The Oxidative Function of Diferric Transferrin

There is evidence for an unexpected role of diferric transferrin as a terminal oxidase for the transplasma membrane oxidation of cytosolic NADH. In the original studies which showed the reduction of iron in transferrin by the plasma membranes NADH oxidase, the possible role of the reduction on iron uptake was emphasized. The rapid reoxidation of transferrin iron under aerobic conditions precludes a role for surface reduction at neutral pH for release of iron for uptake at the plasma membrane. The stimulation of cytosolic NADH oxidation by diferric transferrin indicates that the transferrin can act as a terminal oxidase for the transplasma membrane NADH oxidase or can bind to a site which activates the oxidase. Since plasma membrane NADH oxidases clearly play a role in cell signaling, the relation of ferric transferrin stimulation of NADH oxidase to cell control should be considered, especially in relation to the growth promotion by transferrin not related to iron uptake. The oxidase can also contribute to control of cytosolic NAD concentration, and thereby can activate sirtuins for control of ageing and growth.

Related papers:

title: Mitochondrial free radical generation, oxidative stress, and aging.
abstract: Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.

title: The biomolecule ubiquinone exerts a variety of biological functions
abstract: The chemistry of ubiquinone allows reversible addition of single electrons and protons. This unique property is used in nature for aerobic energy gain, for unilateral proton accumulation, for the generation of reactive oxygen species involved in physiological signaling and a variety of pathophysiological events. Since several years ubiquinone is also considered to play a major role in the control of lipid peroxidation, since this lipophilic biomolecule was recognized to recycle α‐tocopherol radicals back to the chain‐breaking form, vitamin E. Ubiquinone is therefore a biomolecule which has increasingly focused the interest of many research groups due to its alternative pro‐ and antioxidant activity. We have intensively investigated the role of ubiquinone as prooxidant in mitochondria and will present experimental evidences on conditions required for this function, we will also show that lysosomal ubiquinone has a double function as proton translocator and radical source under certain metabolic conditions. Furthermore, we have addressed the antioxidant role of ubiquinone and found that the efficiency of this activity is widely dependent on the type of biomembrane where ubiquinone exerts its chain‐breaking activity.

title: Redox signaling: hydrogen peroxide as intracellular messenger
abstract: Although superoxide anions (O2.-) and H2O2 are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. In mammalian cells, a variety of extracellular stimuli have recently been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of stimulant-dependent signaling. In the next few years, therefore, a flurry of research activity is expected in relation to the elucidation of ROS production in response to receptor stimulation, identification of ROS target molecules, and investigation of ROS elimination. The goal of this report is to review our current knowledge of ROS-regulated signal transduction and propose future directions.

title: The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems.
abstract: The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.

title: The NADPH Oxidase of Phagocytic Leukocytes a
abstract: Phagocytic leukocytes (e.g. neutrophils, monocytes, macrophages, eosinophils) are essential for immunity to infection by bacteria and fungi. These organisms are engulfed into a “phagocytic vacuole,” an invagination of the plasma membrane, the neck of which fuses to form a closed compartment. Cytoplasmic granules containing an array of proteases and other antimicrobial compounds fuse with this vacuole and discharge their contents into the vacuolar lumen, in which the ingested microbe is killed and digested. Phagocytosis, as well as a wide variety of artificial stimuli, induces a burst of oxygen consumption, known as the “respiratory burst.’’ I This process involves the one electron reduction of oxygen to superoxide (Oz-) and is essential for the efficient killing of the ingested microorganism. Defects in the superoxide-generating system are associated with the syndrome chronic granulomatous disease (CGD) in which there is a profound and often fatal predisposition to bacterial and fungal infections.’ The various mutations in these patients leading to abnormalities in the different components of the NADPH oxidase have provided valuable tools with which to dissect out and understand the proteins and cofactors involved.z

title: Superoxide generation of phagocytes and nonphagocytic cells
abstract: SummaryPhagocytes release high amounts of reactive oxygen intermediates upon contact with appropriate stimuli into the environment as an important function in the immune defence against infectious agents. On the other hand nonphagocytic cells release low amounts upon stimulation, which have important functions in the inter- and intracellular signal transduction. Both systems represent a class of isoenzymes making a specific regulation possible. Fibroblasts, hepatocytes, and HeLa cells possess a superoxide system which shows higher activity with NADH than NADPH. The enzyme was purified to homogeneity in an active form, showed a molecular mass of 84 kDa and possessed a flavin and low-potential cytochromeb558.

title: NADPH oxidases: new regulators of old functions.
abstract: This issue of Antioxidants and Redox Signaling focuses on the biochemistry, physiology, and pathophysiology of the NADPH oxidase family of enzymes. Historically, NADPH oxidases were viewed as enzymes expressed in neutrophils that play a vital role in host defense against invading bacteria. Although this is clearly an important function of these proteins, we now know that there is an entire family of NADPH oxidases that mediate diverse functions in the organism. NADPH oxidase catalytic subunits (Nox) have been found in virtually all tissues (5), and have been shown to mediate a variety of responses, including cell growth and apoptosis, innate immunity, angiogenesis, regulation of the extracellular matrix, and thyroid hormone biosynthesis (16). They do so by modulating well-defined signaling pathways that are sometimes shared across tissues, but are sometimes tissue-specific. The articles included in this forum address many of these issues and highlight the current controversies and knowledge of the function of these important enzymes. 
 
Following the discovery of the first gp91phox homologue, Nox1 (2, 28), additional family members were rapidly identified. Known NADPH oxidases now include Nox1, found in colon and vascular cells, which functions in host defense and cell growth; Nox2, the phagocytic catalytic component of the respiratory burst oxidase that is widely distributed in other tissues as well; Nox3, which resides in the inner ear and has been shown to be involved in otoconia morphogenesis; Nox4, a widely distributed enzyme of particular abundance in kidney, bone, and vascular cells; Nox5, a calcium-dependent homologue that is mainly expressed in lymphoid tissues and testis; Duox1, a dual oxidase that contains a putative peroxidase domain, is found in thyroid and respiratory epithelial cells and functions in thyroid hormone synthesis; and Duox2, found in the thyroid and in gastrointestinal glandular epithelia (15). Understanding the regulation and function of each of these family members has been an enormous undertaking, but progress has been swift. We now recognize that reactive oxygen species derived from the Nox enzymes are critical for normal physiological responses, but also contribute to a growing number of diseases, putatively including atherosclerosis, hypertension, arthritis, Alzheimer’s disease, cancer, and respiratory syndromes. 
 
This forum contains five original articles, eight review articles, and one hybrid by some of the leaders in the NADPH oxidase field. The original articles focus mainly on the biological consequences of NADPH oxidase activation in different cell types. The hybrid article, by van Manen et al. (30), deals with new techniques to image the phagocyte oxidase at the single cell level, and contains original, previously unpublished images obtained using Raman spectroscopy. The review articles were selected to highlight areas of intense research in this field, and cover biochemical, molecular, and cell biological studies, as well as discussions of the impact of the NADPH oxidases on the normal function of various organs, including the kidney, brain, thyroid and vascular systems, and the GI tract. Each of these latter articles also reviews the literature on the pathophysiological consequences of dysregulated oxidase activation in disease. Recent identification of new regulatory homologues of the phagocyte oxidase components p47phox and p67phox has opened new vistas in our understanding of cell- and agonist-specific oxidase regulation. The biochemical regulatory mechanisms used by this family of enzymes are covered in a comprehensive article by Takeya and Sumimoto (29). Additionally, Bokoch and Zhao (4) have provided an outstanding overview of the detailed molecular mechanisms by which the small molecular weight G-protein Rac regulates the phagocyte NADPH oxidase. 
 
The first article in this volume, by Ranjan et al. (23), provides new data that address what has become a controversy in the field. The original report on Nox1 suggested that it was a mitogenic oxidase, based on studies in NIH3T3 fibroblasts transfected with a Nox1 plasmid (28). Later, it became apparent that the cells used in this study were already transformed by Ras (16), casting doubt on this conclusion. Other laboratories have suggested that Nox1 plays a role in host defense in the colon, as elegantly reviewed in this issue by Leto and Geiszt (17). New evidence, however, provided by Ranjan et al. (23), clearly shows that expression of Nox1 in mouse lung epithelial cells enhances growth by increasing cyclin D1 expression, and regulates cell cycle reentry by controlling transcriptional regulation of the Fos family genes. Together with previous data showing a relationship between Nox1 and angiogenesis (1), and the ability of Nox1 antisense to inhibit vascular smooth muscle growth (28), these new data argue that at least in some systems, Nox1 may be involved in cell growth. 
 
Another area of intense investigation revolves around the physiological function of Nox4. Nox4 was originally associated with cell senescence (7), and was believed to be constitutively active. Recent work, however, has implicated Nox4 in cell differentiation (6), apoptosis (21), and growth (20, 27), and has clearly shown that it can be activated by agonists such as TGF-β (6, 27) and insulin (18). Two new papers in this forum add to the potential functions and activators of Nox4 by showing an association between Nox4 and collagenase expression in chondrocytes stimulated with IL-1β (Grange et al., 10) and a role for Nox4 in endothelial cell proliferation (Petry et al., 22). This latter paper also demonstrates that two different NADPH oxidases, Nox2 and Nox4, expressed in the same cells, can act together to regulate physiological responses. In addition, the article by Block et al. (3) provides clear evidence that a p22phox-based oxidase, previously shown by this group to include Nox4 (9), mediates angiotensin II-induced mesangial cell hypertrophy. Taken together, the apparent opposing functions of Nox4 (senescence, apoptosis, proliferation, differentiation) appear to be dependent on the cell type and environmental context. As we learn more about the factors that regulate this Nox homologue, this paradox should resolve. 
 
A third theme that derives from this issue is the sensitivity of Nox proteins to mechanical forces. Guest et al. (11) show that cyclic strain stimulates ERK1/2 and expression of the proinflammatory gene monocyte chemoattractant protein-1 (MCP-1) in a manner dependent on the generation of reactive oxygen species derived from Nox1. NADPH oxidases have previously been shown to be activated by shear stress (12, 26), although the sensitivity of various oxidases (Nox1, 2, and 4) to different types of shear stress is controversial. Recent work has implicated bone morphogenic protein-4 (BMP-4) as an important regulator of this mechanical response. A thorough review of research in this area is provided in this issue by Jo et al. (14). 
 
As noted above, many of the reviews in this forum are devoted to the role of NADPH oxidases in specific organ systems, both physiologically and in disease development. Stalpers (24) has thoroughly reviewed the literature on the role of the duoxes in thyroid hormone synthesis. Importantly, she summarizes new evidence that mutations in Duox2 are linked to congenital hypothyroidism. Rokutan et al. (25) describe the role of Nox1 in host defense in the gastrointestinal epithelium, and evaluate the data for its involvement in inflammation-associated tumor development. Infanger et al. (13) provide an extensive review of the distribution, regulation, and function of NADPH oxidases in the brain. They discuss the role of Nox proteins in neuronal signaling, memory, and central cardiovascular homeostasis, and review the evidence for overproduction of reactive oxygen species in neurodegeneration and cardiovascular disease. Finally, Gill et al. (8) summarize the extensive literature on NADPH oxidases in the kidney. This group has been instrumental in identifying subunit expression in this tissue and in demonstrating a role for these oxidases in renovascular hypertension. The breadth of these articles clearly demonstrates the critical role of NADPH oxidases in multiple aspects of physiology and pathophysiology. 
 
Despite the recent enormous progress in NADPH oxidase research, many avenues remain to be explored. Much is still to be discovered regarding the function of the individual Nox proteins in particular cells, and the specific role of multiple Noxes expressed in individual cells. We also require a better understanding of the molecular mechanisms regulating Nox expression, how compartmentalization of Nox proteins mediates specificity, and the regulatory mechanisms responsible for oxidase activation in specific cells. This latter goal is particularly important for Nox4, which does not require the classical or novel regulatory subunits (9). Basic work in Drosophila and Dictyostelium can help to identify new downstream molecular targets of Nox enzymes. In addition, genetic association studies will provide insight into the role of these enzymes in development and disease. The development of new molecular tools, as well as transgenic and knockout animals, will greatly facilitate our understanding of the critical pathophysiological roles of this newly-defined protein family. The future is indeed full of promise.

title: Role of Nox family NADPH oxidases in host defense.
abstract: The phagocytic NADPH oxidase is recognized as a critical component of innate immunity, responsible for generation of microbicidal reactive oxygen species (ROS). This enzyme is one representative of the Nox family of oxidases (Nox1-Nox5, Duox1, and Duox2) that exhibit diverse expression patterns and appear to serve a variety of functions related to ROS generation. Mounting evidence now suggests that several of these novel oxidases also serve in host defense, particularly those showing high expression along epithelial surfaces exposed to the external environment. Within these sites, Nox enzymes tend to be located on apical cell surfaces and release ROS into extracellular environments, where they can be used by known antimicrobial peroxidases. Moreover, microbial factors were shown in several cases to cause higher ROS production, either by direct oxidase activation or by inducing higher oxidase expression. Several oxidases are also induced by immune cytokines, including interferon-gamma, interleukin (IL)-4, and IL-13. Although most of the evidence supporting host defense roles for mammalian nonphagocytic oxidases remains circumstantial, recent evidence indicates that Drosophila Duox plays a role in host resistance to infection. Finally, oxidative defense against invading pathogens appears to be an ancient protective mechanism, because related oxidases are known to participate in disease resistance in plants.

title: Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense
abstract: Lactoperoxidase (LPO) is an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions. Although the formation of microbicidal oxidants by LPO has been recognized for some time, the source of hydrogen peroxide (H2O2) for LPO‐catalyzed reactions remains unknown. Reactive oxygen species produced by the phagocyte NADPH oxidase (phox) play a critical role in host defense against pathogens; however, analogous oxidant‐generating systems in other tissues have not been associated with antimicrobial activity. Several homologues of gp91phox, the catalytic core of this enzyme, were described recently; dual oxidase (Duox)1/thyroid oxidase 1 and Duox2/thyroid oxidase 2 were identified in the thyroid gland and characterized as H2O2 donors for thyroxin biosynthesis. We examined Duox1 and Duox2 expression in secretory glands and on mucosal surfaces and give evidence for their presence and activity in salivary glands, rectum, trachea, and bronchium. Epithelial cells in salivary excretory ducts and rectal glands express Duox2, whereas tracheal and bronchial epithelial cells express Duox1. Furthermore, we detected Duox1‐dependent H2O2 release by cultured human bronchial epithelial cells. Our observations suggest that Duox1 and Duox2 are novel H2O2 sources that can support LPO‐mediated antimicrobial defense mechanisms on mucosal surfaces.

title: NADH oxidation in liver and fat cell plasma membranes
abstract: 1.  Evidence that transformed cells have an increased NADH/NAD ratio [I] and decreased adenylate cyclase [2] and evidence that NADH will inhibit the AMP cyclase of plasma membrane  has brought up the question of a sensor of NADH in the plasma membrane. The fact that the flavin antagonist atebrin inhibits cyclase suggested that an NADH dehydrogenase might be the site of the NADH inhibition  . There have been reports of NADH dehydrogenase activity in plasma membrane preparations.

title: A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.
abstract: NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.

title: Intracellular redox status affects transplasma membrane electron transport in pulmonary arterial endothelial cells.
abstract: Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-D-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD(+), NADPH, and NADP(+) were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD(+) ratio to 34% of the control level, without a significant change in the NADPH/NADP(+) ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD(+) and NADPH/NADP(+) ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP(+) ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD(+) redox pair.

title: Cell Surface NADH Oxidases (ECTO-NOX Proteins) with Roles in Cancer, Cellular Time-keeping, Growth, Aging and Neurodegenerative Diseases
abstract: ECTO-NOX (because of their cell surface location) proteins comprise a family of NAD(P)H oxidases of plants and animals that exhibit both oxidative and protein disulfide isomerase-like activities. The two biochemical activities, hydroquinone [NAD(P)H] oxidation and protein disulfide-thiol interchange alternate, a property unprecedented in the biochemical literature. A tumor-associated ECTO-NOX (tNOX) is cancer-specific and drug-responsive. The constitutive ECTO-NOX (CNOX) is ubiquitous and refractory to drugs. The physiological substrate for the oxidative activity appears to be hydroquinones of the plasma membrane such as reduced coenzyme Q10. ECTO-NOX proteins are growth-related and drive cell enlargement. Also indicated are roles in aging and in neurodegenerative diseases. The regular pattern of oscillations appears to be related to α-helix-β-structure transitions and serves biochemical core oscillator of the cellular biological clock. Period length is independent of temperature (temperature compensated) and synchrony is achieved through entrainment.

title: The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity.
abstract: The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.

title: A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane.
abstract: A 34 kDa coenzyme Q reductase has been solubilized and purified from pig liver plasma membranes. The solubilized enzyme reduced coenzyme Q0 with NADH. Ubiquinones with longer isoprenoid side chain such as Q2 and Q10 were also reduced when the quinones and the enzyme were reconstituted into phospholipid liposomes. N-terminal sequencing of an internal peptide showed identity to bovine NADH-cytochrome b5 reductase. Biochemical characterization of the purified enzyme indicated that the coenzyme Q reductase corresponds to an unusual form of NADH-cytochrome b5 reductase.

title: Requirement for coenzyme Q in plasma membrane electron transport.
abstract: Coenzyme Q is required in the electron transport system of rat hepatocyte and human erythrocyte plasma membranes. Extraction of coenzyme Q from the membrane decreases NADH dehydrogenase and NADH:oxygen oxidoreductase activity. Addition of coenzyme Q to the extracted membrane restores the activity. Partial restoration of activity is also found with alpha-tocopherylquinone, but not with vitamin K1. Analogs of coenzyme Q inhibit NADH dehydrogenase and oxidase activity and the inhibition is reversed by added coenzyme Q. Ferricyanide reduction by transmembrane electron transport from HeLa cells is inhibited by coenzyme Q analogs and restored with added coenzyme Q10. Reduction of external ferricyanide and diferric transferrin by HeLa cells is accompanied by proton release from the cells. Inhibition of the reduction by coenzyme Q analogs also inhibits the proton release, and coenzyme Q10 restores the proton release activity. Trans-plasma membrane electron transport stimulates growth of serum-deficient cells, and added coenzyme Q10 increases growth of HeLa (human adenocarcinoma) and BALB/3T3 (mouse fibroblast) cells. The evidence is consistent with a function for coenzyme Q in a trans-plasma membrane electron transport system which influences cell growth.

title: NADH diferric transferrin reductase in liver plasma membrane.
abstract: Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.

title: NADH Oxidase: A Multifunctional Ectoprotein of the Eukaryotic Cell Surface
abstract: The hormone-responsive external plasma membrane NADH oxidase (NOX) was first described for right side-out vesicles of plasma membranes isolated from dark-grown seedlings of soybean (Morre et al., 1986) in response to the auxin regulator of plant growth, 2,4-dichlorophenoxyacetic acid (2,4-D). A comparable hormone- and growth factor responsive NADH oxidase was subsequently described for isolated plasma membranes of rat liver. When hepatoma plasma membranes were examined, an NADH oxidase activity was observed but the activity was constitutively activated and no longer growth factor responsive (Bruno et al., 1992). Subsequent work has focused largely on the growth factor-insensitive activity of transformed cells grown in culture (Morre et al., 1995a). The latter has now been extensively characterized and is distinguished from that of non-transformed cells and tissues by being inhibited by both thiol reagents and several antitumor or potential antitumor drugs.

title: The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.
abstract: The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

title: Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver
abstract: Aging is associated with increased production of reactive oxygen species and oxidation-induced damage to intracellular structures and membranes. Caloric restriction (CR) is the only non-genetic method proven to extend lifespan in mammals. Although the mechanisms of CR remain to be clearly elucidated, reductions in oxidative stress have been shown to increase lifespan in several model systems. Oxidative stress can be attenuated by CR. Mitochondria and plasma membrane (PM) are normal sources of free radicals. The PM has a trans-membrane redox system that provides electrons to recycle lipophilic antioxidants, such as alpha-tocopherol and coenzyme Q (CoQ). The idea developed in this study is that the PM is intimately involved in cellular physiology controlling the relationship of the cell to its environment. PM is the key for protecting cellular integrity during aging. Specifically, we have investigated age-related alterations and the effects of CR in the trans-PM redox (antioxidant) system in rat liver. We found that age-related declines in the ratio of CoQ(10)/CoQ(9) and alpha-tocopherol in liver PM were attenuated by CR compared to those fed ad libitum (AL). CoQ-dependent NAD(P)H dehydrogenases were increased in CR old rat liver PMs. As a consequence, the liver PM of CR old rats was more resistant to oxidative stress-induced lipid peroxidation than AL rats. Thus, our results suggest that CR induces a higher capacity to oxidize NAD(P)H in the PM of old rat livers and as a result, a higher resistance to oxidative stress-induced damage.

title: Iron reverses impermeable chelator inhibition of DNA synthesis in CCl 39 cells.
abstract: Treatment of Chinese hamster lung fibroblasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.

title: Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells
abstract: Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inKm andVmax for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.

title: Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system.
abstract: Intact cells are known to reduce external, impermeable electron acceptors. We now show that cells can reduce the iron in diferric transferrin at the cell surface and that this reduction reaction depends on the transferrin receptor as well as the transmembrane electron transport system. Reduction of external diferric transferrin is accompanied by oxidation of internal NADH which indicates that the transmembrane enzyme is an NADH diferric transferrin reductase. Highly purified liver plasma membranes have NADH diferric transferrin reductase activity which shows properties similar to the diferric transferrin reductases activity of intact cells. Cell growth stimulation by diferric transferrin and other impermeable oxidants which can react with the diferric transferrin reductase can be based on electron transport through he plasma membrane.

title: Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.
abstract: Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.

title: Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.
abstract: Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.

title: Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase.
abstract: The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophenanthroline disulfonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.

title: The role of transferrin in the mechanism of cellular iron uptake.
abstract: The role of transferrin and its cell surface receptor in cellular iron metabolism has received considerable interest during the last decade. In this review wa attempt to summarize the new knowledge and controversies encountered during the last 4-5 years. The focus is on iron metabolism by the hepatocyte. We also view the concept of transferrin-cell interactions from angles which previously may not have been given particular attention, and we include some aspects of iron metabolism not normally covered by reviews of this type.

title: Transplasma Membrane Electron Transport Functions as a Ferric Reductase
abstract: We have previously presented evidence that ferric ions and iron in diferric transferric can be reduced at the cell surface by the transplasma membrane electron transport system acting in conjuction with the transferrin receptor 1,2. In this paper we examine the reaction further especially in relation to the inhibition of both ferric citrate and diferric transferrin reduction by apotransferrin. We also consider the difference in activity found with different diferric transferrin preparations containing different amounts of loosely bound ferric iron. We will also consider the effects of argon, and cell transformation on the rate of ferric iron reduction in the presence and absence of diferric transferrin. The objective is to evaluate the extent to which loosely bound iron on transferrin can contribute to the formation of ferrous bathophenanthroline disulfonate (Fe(BPS)3) through the transmembrane electron transport system. We also show that iron reduction at acidic pH by the transmembrane enzyme is not inhibited by apotransferrin which would favor its role for ferric reduction in acidic endosomes.

title: Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor.
abstract: Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.

title: NADH oxidase of liver plasma membrane stimulated by diferric transferrin and neoplastic transformation induced by the carcinogen 2-acetylaminofluorene.
abstract: NADH oxidase of purified plasma membranes (electron transfer from NADH to oxygen) was stimulated by the growth factor diferric transferrin. This stimulation was of an activity not inhibited by cyanide and was not seen in plasma membranes prepared from hyperplastic nodules from liver of animals fed the hepatocarcinogen, 2-acetylaminofluorene, nor was it due to reduction of iron associated with diferric transferrin. With plasma membranes from nodules, the activity was already elevated and the added transferrin was without effect. The stimulation by diferric transferrin did not correlate with the absence of transferrin receptors which were increased at the nodule plasma membranes. With liver plasma membranes, the stimulation by diferric transferrin raised the plasma membrane NADH oxidase specific activity to approximately that of the nodule plasma membranes. In contrast to NADH oxidase, which was markedly stimulated by the diferric transferrin, NADH ferricyanide oxidoreductase or reduction of ferric ammonium citrate by liver plasma membranes was approximately equal to or slightly greater than that of the nodule plasma membrane and unaffected by diferric transferrin. The results suggest the possibility of coupling of NADH oxidase activity to a growth factor response in mammalian cells as observed previously for this enzyme in another system.

title: Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.
abstract: The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.

title: Evidence for a different mechanism of lactoferrin and transferrin translocation on HT 29-D4 cells.
abstract: The fate of [125I] lactoferrin after binding on its specific receptor on HT29-D4 cells, a clone of HT29 cells, was studied and compared with that of [125I]transferrin. Time courses of protein binding in relation with temperature clearly show that different mechanisms of ligand-transportation for the two glycoproteins exist on these cells: whereas transferrin would enter into the cell probably by a relatively classic pathway of receptor-mediated endocytosis, lactoferrin and its specific receptor would not be internalized. Thus the mechanism by which lactoferrin promotes HT29 cell growth seems quite different from that of transferrin on the same cells.

title: Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide.
abstract: The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.

title: Control of Transplasma Membrane Diferric Transferrin Reductase by Antitumor Drugs
abstract: It is well known that anthracycline antibiotics have been used successfully in the treatment of human cancer. It has long been thought that DNA is the primary target for the cytotoxic action of these drugs on susceptible cells (Chandra, 1987; DiMarco, 1975; Kanter et al., 1979; Ross et al., 1970). The DNA receptor hypothesis is attractive due to a reasonably high affinity between some of these drugs and nucleic acid. However, this hypothesis does not fully explain the antimitogenic effect of these drugs, since significant mitotic inhibition by daunomycin and adriamycin are observed under conditions in which DNA synthesis is unaffected (Silvetrini, 1970,1973). The N-substituted derivatives, such as N-acetyl daunomycin and N-trifluoro-acetyl adriamycin-l4-valerate (AD32) have very low affinity toward DNA, yet they are capable of inhibiting cell mitosis (DiMarco et al., 1965; DiMarco, 1975; Kanter et al., 1979). Similarly, bleomycin and its chelate, Cu-bleomycin, inhibit mitosis equally well although selective strong DNA binding affinity is shown only by bleomycin (Sun and Crane, 1985a; Gutteridge and Chang, 1981). Furthermore, both cis and trans diammine dichloroplatinum II bind equally well with DNA, yet the trans analog is not effective as an antitumor agent (Macquet and Butour, 1985; Salles et al., 1983) and is much less cytotoxic than the cis analog (Pascoe and Roberts, 1974). Therefore, there are reasons to suspect that additional targets other than DNA are involved in killing cancer cells with these drugs.

title: Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1
abstract: SummaryPlasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.

title: Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system
abstract: Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.

title: Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes
abstract: The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H2O2. The rate of H2O2 formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theKm value for NADH was found to be 3×10−6 M. The H2O2-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H2O2 generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H2O2. In view of the growing evidence that H2O2 can be involved in metabolic control, the formation of H2O2 by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.

title: A multifunctional hydroquinone oxidase of the external cell surface and sera
abstract: A multifunctional cell surface protein with NADH oxidase (NOX) activity and capable of oxidizing hydroquinones is located at the exterior of the cell and is shed in soluble form into sera. The oxidase appears to function as a terminal oxidase of a trans plasma membrane electron transport chain consisting of a NAD(P)H‐ubiquinone reductase at the cytosolic membrane surface, possibly a b‐type cytochrome, ubiquinone and the oxidase. Hyperactivity or conditions that interrupt ordered 2H+ + 2e− transport from NAD(P)H or hydroquinone to molecular oxygen and other acceptors at the external cell surface may result in the generation of superoxide. The latter may serve to propagate aging‐related redox changes both to adjacent cells and circulating blood components. A circulating NOX activity form associated with aging and the reduction of cytochrome c by sera of aged patients that is partially inhibited by ubiquinone are described.

title: Diferric transferrin reduction stimulates the Na+/H+ antiport of HeLa cells.
abstract: Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.

title: Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).
abstract: Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

title: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage
abstract: Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFκB activation.

title: Sirtuins, Bioageing, and Cancer
abstract: The Sirtuins are a family of orthologues of yeast Sir2 found in a wide range of organisms from bacteria to man. They display a high degree of conservation between species, in both sequence and function, indicative of their key biochemical roles. Sirtuins are heavily implicated in cell cycle, cell division, transcription regulation, and metabolism, which places the various family members at critical junctures in cellular metabolism. Typically, Sirtuins have been implicated in the preservation of genomic stability and in the prolongation of lifespan though many of their target interactions remain unknown. Sirtuins play key roles in tumourigenesis, as some have tumour-suppressor functions and others influence tumours through their control of the metabolic state of the cell. Their links to ageing have also highlighted involvement in various age-related and degenerative diseases. Here, we discuss the current understanding of the role of Sirtuins in age-related diseases while taking a closer look at their roles and functions in maintaining genomic stability and their influence on telomerase and telomere function.

title: Essential role of copper in the activity and regular periodicity of a recombinant, tumor-associated, cell surface, growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2)
abstract: ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

title: HIF hydroxylation and the mammalian oxygen-sensing pathway.
abstract: Hypoxia Hypoxia refers to below-normal levels of oxygen in air, blood, or tissue. Tissue hypoxia leads to cellular dysfunction and ultimately can lead to cell death. Causes of tissue hypoxia include (a) decreased blood oxygenation (such as occurs in certain pulmonary disorders), (b) altered oxygen release from hemoglobin (associated with some hemoglobinopathies), and (c) impaired blood delivery leading to localized anemia (i.e., ischemia) as a result of low cardiac output or vascular obstruction. In order to adapt to hypoxia, mammals use a number of physiological responses. These include (a) increased production of erythropoietin (EPO), which augments the production of red blood cells; (b) induction of tyrosine hydroxylase, which facilitates the control of ventilation through the carotid body; and (c) the stimulation of new blood vessels by upregulation of VEGF (1). At the cellular level, hypoxia induces a number of metabolic changes that allow for continued energy generation despite decreased oxygen availability.

title: The hypoxia‐inducible‐factor hydroxylases bring fresh air into hypoxia signalling
abstract: Metazoans rapidly respond to changes in oxygen availability by regulating gene expression. The transcription factor hypoxia‐inducible‐factor (HIF), which controls the expression of several genes, ‘senses’ the oxygen concentration indirectly through the hydroxylation of two proline residues that earmarks the HIF‐α subunits for proteasomal degradation. We review the expression, regulation and function of the HIF prolyl hydroxylases or prolyl hydroxylases domain proteins, which are genuine oxygen sensors.

title: Hypoxia signalling in cancer and approaches to enforce tumour regression
abstract: Tumour cells emerge as a result of genetic alteration of signal circuitries promoting cell growth and survival, whereas their expansion relies on nutrient supply. Oxygen limitation is central in controlling neovascularization, glucose metabolism, survival and tumour spread. This pleiotropic action is orchestrated by hypoxia-inducible factor (HIF), which is a master transcriptional factor in nutrient stress signalling. Understanding the role of HIF in intracellular pH (pHi) regulation, metabolism, cell invasion, autophagy and cell death is crucial for developing novel anticancer therapies. There are new approaches to enforce necrotic cell death and tumour regression by targeting tumour metabolism and pHi-control systems.