NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.
The HIV-1 virion infectivity factor (Vif) is required during viral replication to inactivate the host cell anti-viral factor, APOBEC3G (A3G). Vif binds A3G and a Cullin5-ElonginBC E3 ubiquitin ligase complex which results in the proteasomal degradation of A3G. The Vif PPLP motif (amino acids 161-164) is essential for normal Vif function because mutations in this motif reduce the infectivity of virions produced in T-cells. In this report, we demonstrate that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5. We demonstrate that the failure of the Vif mutant to bind A3G resulted in A3G incorporation into assembling virions with loss of viral infectivity.
The cytidine deaminases APOBEC3G and APOBEC3F exert anti–HIV-1 activity that is countered by the HIV-1 vif protein. Based on potential transcription factor binding sites in their putative promoters, we hypothesized that expression of APOBEC3G and APOBEC3F would vary with T helper lymphocyte differentiation. Naive CD4+ T lymphocytes were differentiated to T helper type 1 (Th1) and 2 (Th2) effector cells by expression of transcription factors Tbet and GATA3, respectively, as well as by cytokine polarization. APOBEC3G and APOBEC3F RNA levels, and APOBEC3G protein levels, were higher in Th1 than in Th2 cells. T cell receptor stimulation further increased APOBEC3G and APOBEC3F expression in Tbet- and control-transduced, but not in GATA3-transduced, cells. Neutralizing anti–interferon-γ antibodies reduced both basal and T cell receptor-stimulated APOBEC3G and APOBEC3F expression in Tbet- and control-transduced cells. HIV-1 produced from Th1 cells had more virion APOBEC3G, and decreased infectivity, compared to virions produced from Th2 cells. These differences between Th1- and Th2-produced virions were greater for viruses lacking functional vif, but also seen with vif-positive viruses. Over-expression of APOBEC3G in Th2 cells decreased the infectivity of virions produced from Th2 cells, and reduction of APOBEC3G in Th1 cells increased infectivity of virions produced from Th1 cells, consistent with a causal role for APOBEC3G in the infectivity difference. These results indicate that APOBEC3G and APOBEC3F levels vary physiologically during CD4+ T lymphocyte differentiation, that interferon-γ contributes to this modulation, and that this physiological regulation can cause changes in infectivity of progeny virions, even in the presence of HIV-1 vif.
Abstract-Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 mol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O 2 Ϫ was not significantly reduced by 10 mol/L oxypurinol or 30 mol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 mol/L) as well as the O 2 Ϫ scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 mol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O 2 Ϫ scavenger Tiron (10 mmol/L), concomitant with 1 mol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O 2 Ϫ . Incubation with 100 mol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. Key Words: cyclosporin Ⅲ bradykinin Ⅲ nitric oxide Ⅲ cyclic GMP Ⅲ antioxidants C yclosporin (CsA) is an important immunosuppressant used in improving the chances of whole organ transplant and graft survival. 1,2 However, cyclosporin treatment has been linked to several significant nephrotoxic side effects. The side effects range from afferent arteriolar constriction 3 and a reduction in glomerular filtration rate 4 to interstitial fibrosis 5 and ultimately hypertension. Although the toxic effects are well established, the exact mechanisms that lead to the pathology and hypertension are not agreed on. The proposed mechanisms for the development of hypertension focus on induction of vasoconstrictive pathways as well as obstruction of vasodilative pathways examined in patients, rat models, and endothelial cells. The pathways examined as possibly affected by CsA include the renal sensory nerve endings, 6 the renin-angiotensin system, 7 endothelin-1, 8,9 thromboxane, 9,10 and the renal kallikrein-kinin system. 11,12 Bradykinin is an important vasodilating peptide involved in the renal kallikrein-kinin system. The bradykinin peptide exerts its effects by binding to its receptor, activating a heterotrimeric G protein complex, phospholipase C, and then nitric oxide synthase (eNOS), which generates nitric oxide (NO). NO then binds to the heme group of soluble guanylate cyclase (sGC), thereby activating the enzyme to produce cGMP. 13,14 The NO synthase family and the NO radical have been shown to play an important role in many ...
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