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S Papa

Publications and source records attributed to S Papa.

At least 145 records · Page 8Linked to original sources
PubMed1994

The proton pump of heme-copper oxidases.

Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The CuB-heme a3 (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper: i) the efficiency of coupling in the mitochondrial aa3 cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H+/e- stoichiometry and the role of subunit IV; ii) mutational analysis of the aa3 quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H+/e- coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.

Animals↗
PubMed1994

Mechanistic and phenomenological features of proton pumps in the respiratory chain of mitochondria.

Various direct, indirect (kinetic and thermodynamic), and combined mechanisms have been proposed to explain the conversion of redox energy into a transmembrane protonmotive force (delta p) by enzymatic complexes of respiratory chains. The conceptual evolution of these models is examined. The characteristics of thermodynamic coupling between redox transitions of electron carriers and scalar proton transfer in cytochrome c oxidase and its possible involvement in proton pumping is discussed. Other aspects dealt with in this paper are: (i) variability of <--H+/e- stoichiometries, in cytochrome c oxidase and cytochrome c reductase and its mechanistic implications; (ii) possible models by which the reduction of dioxygen to water at the binuclear heme-copper center of protonmotive oxidases can be directly involved in proton pumping. Finally a unifying concept for proton pumping by the redox complexes of respiratory chain is presented.

Animals↗
PubMed1994

Induction of binucleation in human lymphocytes by 14 synthetic isoindolone derivatives related to cytochalasins.

Fourteen hydrogenated isoindolone derivatives with affinity to cytochalasin B were synthesized and tested for the induction of binucleation in the human lymphocyte micronucleus (MN) assay. The experimental procedure was the one commonly used for the human lymphocyte MN assay. Compounds like cyt-B were added to cultures at 44 h and tested at increasing concentrations, up to 200 mumol/l, in the range commonly used for cyt-B in the MN assay (3 micrograms/ml = 6.25 mumol/l). Induction of cytokinesis-blocked binucleated cells was found for all compounds but, at the same molarity, cyt-B induced a higher percentage of binucleation. Only one of the compounds tested was found to induce micronuclei significantly: MN were induced across the dose range of 25-100 mumol/l.

Cytochalasin B↗
PubMed1994

Age dependent changes in mitochondrial FoF1 ATP synthase in regenerating rat-liver.

The effect of aging on rat liver regeneration and on the FoF1-ATP synthase complex of isolated liver mitochondria was followed after partial (70%) hepatectomy. ATP hydrolase activity in submitochondrial particles prepared from regenerating liver was first depressed; the time needed to reach the lowest activity was age dependent. This decrease was accompanied by parallel decrease of i) the respiratory rate of succinate supplemented mitochondria in state III; ii) the respiratory control index; iii) the rate of synthesis of ATP in succinate supplemented submitochondrial particles. This first phase of liver regeneration, characterized at all ages by a lag phase in the growth, was followed by a second phase in which the tissue mass was restored and the enzyme activities normalized. Immunoblot analysis showed that the changes in the catalytic activities of the FoF1-ATP synthase observed during liver regeneration were accompanied by parallel changes in the amount of subunits of both the catalytic (F1) and the membrane (Fo) sector of the complex.

Aging↗
PubMed1994

The blockage of the human transferrin receptor by a monoclonal antibody, EA.3, induces growth inhibition in leukemia cell lines.

In our program to produce monoclonal antibodies against hemopoietic functional antigens we have obtained an hybridoma cell line EA.3 reacting with human transferrin receptor. This monoclonal antibody is an IgM that displays "in vitro" antiproliferative activity against leukemic cells due to its competition with transferrin on the receptor. Cell cytotoxicity was demonstrated either with the blockage of BrdU incorporation in HL-60 cells and with leukemic CFU inhibition. In contrast, K562 cells were insensitive to the blockage of the transferrin receptor induced by this MAb.

Antibodies, Monoclonal↗
PubMed1994

Inhibition of NK binding to K562 cells induced by MAb saturation of adhesion molecules on target membrane.

Cell to cell interaction play a major role in the induction of a immune response. NK cells represent a special lymphoid subset which displays its cytotoxic function without the engagement of MHC system. In order to investigate the role of different adhesion molecules in the mechanism of binding of the NK cell to the classic tumor target K562 cell, we have employed different unclustered mAbs of the Adhesion session (5th "CD" Workshop, Boston 1993) mostly of the CAM (cell adhesion molecule) subpanel. After their reactivity characterization both on lymphocytes and K562 cells, those that demonstrate reactivity against the tumor target were tested in the binding assay. The target was pretreated with the monoclonal in order to block a possible reacting molecule for the effector. Then, after the incubation of lymphocytes with PE-labelled anti CD16 mAb, their ability to bind to the target was tested in flow. While the majority of the mAbs did not induce any significant change in the binding capacity of the NK subset, few of them did, and precisely anti-CD58 (LFA-3) and anti-CD54 (ICAM-1) which showed different level of inhibition, particularly drastic with S002, S083 and S100. Other mAbs, such as the S011 (anti-CD59), due to the presence of the PI-linked glycoprotein recognized on both target and effector membranes, and to its capacity to stimulate NK activity, produced a total binding of the NK population. The coincubation of targets with anti-CD54 and anti-CD58 allowed to reduce at the lowest level this function. This data seem to support the hypothesis of specific surface molecules involved in the binding process of the NK cell, after recognition of the target.

Antibodies, Monoclonal↗
PubMed1993

Phosphorylation of mitochondrial proteins in bovine heart. Characterization of kinases and substrates.

Protein phosphorylation by [gamma-32P]ATP in total extract and subfractions of bovine heart mitochondria has been studied. The results show that, in addition to pyruvate dehydrogenase, three mitochondrial proteins, with molecular weights of 44,000, 39,000 and 31,000 Da, are phosphorylated by a cAMP-independent mitochondrial protein kinase. Three other proteins associated with mitochondria, with molecular weights of 125,000, 19,000 and 6,500 Da, are phosphorylated by the cytoplasmic cAMP-dependent protein kinase (kinase A).

Animals↗
PubMed1993

Deficiency of pyruvate dehydrogenase complex in tissues of an eight month old infant.

A metabolic investigation was carried out in an eight-month old infant with intrauterine hypotrophia, failure to thrive, psychomotoric retardation and cerebral atrophy, who died after respiratory infections. Blood analysis revealed intermittent lactic acidosis with normal lactate/pyruvate ratio. Activities of cytochrome c oxidase in skeletal muscle, heart, liver and fibroblasts were all in the reference range of controls. Activity of pyruvate dehydrogenase complex (PDH) was decreased in muscle homogenate, heart and liver mitochondria but was normal in cultured skin fibroblasts. Immunodetection of PDH subunits, and assay of El alpha phosphorylation showed in the patient decrease of E1 alpha in skeletal muscle, and enhanced level of E1 alpha phosphorylation in liver mitochondria.

Cells, Cultured↗
PubMed1993

Functional and molecular changes in FoF1 ATP-synthase of cardiac muscle during aging.

We have studied the functional and molecular changes of mitochondrial FoF1 ATP synthase of cardiac muscle during aging. ATP hydrolase activity was lower in sonic submitochondrial particles prepared from hearts of senescent rats (24 months) than in those prepared from hearts of adult rats (12 months). Oligomycin-sensitive proton conduction of cardiac submitochondrial particles was greater in senescent rats than in adult rats. The beta subunit of F1, detected immunologically in submitochondrial particles, was less in senescent rats than in adult rats. Exposure of cardiac submitochondrial particles from adult rats to free radicals, generated by 60Co, resulted in inactivation of ATP hydrolase activity and a decreased content of F1. The structural and functional alterations of mitochondrial FoF1 ATP synthase during aging may be expected to affect energy metabolism, and our results suggest that they could originate from the action of free radicals generated in the inner mitochondrial membrane.

Aging↗
PubMed1992

pH changes associated with cytochrome c oxidase reaction with H2O2. Protonation state of the peroxy and oxoferryl intermediates.

pH changes associated with the mitochondrial cytochrome oxidase reaction with H2O2 have been studied. In the presence of ferricyanide or Tris-phenanthroline complex of CoIII as electron acceptors, reaction of H2O2 with the oxidized cytochrome oxidase is accompanied by a steady proton release with a rate constant of ca. 3 M-1.s-1 at pH 6.8. The acidification is completely inhibited by superoxide dismutase and its pre-steady-state kinetics correlates with that of the oxoferryl compound (F) accumulation. Apparently, the proton release is linked to superoxide generation by cytochrome oxidase under these conditions. In the presence of superoxide dismutase and without the electron acceptors, the H2O2-induced transitions of cytochrome oxidase from the oxidized to the peroxy (P) and from the peroxy to the oxoferryl state are not associated with any significant proton release or uptake. The results point to the following mechanism of O2- generation and protonation states of the cytochrome oxidase compounds P and F: [formula: see text]

Animals↗
PubMed1992

Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.

Animals↗
PubMed1992

Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.

Adenosine Triphosphate↗
PubMed1992

Natural killer function in flow cytometry. III. Surface marker determination of K562-conjugated lymphocytes by dual laser flow cytometry.

The recognition of effector cell populations that are able to actively from conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the binding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8dim) has been studied on both CD3+ and CD16+ lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3+ 8dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.

Antigens, Differentiation↗
PubMed1992

DNA synthesis progression in 3T3 synchronized fibroblasts: a high resolution approach.

In order to investigate at ultrastructural level the mechanism of DNA synthesis progression during the different moments of S-phase a bromodeoxyuridine-anti bromodeoxyuridine (BrdU-anti BrdU) method has been applied to synchronized 3T3 fibroblasts. After 30 min BrdU incorporation, five different labelling patterns can be identified and should be related to early, middle and late S-phase. These patterns are represented mainly by diffuse labelling localized in different nuclear domains and by quite rare cases in which the labelling is limited to isolated clusters of gold particles. After a 5-min pulse with BrdU it is possible to observe isolated clusters of gold particles at each moment of S-phase, which, however, exhibit the same distribution of the five principal labelling patterns observed after 30 min incorporation. In both cases labelling can be detected in the interchromatin regions during early S-phase, at the boundary between interchromatin and heterochromatin during middle S-phase and in the heterochromatin domains during late S-phase. Considering their size, the isolated spots of labelling could be interpreted as single replication units which are subsequently activated throughout the different moments of the S-phase.

3T3 Cells↗
PubMed1992

The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism.

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.

Adult↗