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991.
Colombo S Ronchetti D Thevelein JM Winderickx J Martegani E 《The Journal of biological chemistry》2004,279(45):46715-46722
The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism. 相似文献
992.
Bhushan B Halasz A Thiboutot S Ampleman G Hawari J 《Biochemical and biophysical research communications》2004,316(3):816-821
Cyclic nitramine explosives, RDX, HMX, and CL-20 are hydrophobic pollutants with very little aqueous solubility. In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and distribute heterogeneously in aqueous environments. For efficient bioremediation of these explosives, the microorganism(s) must access them by chemotaxis ability. In the present study, we isolated an obligate anaerobic bacterium Clostridium sp. strain EDB2 from a marine sediment. Strain EDB2, motile with numerous peritrichous flagella, demonstrated chemotactic response towards RDX, HMX, CL-20, and NO(2)(-). The three explosives were biotransformed by strain EDB2 via N-denitration with concomitant release of NO(2)(-). Biotransformation rates of RDX, HMX, and CL-20 by the resting cells of strain EDB2 were 1.8+/-0.2, 1.1+/-0.1, and 2.6+/-0.2nmol h(-1)mgwet biomass(-1) (mean+/-SD; n=3), respectively. We found that commonly seen RDX metabolites such as TNX, methylenedinitramine, and 4-nitro-2,4-diazabutanal neither produced NO(2)(-) during reaction with strain EDB2 nor they elicited chemotaxis response in strain EDB2. The above data suggested that NO(2)(-) released from explosives during their biotransformation might have elicited chemotaxis response in the bacterium. Biodegradation and chemotactic ability of strain EDB2 renders it useful in accelerating the bioremediation of explosives under in situ conditions. 相似文献
993.
Blotting analysis of native IRP1: a novel approach to distinguish the different forms of IRP1 in cells and tissues 总被引:1,自引:0,他引:1
Iron regulatory protein 1 (IRP1) is a bifunctional protein, which either has aconitase activity or binds to specific mRNA structures to regulate the expression of iron proteins. Using recombinant human IRP1, we found that the two functional forms are resolved by nondenaturing polyacrylamide gel electrophoresis and that they are distinguished from IRP1/RNA complexes. This allowed us to use specific antibodies to develop a blotting system that recognized the iron-free and iron-containing IRP1 forms in the soluble fraction and the RNA-bound IRP1 in the high-speed precipitate fraction of cell extracts. The system was used to study IRP1 in HeLa, K562 cells, and monocytes/macrophages before and after treatment with iron salts, iron chelators, or hydrogen peroxide, as well as in stomach and duodenum biopsies. The results showed that iron-bound aconitase IRP1 is by far the prevalent form in most cells and that the major effect of cellular iron modifications is a shift between free and RNA-bound IRP1. The fraction of RNA-bound IRP1 was highly variable among different cells and was often a minor one. Furthermore, blotting showed that electrophoretic mobility shift assay, as commonly used, tends to under-evaluate the amount of total IRP1 and to over-evaluate the actual RNA-binding activity of IRP1. In conclusion, blotting analysis of IRP1 is a new, useful, and convenient method to analyze the amount and conformations of the protein that reveals previously undetected differences in IRP1 compartmentalization among various cell types. 相似文献
994.
995.
Granier S Terrillon S Pascal R Déméné H Bouvier M Guillon G Mendre C 《The Journal of biological chemistry》2004,279(49):50904-50914
In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [3H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs. 相似文献
996.
Dai T Abou-Rjaily GA Al-Share' QY Yang Y Fernström MA Deangelis AM Lee AD Sweetman L Amato A Pasquali M Lopaschuk GD Erickson SK Najjar SM 《The Journal of biological chemistry》2004,279(43):45155-45161
Inactivation of CEACAM1 in L-SACC1 mice by a dominant-negative transgene in liver impairs insulin clearance and increases serum free fatty acid (FFA) levels, resulting in insulin resistance. The contribution of elevated FFAs in the pathogenesis of insulin resistance is herein investigated. Treatment of L-SACC1 female mice with carnitine restored plasma FFA content. Concomitantly, it normalized insulin levels without directly regulating receptor-mediated insulin internalization and prevented glucose tolerance in these mice. Similarly, treatment with nicotinic acid, a lipolysis inhibitor, restored insulin-stimulated receptor uptake in L-SACC1 mice. Taken together, these data suggest that chronic elevation in plasma FFAs levels contributes to the regulation of insulin metabolism and action in L-SACC1 mice. 相似文献
997.
Scaling techniques were used to analyze the fractal nature of colonies of 15 cell lines growing in vitro as well as of 16 types of tumor developing in vivo. All cell colonies were found to exhibit exactly the same growth dynamics-which correspond to the molecular beam epitaxy (MBE) universality class. MBE dynamics are characterized by 1), a linear growth rate, 2), the constraint of cell proliferation to the colony/tumor border, and 3), surface diffusion of cells at the growing edge. These characteristics were experimentally verified in the studied colonies. That these should show MBE dynamics is in strong contrast with the currently established concept of tumor growth: the kinetics of this type of proliferation rules out exponential or Gompertzian growth. Rather, a clear linear growth regime is followed. The importance of new cell movements-cell diffusion at the tumor border-lies in the fact that tumor growth must be conceived as a competition for space between the tumor and the host, and not for nutrients or other factors. Strong experimental evidence is presented for 16 types of tumor, the growth of which cell surface diffusion may be the main mechanism responsible in vivo. These results explain most of the clinical and biological features of colonies and tumors, offer new theoretical frameworks, and challenge the wisdom of some current clinical strategies. 相似文献
998.
In our earlier communication we have shown that Lupeol inhibits early responses of tumour induction in murine skin. The free radical mediated damage to the cellular macromolecules such as DNA, proteins, lipids and alteration in the activities of quinone reductase and xanthine oxidase are important biochemical parameters of tumor development. The suppression of free radical mediated damage to cellular macromolecules and induction of quinone reductase along with depletion of xanthine oxidase are prominent characteristics of chemopreventive agents. In the present investigation, we have elucidated the mechanism of action of lupeol (Lup-20 (29)-en-3beta-ol), a triterpene found in moderate amount in many vegetables, fruits and anti-tumor herbs. In the present investigation, lupeol significantly reduced the free radical mediated DNA-sugar damage and microsomal lipid peroxidation in an iron/ascorbate free radical generating system in vitro. Benzoyl peroxide, a known free radical generating tumor promoter mediated oxidation of proteins and modulation in the activities of quinone reductase as well as xanthine oxidase was significantly prevented by lupeol when tested on murine skin in vivo. It was concluded from this study that lupeol acts as an effective chemopreventive agent against cutaneous toxicity. 相似文献
999.
Longhi S Receveur-Bréchot V Karlin D Johansson K Darbon H Bhella D Yeo R Finet S Canard B 《The Journal of biological chemistry》2003,278(20):18638-18648
The nucleoprotein of measles virus consists of an N-terminal moiety, N(CORE), resistant to proteolysis and a C-terminal moiety, N(TAIL), hypersensitive to proteolysis and not visible as a distinct domain by electron microscopy. We report the bacterial expression, purification, and characterization of measles virus N(TAIL). Using nuclear magnetic resonance, circular dichroism, gel filtration, dynamic light scattering, and small angle x-ray scattering, we show that N(TAIL) is not structured in solution. Its sequence and spectroscopic and hydrodynamic properties indicate that N(TAIL) belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The same epitopes are exposed in N(TAIL) and within the nucleoprotein, which rules out dramatic conformational changes in the isolated N(TAIL) domain compared with the full-length nucleoprotein. Most unstructured proteins undergo some degree of folding upon binding to their partners, a process termed "induced folding." We show that N(TAIL) is able to bind its physiological partner, the phosphoprotein, and that it undergoes such an unstructured-to-structured transition upon binding to the C-terminal moiety of the phosphoprotein. The presence of flexible regions at the surface of the viral nucleocapsid would enable plastic interactions with several partners, whereas the gain of structure arising from induced folding would lead to modulation of these interactions. These results contribute to the study of the emerging field of natively unfolded proteins. 相似文献
1000.
van Oostrom AJ Sijmonsma TP Verseyden C Jansen EH de Koning EJ Rabelink TJ Castro Cabezas M 《Journal of lipid research》2003,44(3):576-583
Atherosclerosis is a low-grade inflammatory disease involving leukocytes, lipids, and glucose leading to endothelial dysfunction. Since activation of neutrophils by triglycerides and glucose has been described in vitro, we hypothesized that the postprandial phase is an inflammatory state affecting leukocytes, possibly contributing to endothelial dysfunction. We measured postprandial blood leukocyte counts, cytokines, hydroperoxides (HPOs), and flow-mediated vasodilation (FMD) in eight healthy males (age 23 +/- 2 years) after a FAT (50 g/m2) and GLUCOSE challenge (37.5 g/m2), a combination of both (MIXED test), and after WATER. All tests, except WATER, resulted in significantly impaired FMD (10% reduction) between t = 1 h and t = 3 h, accompanied by a significant increase of neutrophils (59% after FAT and 28% after GLUCOSE and MIXED), total plasma HPOs (15 to 31% increase), and plasma interleukin-8 (IL-8) (50-130% increase). WATER did not affect FMD, neutrophils, HPOs, or IL-8. Lymphocytes increased gradually in all tests (40-70% increase at t = 10 h compared with t = 0; P < 0.005), paralleling a gradual 3- to 5-fold interleukin-6 increase. Monocyte and erythrocyte counts did not change in any test. In conclusion, the neutrophil increment during postprandial lipemia and glycemia with concomitant IL-8 and HPO increases may contribute to endothelial dysfunction. Lymphocyte increment is a nonspecific diurnal process. Postprandial intravascular inflammatory changes may be relevant for the pathogenesis of atherosclerosis. 相似文献