Clinical Significance of Serum Kallistatin and ENOX1 Levels in Patients with Coronary Heart Disease.

BACKGROUND: Kallistatin and ENOX1 are regulators of inflammation and oxidative stress which are typical pathological reactions in atherosclerosis. However, there is limited information of kallistatin and ENOX1 in coronary heart disease (CHD). METHODS: Fifty healthy controls, 56 stable angina pectoris (SAP) patients, and 47 acute coronary syndrome (ACS) patients were included in this study. Levels of kallistatin and ENOX1 in serum were measured by ELISA. χ2 test was performed to analyze categorical data. ANOVA, Pearson correlation analysis, and multiple linear regression were performed to analyze the numerical data. Finally, receiver operating characteristic (ROC) curve was applied to assess the diagnostic value of kallistatin in CHD. RESULTS: Among the 153 participants, 59.5% were male and the average age was 63.8 ± 11.39 years. Compared with the control group, kallistatin expression was decreased in the SAP and ACS groups while expression of ENOX1 was increased in the ACS group (p < 0.05). Pearson correlation analysis showed that the kallistatin level was negatively correlated with the Gensini score (r = -0.210, p < 0.01), white blood cell (WBC) count (r = -0.283, p < 0.001), and triglyceride levels (r = -0.242, p < 0.01) and positively correlated with age (r = 0.353, p < 0.001) and high-density lipoprotein cholesterol (r = 0.310, p < 0.001). ENOX1 expression was positively correlated with WBC count (r = 0.244, p < 0.01), international normalized ratio (r = 0.177, p < 0.05), and Gensini score (r = 0.201, p < 0.05). Multiple linear regression showed that Cr, alanine transaminase, glucose, and kallistatin are independent predictors for Gensini score. The ROC curve showed that kallistatin had the highest diagnostic significance (p = 0.007) when the area under curve was 0.636, with a sensitivity of 0.735 and a specificity of 0.495. CONCLUSION: Expression of kallistatin was decreased in CHD patients and that of ENOX1 was increased in ACS patients. Kallistatin and ENOX1 were closely connected with the severity of CHD, and kallistatin may be helpful in the diagnosis of CHD.

ENOX2 (or tNOX): a new and old molecule with cancer activity involved in tumor prevention and therapy.

Cancer includes a number of related diseases due to abnormal cell proliferation that spreads to nearby tissues. Many compounds (physical, chemical and biological) have been used to try to halt this abnormal proliferation, but the therapeutic results are poor, due also to the side effects. It has been reported that ecto-nicotinamide adenine dinucleotide oxidase di-sulfide-thiol exchanger 2 (ENOX2), also known as tumor-associated nicotinamide adenine dinucleotide oxidase (tNOX), was found to be located on the cancer cell surface, essential for cancer cell growth. Capsaicin and other anti-oxidants are capable of inhibiting tNOX, causing apoptosis of cells, exerting anti-tumor activity. It is interesting that some authors reported that ENOX2 is present in the serum of cancer patients several years before the clinical symptoms of the tumor. However, this result has to be confirmed. In this article we discuss ENOX2 and its inhibition as a hope of improving cancer therapy.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

Impact of allyl disulfide on oxidative damage and liver regeneration in an experimental hepatectomy model.

BACKGROUND: We investigated the effects of allyl disulfide (agarlic extract) on tissue damage, regeneration, proliferation and oxidative damage in an experimental liver resection model. MATERIALS AND METHODS: In the study, 24 female Wistar albinorats weighing approximately 200-250 g were used. Group 1:The rats in the experimental group all received a 70%hepatectomy and were fed an Allyl disulfide (30 λg kg day,Allyl disulfide, Sigma-Aldrich, formula: C6H10S2, CASNumber: 2179-57-9, formula weight: 146.27 g mol) in supplement to a regular diet for 1 week both preoperatively and postoperatively. Group 2: The rats in the control group also underwent a 70% hepatectomy and were given regular food and water for 1 week both preop and postop. Group 3: In the sham group, all rats were sacrificed 7 days after surgery. Forbiochemical evaluation, SGOT, SGPT, bilirubin, CRP and MDA were studied. In a histopathological examination, the fattening of the liver tissue, existence of (macro-micro vesicular),fibrosis, pleomorphism at hepatocyte nuclei, portal inflammation, existence of intralobular inflammatory cells,dilation at sinusoids, congestion, congestion at the central vein, regeneration, existence of Kupffer cells in the sinu soidallumen and ki-67 proliferation index at hepatocytes were examined. RESULTS: A significant difference between group 1 and group2 was observed regarding the existence of regeneration,(p:0.06), the occurrence of nuclear pleomorphisms (p:0,001)and the fibroblast activity status (p:0.001). Significant differences were found between the experimental groups in regard to Kupffer cell increase and dilation and the hyperemiastatus in the sinusoid lumens (p:0.013 and p:0.001,respectively). In the Allyl disulfide group, the proliferation index was significantly higher than that of the other groups(p:0,001), while the average plasma MDA value was lower than that of the other groups (p: 0,042). No significant differences were found among the groups with respect to tissue MDA values (p:0,720). No significant difference was found for SGPT (ALT) and SGOT (AST) levels between Group 1 and the other groups (p:0.247 and p:0.539, respectively).The average total bilirubin (T. Bili) values were 0,12,0,08 and 0,04 in the allyl disulfide group, control group andSham group, respectively. This difference among the groups is statistically significant (p:0.001). The average direct bilirubin (D. Bili) values were 0,06, 0,02 and 0,02 in the allyl disulfide group, control group and Sham group, respectively.This variation among the groups is also statistically significant (0.001). CONCLUSION: We observed that the use of Allyl disulfide supplementation after major hepatectomy has a positive impact on liver regeneration, proliferation and oxidative damage. ABBREVIATIONS: Postop: post-operative, Preop: pre-operative,SGOT(AST): serum glutamic oxaloacetic transaminase,SGPT(ALT): serum glutamate-pyruvate transaminase, CRP:C- Reactive protein, MDA: Malondialdehyde, DAS: Garlicextract diallyl sulfide, AGE: aged garlic extract.

arNOX: generator of reactive oxygen species in the skin and sera of aging individuals subject to external modulation.

An aging-related cell-surface oxidase (aging-related NADH oxidase, arNOX) generating superoxide and other reactive oxygen species is shed from the cell surface and is found in saliva, urine, perspiration, and interstitial fluids that surround the collagen and elastin matrix underlying dermis. arNOX activity correlates with age and reaches a maximum at about age 65 in males and 55 in females. arNOX activities are highly correlated with values of human skin where a causal relationship is indicated. Ongoing efforts focus on cloning arNOX proteins and development of antiaging formulas based on arNOX inhibition (intervention).

Aging-related nicotinamide adenine dinucleotide oxidase response to dietary supplementation: the French paradox revisited.

Aging-related cell-surface NADH oxidase (arNOX)-specific activities increase with age between age 30 and ages 50-65. The protein is shed and circulates. Activity correlates with a number of aging-related disorders including low-density lipoprotein (LDL) oxidation as a precondition to atherosclerosis as well as oxidation of collagen and elastin as a major contributor to skin aging. arNOX inhibitors formulated for sustained release are capable of maintaining circulating arNOX at low levels with regular use as food supplements formulated with natural compounds. Among the best sources are certain culinary seasonings, all of which are ingredients used extensively in the French kitchen. Their regular use may contribute to an understanding of the nutritional basis for the French Paradox.

Oxidative stress expression status associated to Helicobacter pylori virulence in gastric diseases.

OBJECTIVES: To analyze the status of expression of inflammation markers, antioxidant and oxidant enzymes in biopsies from patients diagnosed with gastritis, gastric ulcer (GU) and gastric cancer (GC) and the Helicobacter pylori virulence from these isolated biopsies in order to evaluate a possible association among these factors. METHODS: H. pylori genotype from isolated biopsies was performed by PCR. The pattern of expression of inflammation (TNF-alpha, IL-1beta, IL-8, IL-10 and IL-12), oxidant (iNOS and Nox1) and antioxidant markers (MnSOD, GPX and CAT) of biopsies from gastritis, GU, GC and control groups was performed by RT-PCR. RESULTS: Different from other gastric diseases studied here, gastritis is characterized by an oxidative stress with significant expression of TNF-alpha, IL-8, IL-12, iNOS and Nox and significant absence of MnSOD and GPX expression. Gastritis was the only condition where there was an association between TNF-alpha or IL-8 expression and H. pylori cagA+/vacAs1 genotype. In this case, TNF-alpha expression was about 3 times higher when compared to control subjects. CONCLUSION: In this study, only gastritis was found to be associated with significant oxidative stress marker expression of TNF-alpha and IL-8 that was also related to H. pylori virulence, suggesting that they are the main oxidant stress markers responsible to trigger an increase in ROS level that contributes to decrease the expression of the MnSOD and GPX.

Effects of vitamin C infusion and vitamin E-coated membrane on hemodialysis-induced oxidative stress.

Chronic hemodialysis (HD) patients manifest anemia and atherosclerosis with associated oxidative stress. We explored whether intravenous infusion of vitamin C (VC) and/or use of vitamin E (VE)-coated dialysis membrane could palliate HD-evoked oxidative stress. Eighty patients undergoing chronic HD were enrolled and randomly assigned into four groups: HD with intravenous VC (n=20), HD with VE-coated dialyzer (n=20), HD with both (n=20), and HD with neither (n=20). We evaluated oxidative stress in blood and plasma, erythrocyte methemoglobin/ferricyanide reductase (red blood cells (RBC)-MFR) activity, plasma methemoglobin, and pro-inflammatory cytokines in these patients. All patients showed marked increases (14-fold) in blood reactive oxygen species (ROS) after HD. The types of ROS were mostly hydrogen peroxide, and in lesser amounts, O2*- and HOCl. HD resulted in decreased plasma VC, total antioxidant status, and RBC-MFR activity and increased plasma and erythrocyte levels of phosphatidylcholine hydroperoxide (PCOOH) and methemoglobin. Intravenous VC significantly palliated HD-induced oxidative stress, plasma and RBC levels of PCOOH, and plasma methemoglobin levels and preserved RBC-MFR activity. The VE-coated dialyzer effectively prevented RBCs from oxidative stress, although it showed a partial effect on the reduction of total ROS activity in whole blood. In conclusion, intravenous VC plus a VE-coated dialyzer is effective in palliating HD-evoked oxidative stress, as indicated by hemolysis and lipid peroxidation, and by overexpression of proinflammation cytokines in HD patients. Using VE-coated dialyzer per se is, however, effective in reducing lipid peroxidation and oxidative damage to RBCs.

NAD(P)H-dependent superoxide production in platelets: the role of angiotensin II and protein kinase C.

OBJECTIVES: Vascular NAD(P)H oxidase represents a major source for excessive superoxide production in hypertension. Angiotensin II (AngII) can activate NAD(P)H oxidase via the angiotensin II type 1 (AT1) receptor and protein kinase C (PKC). Platelets possess AT1 receptors and all the components of the NAD(P)H oxidase system. We employed this tissue model to explore mechanisms involved in AngII-mediated superoxide production. DESIGN AND METHODS: Platelet suspensions from hypertensive patients' blood were activated with AngII or phorbol 12-myristate 13-acetate (PMA). Inhibitors of NAD(P)H oxidase, PKC, and the AT1 receptor were employed to study their effects on superoxide production. RESULTS: Superoxide production was stimulated by AngII and PMA and attenuated by AT1 receptor antagonists (mean percentage reduction 80.2%, P<0.01) and inhibitors of PKC (mean reduction 94.8%, P<0.001) and NAD(P)H oxidase (mean reduction 100%, P< 0.001). CONCLUSIONS: AngII stimulates platelet superoxide production through activation of vascular NAD(P)H oxidase via the AT1 receptor and PKC.

Sera from cancer patients contain two oscillating ECTO-NOX activities with different period lengths.

ECTO-NOX protein's are cell surface-associated and growth-related hydroquinone oxidases with both protein disulfide-thiol interchange activity and the capacity to oxidize NAD(P)H. The activities of these ECTO-NOX proteins are not steady state but fluctuate to create a repeating pattern of oscillations. Two forms of ECTO-NOX activities have been distinguished. The constitutive ECTO-NOX (CNOX), is hormone responsive and refractory to quinone-site inhibitors. A tumor-associated NOX (tNOX) is unregulated, refractory to hormones and growth factors and responds to quinone-site inhibitors. CNOX proteins are widely distributed and exhibit oscillations in enzymatic activity with a period length of 24 min. tNOX proteins are cancer specific and exhibit oscillations with a period length of about 22 min. Our findings now demonstrate the presence of the novel oscillating tNOX activity in sera of patients with cancer whereas the constitutive NOX of non-cancer cells is present in sera of both cancer patients and healthy volunteers. We conclude that ECTO-NOX proteins in sera exhibit oscillatory characteristics similar to those of ECTO-NOX forms of the cell surface.

NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patients.

It is believed that the enhanced capability of activated macrophages to resist infection is related to the remarkable increase in the production of oxygen metabolites in response to phagocytosis. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. Low NADH-oxidase, NADPH-oxidase and myeloperoxidase activity were observed in monocytes of patients with active visceral leishmaniasis as compared with healthy controls. These results suggest that low NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for persistence of Leishmania parasites in visceral leishmaniasis.

Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

Enhanced activity of the plasma membrane oxidoreductase in circulating lymphocytes from insulin-dependent diabetes mellitus patients.

Circulating human lymphocytes contain a transmembrane oxidoreductase (PMOR) capable of reducing dichlorophenol indophenol (DCIP) by endogenous reductants, presumably NADH. Membranes from lymphocytes obtained from buffy coats contain a NADH DCIP reductase having a K(m) of about 1 microM and almost insensible to dicoumarol. The PMOR of lymphocytes from insulin-dependent diabetic patients is higher than that from age-matched controls and, in addition, has a dicoumarol-sensitive component, lacking in most controls, presumably due to membrane association of DT-diaphorase. The increase of PMOR in diabetes is likely due to overexpression of the enzyme, in view of the very low K(m) for NADH indicating that, in intact cells, the enzyme is practically saturated with the reductant substrate.

Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity.

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O(-)(2) at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47(phox) in vitro. Phosphoamino acid analysis of phosphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47(phox). Dependence of phosphorylation on the conformation of p47(phox) is supported by the finding that p47(phox) undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47(phox). In addition, 5,6-dichloro-1-beta-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase.

Abnormalities of flavin monooxygenase as an etiology for sideroblastic anemia.

We postulated that a deficiency of flavin monooxygenase (FMO)-a ferrireductase component of cells-could produce sideroblastic anemia. FMO is an intracellular ferrireductase which may be responsible for the obligatory reduction of ferric to ferrous iron so that reduced iron can be incorporated into heme by ferrochelatase. Abnormalities of this mechanism could result in accumulation of excess ferric iron in mitochondria of erythroid cells to produce ringed sideroblasts and impair hemoglobin synthesis. To investigate this hypothesis we obtained blood from patients with sideroblastic anemia and normal subjects. Extracts of peripheral blood lymphocytes were used to measure ferrireduction by utilization of NADPH. Lymphoid precursors are reported to accumulate iron in mitochondria similarly to erythroid precursors. Utilization of lymphoid precursors avoided the need for bone marrow aspirations. We studied three patients with sideroblastic anemia. One patient and his asymptomatic daughter had a significant decrease in ferrireductase activity. They also had markedly diminished concentrations of FMO in lymphocyte protein extracts on Western blots. This was accompanied by increased concentration of mobilferrin in the extracts. These results suggest that abnormalities of FMO and mobilferrin may cause sideroblastic anemia and erythropoietic hemochromatosis in some patients.

Insulin and high glucose modulation of phosphatase and reductase enzymes in the human erythrocytes: a comparative analysis in normal and diabetic states.

The ability of insulin to influence activities of various protein kinases and protein phosphatases, that are thought to mediate insulin action, are limited in patients with insulin resistance. Because numerous responses to insulin are affected, we undertook studies to determine whether protein tyrosine phosphatases (PTPs) activities are altered in patients with diabetes syndrome. In order to evaluate abnormal PTP activities, we done a comparative study using erythrocytes from normal and diabetic patients. We determined the activity of the cytosolic acid PTP in basal and insulin-dependent states. Mean basal PTP activities, were found to be significantly higher in diabetics than in normal subjects (type 1 diabetics: 0.36 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g hemoglobin (Hb), P < 0.001; type 2 diabetics: 0.35 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g Hb, P < 0.001). Insulin, at concentrations above physiological levels (1 mIU/ml), inhibited the PTP activities in erythrocytes from normal subjects (-15 +/- 4.1%, P < 0.01). Insulin could also modulate glycolysis, probably as a consequence of receptor tyrosine kinase activation, inducing phosphorylation of protein band 3 and hence the release of glycolytic enzymes. We have previously reported that a reductase enzyme in human erythrocytes is dependent on glycolysis being significantly activated (+28 +/- 3.1%, P < 0.001) by high insulin levels (1 mIU/ml). Mean basal reductase activities were found to be significantly lower in diabetics than in normal subjects (type 1 diabetics: 0.77 +/- 0.03 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001; type 2 diabetics: 0.77 +/- 0.04 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001), indicating altered erythrocyte metabolism in the diabetic patients. High glucose levels were used to mimic hyperglycemia condition, using erythrocytes from normal subjects. At 30 mM glucose, erythrocytic phosphatase activity was stimulated (+32 +/- 4.2%, P < 0.0001), although no effect was observed on the reductase enzyme at the same glucose levels. Results indicated that diabetic disorders appear to be associated with quantitative alterations of erythrocyte acid phosphatase activity and other enzymes that depend on the glycolytic rate (reductase). The overall data suggest that erythrocyte acid phosphatase may have a role in the modulation of glycolytic rates through the control of insulin receptor phosphorylation.

Gene therapy for chronic granulomatous disease.

Recent progress in the development of gene therapy for chronic granulomatous disease (CGD), an inherited immunodeficiency syndrome, is reviewed. This disorder results from defects in any of the four genes encoding essential subunits of respiratory burst oxidase, the superoxide-generating enzyme complex in phagocytic leukocytes. The absence of respiratory burst oxidants results in recurrent bacterial and fungal infections and can also be complicated by the formation of inflammatory granulomas. Although current management, including prophylactic use of antimicrobial agents and interferon-gamma, has significantly improved its prognosis, CGD continues to be associated with significant morbidity and mortality from life-threatening infections and complications. Allogeneic bone marrow transplantation can provide a life-long cure of the disease, but difficulty in finding suitable donors and risks associated with this procedure have limited its application. Recently CGD has emerged as a promising candidate for gene therapy targeted at the hematopoietic system. CGD mouse models have been developed with gene targeting technology, and preclinical studies in these animals with recombinant retroviral vectors have demonstrated the appearance of functionally normal neutrophils and increased resistance against pathogens such as Aspergillus. Although the murine studies have provided a promise of long-term cure of patients by gene transfer, phase I clinical studies in a limited number of patients with CGD with such vectors have yet to produce a clinically relevant number of corrected neutrophils for extended time periods. Efforts are ongoing to improve gene transfer efficiency into human hematopoietic stem/progenitor cells and to achieve better engraftment of the gene-corrected stem cells.

High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane, cell surface, and serum NADH-oxidases.

The high capacity of proliferating mammalian cells to transfer electrons from cytosolic NADH to extracellular electron acceptors like oxygen is poorly understood and not widely recognized. Nevertheless, trans-plasma membrane electron transport (plasma membrane redox control) probably ranks alongside the Na+/H+ antiport system (pH control) and glucose transport in facilitating cellular responses to physiological stimuli. These plasma membrane transport systems are acutely responsive to receptor ligation by growth factors, polypeptide hormones, and other cell activators. A novel tetrazolium-based cell proliferation assay that we have shown to measure an NADH-oxidoreductase component of the trans-plasma membrane electron transport system has allowed direct comparisons with NADH:ferricyanide-oxidoreductase and respiratory burst NADPH-oxidoreductase. In addition, an NAD(P)H-oxidase at the cell surface and an NADH-oxidase activity in body fluids can be measured by modifying the basic cell proliferation assay. As determined by reduction of the cell-impermeable tetrazolium reagent, WST-1, electron transfer across the plasma membrane of dividing cells can exceed that of fully activated human peripheral blood neutrophils. Cellular reduction of WST-1 is dependent on the presence of an intermediate electron acceptor and is inhibited by superoxide dismutase (SOD) and by oxygen, implying indirect involvement of superoxide in WST-1 reduction. Cell-surface NAD(P)H-oxidase and serum NADH-oxidase are shown to be distinct from trans-plasma membrane NADH-oxidoreductase by their differential sensitivity to capsaicin and pCMBS. The glycolytic metabolism of cancer cells may be linked to changes in trans-plasma membrane NADH:WST-1-oxidoreductase activity and to increased serum NADH-oxidase in cancer.

Quantitative study of mitochondrial complex I in platelets of parkinsonian patients.

Activity of mitochondrial enzyme complex I (NADH-ubiquinone oxidoreductase) is reduced in the substantia nigra of patients with Parkinson's disease (PD). A less pronounced decrease in the activity of this enzyme has also been reported in platelets of PD patients. To obtain quantitative information on platelet complex I in PD, we studied platelet complex I in 16 PD patients and 16 age-matched controls by using a newly developed technique based on the binding of [3H]dihydrorotenone ([3H]DHR), an analog of the pesticide rotenone, to complex I. We also investigated the inhibitory effect of MPP+ (1-methyl-4-phenyl-pyridinium) on [3H]DHR specific binding to platelet complex I. PD patients and controls showed similar levels of [3H]DHR specific binding; preincubation of platelets with MPP+ caused the same degree of inhibition of [3H]DHR specific binding in the two groups. In PD patients, we observed a direct correlation between MPP+-induced inhibition of [3H]DHR specific binding and the daily intake of levodopa, which may be related to drug-induced changes in the transport of MPP+ into the platelet or in its binding to complex I. These findings demonstrate that the reported reduction in complex I activity in platelets of PD patients can not be accounted for by an abnormality at the level of the rotenone binding site (putatively the ND-1 gene product), although they do not exclude differences in complex I activity between PD patients and controls.