共查询到20条相似文献,搜索用时 15 毫秒
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In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11 alpha-hydroxy groups (11 alpha-OH-testosterone and 11 alpha-OH-cortisol) decreased leakage, and those with 11 beta-OH groups (11 beta-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells. 相似文献
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J Rucker AL Edinger M Sharron M Samson B Lee JF Berson Y Yi B Margulies RG Collman BJ Doranz M Parmentier RW Doms 《Canadian Metallurgical Quarterly》1997,71(12):8999-9007
Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use. 相似文献
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The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks in vitro. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface. 相似文献
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The evaluation of ultrafiltration failure is embarked upon when a patient has persistent problems with symptoms and signs of fluid overload. Fluid overload is a common problem in peritoneal dialysis (PD) patients and the risk of its occurrence increases with time on dialysis. Although often attributed to changes in peritoneal membrane function (membrane failure), there are a number of potential, and frequently more common factors that can contribute to the failure of adequate fluid removal in patients on PD. Many of the causes of ultrafiltration failure may be apparent after an initial informal evaluation. However, if after this the etiology remains unexplained, a systematic approach to the differential diagnosis of this problem can be utilized with the use of the peritoneal equilibration test. Once a diagnosis is confirmed, a logical therapeutic plan can be formulated. 相似文献
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The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba. 相似文献