Biomedical subjects

D J Morré

Publications and source records attributed to D J Morré.

At least 19 recordsLinked to original sources

PubMed2005

Benzo[b]thiophenesulphonamide 1,1-dioxide derivatives inhibit tNOX activity in a redox state-dependent manner.

Benzo[b]thiophenesulphonamide 1,1-dioxide (BTS) derivatives are strong cytotoxic agents that induce reactive oxygen species (ROS) overproduction and apoptosis in tumour cells. Although the precise origin of BTS-induced ROS is not known, a clear correlation between their cytotoxic effect and ability to inhibit a tumour-associated NADH oxidase (tNOX) activity of the plasma membrane has been described. To analyse the putative implication of tNOX in BTS-induced ROS generation, in this work we have synthesised and tested a new BTS derivative, the 6-[N-(2-phenylethyl)]benzo[b]thiophenesulphonamide 1,1-dioxide. According to its high lipophilicity, this compound showed a strong cytotoxic activity against a panel of six human tumour cell lines, including two human leukaemia (K-562 and CCRF-CEM) and four human solid tumours (HT-29, HTB54, HeLa and MEL-AC). We also tested the ability of this compound to inhibit the tNOX activity and we found an absolute dependence of this inhibition on the redox state of the tNOX: while under reducing conditions, that is, 100 mM GSH, the drug inhibits strongly the NOX activity with an EC(50) of about 0.1 nM, under oxidising conditions, there is no effect of the drug or just a slight stimulation of activity.

Antineoplastic Agents↗

PubMed2001

Surface NADH oxidase of HeLa cells lacks intrinsic membrane binding motifs.

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37 degrees C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.

Blotting, Western↗

PubMed2001

Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.

Amino Acid Sequence↗

PubMed2001

Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.

Animals↗

PubMed2001

NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms.

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

Activity Cycles↗

PubMed2001

Is the cancer protective effect correlated with growth inhibitions by green tea (-)-epigallocatechin gallate mediated through an antioxidant mechanism?

The preferential inhibition by (-)-epigallocatechin gallate (EGCg) of growth of cancer cells (e.g. HeLa) in culture correlates with the ability of EGCg to inhibit a growth-related, cell surface hydroquinone oxidase with protein disulfide-thiol interchange activity (tNOX) measured as an NADH oxidase and specifically associated with tumorigenically-transformed cells and tissues. tNOX is reduced or absent from the surface of non-cancer cells. Various oxidizing conditions known to render other antioxidants such as thiols, ascorbate and vitamin E ineffective did not reduce the effectiveness of EGCg in inhibiting either the tNOX activity or the growth of HeLa cells. Only after Fenton reaction with iron catalysis in the presence of hydrogen peroxide was the effectiveness of the EGCg reduced. We conclude that it is unlikely that the anticancer action of green tea EGCg on the tNOX protein is mediated through antioxidant properties of EGCg.

Anticarcinogenic Agents↗

PubMed2000

CHO cell enlargement oscillates with a temperature-compensated period of 24 min.

The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14 degrees C, 24 degrees C or 34 degrees C) while the rate of cell enlargement increased with temperature over this same range of temperatures.

Animals↗

PubMed2000

Periodic NADH oxidase activity associated with an endoplasmic reticulum fraction from pig liver. Response to micromolar concentrations of retinol.

An endoplasmic reticulum fraction from pig liver enriched in transitional endoplasmic reticulum vesicles capable of forming 50-60 nm buds in the presence of ATP and retinol was assayed for retinol-responsive oxidation of NADH and cleavage of a dithiodipyridine (DTDP) protein disulfide-thiol interchange substrate. Maxima for the two activities alternated giving rise to a 24 min period. The NADH oxidase activity was inhibited by micromolar and submicromolar concentrations of retinol. Retinol at 0.1 mM stimulated the activity. The inhibition was confined to two activity maxima separated in time by about 5 min. In contrast, with the DTDP substrate, the activity was stimulated by retinol and the stimulations were in the part of the oscillatory pattern where retinol inhibition of NADH oxidation was observed. The findings support an earlier proposed mechanism whereby retinol exerted opposing effects on NADH oxidation and protein disulfide reductions.

Animals↗

PubMed2000

Preferential inhibition by (-)-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture.

A drug-responsive and cancer-specific NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa and human mammary adenocarcinoma by the naturally-occurring catechin of green tea, (-)-epigallocatechin-3-gallate (EGCg). With cells in culture, EGCg preferentially inhibited growth of HeLa and mammary adenocarcinoma cells compared with growth of mammary epithelial cells. Inhibited cells became smaller, and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. Mammary epithelial cells recovered from EGCg treatment even at 50 microM, whereas growth of HeLa and mammary adenocarcinoma cells was inhibited by EGCg at concentrations as low as 1 microM with repeated twice-daily additions and did not recover from treatment with 50 microM EGCg. The findings correlate inhibition of cell surface NADH oxidase activity and inhibition of growth with EGCg-induced apoptosis.

Apoptosis↗

PubMed2000

Applications of aqueous two-phase partition to isolation of membranes from plants: a periodic NADH oxidase activity as a marker for right side-out plasma membrane vesicles.

Phase separations using standardized mixtures of polyethylene glycol, dextran and potassium phosphate are used widely to prepare highly purified plasma membranes from plants and in the preparation of chloroplast subfractions. Other uses include the removal of right side-out plasma membrane vesicles as contaminants from Golgi apparatus, endoplasmic reticulum and tonoplast (vacuole membrane) fractions and separation of right side-out and inside-out plasma membrane vesicles. The higher degree of separation between plasma membranes into the upper phase and internal membranes into the lower phase is in large measure due to the fact that only plasma membranes are oriented cytoplasmic side in. Most other membranes are oriented cytoplasmic side-out. This property extends to separations of right side-out and inside-out plasma membrane vesicles and to the separation of right side-out and inside-out sub-mitochondrial particles. The inside-out vesicles partition into the lower phase whereas the right side-out vesicles remain in the upper phase. The lack of efficacy of aqueous two-phase partitioning in other types of membrane separations is apparently due to the fact that surface characteristics that may distinguish different internal membranes are not located at the cytosolic membrane surface. At present there are no direct enzymatic markers for right side-out plasma membrane vesicles from plants. Demonstrations of sidedness and estimates of fraction purity are based on measurements of latency of marker enzymes, e.g., ATPases, at the cytosolic surface. This report describes a periodic NADH oxidase as an enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions of vesicles for measurement of activity.

Cell Fractionation↗

PubMed2000

The plasma membrane NADH oxidase of soybean has vitamin K(1) hydroquinone oxidase activity.

Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K(1) hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K(1) (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K(1) reduction or oxygen consumption. The K(m) for reduced K(1) was 350 microM. With the partially purified enzyme, reduced vitamin K(1) was oxidized at a rate of about 600 nmol/min/mg protein and the K(m) was 400 microM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 microM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2, 4-dichlorophenoxyacetic acid. 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 reduced vitamin K(1) to acceptors (molecular oxygen or protein disulfides) at the cell surface.

Cell Membrane↗

PubMed2000

Cell surface NADH oxidase activity of brine shrimp oscillates with a period of 25 min and is entrained by light.

Plants have a surface NADH oxidase that measures time by oscillating with a 24-min period. The period is synchronized by light. With plants, a new maximum is observed exactly 12 min after the beginning of the light exposure. These experiments were to determine if animals exhibited a cell surface NADH oxidase having a similar periodicity and to answer the question, does the periodicity in animals respond to light? Using brine shrimp as a model, the findings show that plants and animals exhibit similar oscillating NADH oxidase activity and that the periodicity in this invertebrate animal does respond to light. Brine shrimp were grown for two to three days and transferred to darkness for 45 min. After return to light for one min, NADH was added and measurements of NADH oxidation were recorded over 50 min. The brine shrimp exhibited a cell surface NADH oxidase that oscillated with a period of 25 min. After being subjected to light, the brine shrimp showed a new maximum in NADH oxidation between 12 to 13 min after the beginning of the light exposure and again at 37 min and at 25 min intervals thereafter. The findings demonstrate that the periodic oscillations in NADH oxidation of brine shrimp are light entrainable.

Animals↗

PubMed2000

A light-responsive and periodic NADH oxidase activity of the cell surface of Tetrahymena and of human buffy coat cells.

Oxidation of external NADH (NADH is an impermeant substrate) by cells of Tetrahymena pyriformis oscillated with a period of 24-26 min. The period length in darkness (25.6 min) appeared to be slightly longer than the period in light (approximately 24 min). When Tetrahymena were placed in darkness for 30-50 min and then returned to light, a new maximum in the rate of NADH oxidation was observed 36-38 min (13 + 24) min after the beginning of the light treatment. The cell-surface NADH oxidase of human buffy coats (a mixture of white cells and platelets) also was periodic and light responsive.

Animals↗

PubMed2000

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum.

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

Adenosine Triphosphatases↗

PubMed2000

Surface oxidase and oxidative stress propagation in aging.

This report summarizes new evidence for a plasma-membrane-associated hydroquinone oxidase designated as CNOX (constitutive plasma membrane NADH oxidase) that functions as a terminal oxidase for a plasma membrane oxidoreductase (PMOR) electron transport chain to link the accumulation of lesions in mitochondrial DNA to cell-surface accumulations of reactive oxygen species. Previous considerations of plasma membrane redox changes during aging have lacked evidence for a specific terminal oxidase to catalyze a flow of electrons from cytosolic NADH to molecular oxygen (or to protein disulfides). Cells with functionally deficient mitochondria become characterized by an anaerobic metabolism. As a result, NADH accumulates from the glycolytic production of ATP. Elevated PMOR activity has been shown to be necessary to maintain the NAD(+)/NADH homeostasis essential for survival. Our findings demonstrate that the hyperactivity of the PMOR system results in an NADH oxidase (NOX) activity capable of generating reactive oxygen species at the cell surface. This would serve to propagate the aging cascade both to adjacent cells and to circulating blood components. The generation of superoxide by NOX forms associated with aging is inhibited by coenzyme Q and provides a rational basis for the anti-aging activity of circulating coenzyme Q.

Animals↗

PubMed1999

Drug-antibody conjugates with anti-HIV activity.

Human immunodeficiency virus (HIV)-specific peptide antibody-brefeldin A conjugates and antibody-glaucarubolone conjugates directed to cell surface viral glycoprotein epitopes were prepared and tested for antiviral activity. A selective response was observed both on survival of cell lines permanently infected with lentiviruses and on HIV infectivity. With human peripheral blood mononuclear cells (PBMCs), the conjugate also was effective in reducing virus titers. The effectiveness of an HIV-specific peptide antibody-brefeldin A conjugate was enhanced by combination with 3'-azido-3'-deoxythymidine (AZT) and was effective against AZT-resistant isolates in combination with AZT. The conjugates reduced virus production in MOLT-4 cells and in HIV-1-infected PBMCs without affecting the viability of uninfected cells.

Amino Acid Sequence↗

PubMed1999

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.

Adenosine Triphosphate↗

PubMed1999

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity.

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.

Cell Membrane↗