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Biomedical subjects

J M Villalba

Publications and source records attributed to J M Villalba.

At least 19 recordsLinked to original sources
PubMed1997

Ascorbate stabilization is stimulated in rho(0)HL-60 cells by CoQ10 increase at the plasma membrane.

Long-term treatment with ethidium bromide of HL-60 cells induced a mitochondria-deficient rho degree cell line, where mitochondrial DNA can not be identified by PCR and cytochrome c oxidase activity was 80% decreased. These cells showed a progressive increase of ascorbate stabilization which was 52% higher in the established rho degree HL-60 cells. Both CoQ10 and NADH-ascorbate free radical reductase of the plasma membrane were increased in rho(0)HL-60 cells compared to parental cells, while NADH-cytochrome c reductase was unchanged. CoQ10 is a component of the ascorbate stabilization activity in the plasma membrane that would provide both a mechanism to deplete the excess of NADH produced in rho(0)HL-60 cells and for resistance to oxidative stress.

Ascorbic Acid↗
PubMed1997

Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane.

Cytochrome b5 reductase purified from liver plasma membrane reduces coenzyme Q (CoQ) in reconstituted liposomes in the absence of cytochrome b5. Both CoQ and its reductase are responsible for the reduction of the ascorbate free radical at the cell surface. Thus, NADH-CoQ reductase represents a partial reaction of NADH-AFR reductase in the plasma membrane. Cytochrome b5 reductase maintains CoQ and ascorbate in their reduced state to support antioxidations. Reduced CoQ prevents lipid peroxidation in liposomes and plasma membranes. Also, oxidized CoQ can prevent lipid peroxidations in the presence of cytochrome b5 reductase and NADH. Addition of CoQ to intact cells prevents serum withdrawal-induced lipid peroxidation and apoptosis. The prevention of apoptosis by CoQ is independent of the bcl-2 protein content in the cell. Antioxidants that act at the plasma membrane as CoQ and ascorbate would represent a first barrier to protect lipids from oxidative stress and subsequent apoptosis. Cytochrome b5 reductase is then an enzyme leading this function at the plasma membrane. These data support the idea that when the plasma membrane barrier fails, bcl-2 protein would be required to prevent cell death.

Animals↗
PubMed1997

Antioxidant ascorbate is stabilized by NADH-coenzyme Q10 reductase in the plasma membrane.

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ10 and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ10 reductase activity and its internal sequence being identical to cytochrome b5 reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ10 content in isolated plasma membranes. We show here the role of CoQ10 and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b5-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ10 and its NADH-dependent reductase.

Animals↗
PubMed1997

Plasma membrane ubiquinone controls ceramide production and prevents cell death induced by serum withdrawal.

Serum provides cultured cells with survival factors required to maintain growth. Its withdrawal induces the development of programmed cell death. HL-60 cells were sensitive to serum removal, and an increase of lipid peroxidation and apoptosis was observed. Long-term treatment with ethidium bromide induced the mitochondria-deficient rho(o)HL-60 cell line. These cells were surprisingly more resistant to serum removal, displaying fewer apoptotic cells and lower lipid peroxidation. HL-60 cells contained less ubiquinone at the plasma membrane than rho(o)HL-60 cells. Both cell types increased plasma membrane ubiquinone in response to serum removal, although this increase was much higher in rho(o) cells. Addition of ubiquinone to both cell cultures in the absence of serum improved cell survival with decreasing lipid peroxidation and apoptosis. Ceramide was accumulated after serum removal in HL-60 but not in rho(o)HL-60 cells, and exogenous ubiquinone reduced this accumulation. These results demonstrate a relationship between ubiquinone levels in the plasma membrane and the induction of serum withdrawal-induced apoptosis, and ceramide accumulation. Thus, ubiquinone, which is a central component of the plasma membrane electron transport system, can represent a first level of protection against oxidative damage caused by serum withdrawal.

Apoptosis↗
PubMed1996

Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H+-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H+-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H+-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H+-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H+-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.

Amino Acid Sequence↗
PubMed1996

Modified plant plasma membrane H(+)-ATPase with improved transport coupling efficiency identified by mutant selection in yeast.

Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H(+)-ATPase). Random mutants of Arabidopsis H(+)-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H(+)-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced gy site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200-500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different specifies of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H(+)-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plant transporters.

Adenosine Triphosphate↗
PubMed1995

Topography of the 27- and 31-kDa electron transport proteins in the onion root plasma membrane.

Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane.

Allium↗
PubMed1995

A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane.

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.

Animals↗
PubMed1995

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.

Animals↗
PubMed1995

The effect of amphotericin B on the viability of cryopreserved human skin.

To evaluate whether amphotericin B has a detrimental effect on the viability of cryopreserved skin, human skin samples were incubated during 7 and 24 h in three different media: 0.9% phosphate-buffered saline and Medium 199 with and without 10 micrograms/ml amphotericin B. The viability of the tissue was assessed by measuring the tetrazolium reductase activity before and after cryopreservation. A significant decrease in cryopreserved human skin viability was observed after 7 and 24 h incubation with amphotericin B. These results indicate that in the processing of skin preservation, the beneficial effect of using amphotericin B as an antifungal agent may be reduced by its negative effect on tissue viability.

Amphotericin B↗
PubMed1995

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.

Amino Acid Sequence↗
PubMed1994

Cyclic AMP-plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes.

Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the Km for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37 degrees C with cyclic AMP (0.1-10 nM) and ATP (1-100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the Km for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of Km and Vmax as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1-2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4 degrees C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4 degrees C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37 degrees C or after storage at 4 degrees C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in Km, cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.

Adenosine Triphosphate↗
PubMed1994

Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

Animals↗
PubMed1994

Two distinct NAD(P)H-dependent redox enzymes isolated from onion root plasma membranes.

Plasma membranes purified by two-phase partition from onion roots catalyzed the NAD(P)H-dependent reduction of a variety of electron acceptor such as ferricyanide, quinones, dyes and ascorbate free radical. Among these, NAD(P)H-ferricyanide and -quinone oxidoreductase activities were effectively solubilized by Triton X-100. Both oxidoreductase activities were bound to an affinity column of Blue-Sepharose CL 6B. NADH eluted a redox enzyme showing more juglone than ferricyanide-dependent activity. Ulterior unspecific elution with salt allowed us to the partial purification of a different redox enzyme of about 31 kDa that reduced better ferricyanide than quinones and constituted the bulk of solubilized redox activity.

Allium↗
PubMed1993

Transplasma membrane redox system of HL-60 cells is controlled by cAMP.

Transplasma membrane redox activity of HL-60 cells was determined by measuring the prevention of ascorbate chemical oxidation. The ascorbate free radical produced as the first step of ascorbate oxidation was reduced back by the transplasma membrane electron transport system, causing then the regeneration of extracellular ascorbate. Agents that increase intracellular cAMP, such as forskolin and dibutyryl cAMP (db-cAMP), increased the rate of ascorbate regeneration by HL-60 cells. Also, the phosphodiesterase-resistant cAMP analogue Sp-cAMP-S (agonist of the protein kinase A) increased the electron flow to the ascorbate free radical at the plasma membrane. Rp-cAMP-S, antagonist of the protein kinase A, partially inhibited the redox activity of cells and abolished the effect of Sp-cAMP-S. Inhibition obtained after preincubation of cells in Rp-cAMP-S was reversed by Sp-cAMP-S. Tunicamycin, a compound that inhibited the electron flow to the ascorbate free radical at the plasma membrane, also reduced the response of transplasma membrane redox system to Sp-cAMP-S. Lactate slightly affected the ascorbate regeneration in nonstimulated cells, but showed a significant effect on Sp-cAMP-S-stimulated plasma membrane electron flow. We show here a role for cAMP in the short-term modulation of transplasma membrane redox system measured as the regeneration of ascorbate at the cell surface of HL-60 cells, probably mediated by cAMP-dependent protein kinases.

Ascorbic Acid↗