion indices allowed for identification of underlying pathophysiology and risk factors. Age independently affected insulin secretion, resulting in a relative risk for prediabetes of 2.95 (95%CI 1.38–4.83) with a cut-off at 48 years. Body mass index independently affected insulin sensitivity as a continuous variable.

Conclusions

The prevalence of DM or prediabetes on kidney transplant waiting list is as high as 55%, with more than one third of patients previously undiagnosed. Oral glucose tolerance test is mandatory to detect all patients at risk. Metabolic phenotyping allows for differentiation of underlying pathophysiology and provides a basis for early individual risk stratification and specific intervention to improve patient and allograft outcome.