Biomedical subjects

A L Moore

Publications and source records attributed to A L Moore.

At least 91 records · Page 5Linked to original sources

PubMed1989

Regulation of alternative pathway activity in plant mitochondria: nonlinear relationship between electron flux and the redox poise of the quinone pool.

The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.

Electron Transport↗

PubMed1989

Role of nonohmicity in the regulation of electron transport in plant mitochondria.

The relationship between the respiratory rate and the membrane ionic current on the protonmotive force has been investigated in percoll purified potato mitochondria. The dependence of the membrane ionic current on the membrane potential was monitored using a methyltriphenylphosphonium-sensitive electrode and determining the maximal net rate of depolarization following the addition of a respiratory inhibitor. We have confirmed that a nonohmic relationship exists between the ionic conductance and membrane potential. Addition of ATPase inhibitors markedly increased the initial rate of dissipation suggesting that in their absence the dissipation rate induced by respiratory inhibitors is partially offset by H(+)-efflux due to the hydrolysis of endogenous ATP. This was corroborated by direct measurement of endogenous ATP levels which decreased significantly following dissipation of the membrane potential. Results are discussed in terms of the regulation of electron transport in plant mitochondria in vivo.

Journal Article↗

PubMed1989

Increased susceptibility to inoculated Lewis lung carcinoma (3LL) but unaltered tumor growth in mice treated with monoclonal antibody to L3T4 on mouse T-helper cells.

The importance of different lymphocyte cell populations in early recognition and destruction of tumor cells has not been fully established. Certainly natural killer cells and cytotoxic T lymphocytes are involved. Using a recently developed monoclonal antibody (GK 1.5) that has been shown to have in vivo cytotoxic activity directed at L3T4-bearing T cells, we provide in these experiments evidence that T-helper cells are also important in early antitumor immunity. Development of progressive tumor growth after the subcutaneous inoculation of 10(5) Lewis lung carcinoma (3LL) cells was greater in antibody-treated mice (13 of 20 treated mice vs. 6 of 21 controls). Nevertheless, in those animals that developed tumors, the latentcy period (time from tumor cell inoculation until tumor first palpable) and tumor growth rate were no different in antibody-treated mice when compared with control animals. In subsequent experiments, animals were exposed to irradiated tumor cells in Freund's adjuvant on three occasions and tumor growth was then assessed. Growth was slower in the sensitized group. Administration of GK 1.5, however, did not enhance the tumor growth rates in either the previously sensitized or control groups. The results suggest that T-helper cells might be of greater functional importance in early tumor cell recognition and destruction and of lesser importance in the restraint of tumor growth, once the tumor has become established.

Animals↗

PubMed1988

Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision.

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.

Cytochrome Reductases↗

PubMed1988

Immunologic enhancement of B16 melanoma growth.

In certain experimental tumor models, tumor growth is less pronounced in immune deficient animals. Characteristically, tumors such as the murine B16 melanoma and Lewis lung carcinoma (3LL) are weakly antigenic. We proposed that with such tumors that are weakly antigenic, growth is enhanced by T-cell factors. Young mice were inoculated with irradiated B16 cells in complete Freunds adjuvant (CFA) on three occasions, each separated by 2 weeks. Specific antibody (IgG) to B16 membrane antigens was detected by an enzyme-linked immunosorbent assay (ELISA) after the first injection, and it continued to rise for 6 weeks. B16 growth was compared in 20 mice that had received irradiated B16 in CFA or CFA alone by the same schedule previously. Despite the previous sensitization, the rates of tumor appearance and growth were similar. In an additional experiment involving 23 mice that had received B16 immunization, the period of time in which a palpable tumor developed after the injection of viable B16 cells did not correlate with anti-B16 antibody level. It appeared that detectable antibody to B16 antigens was of little consequence. To explain why B16 primary growth and metastases were reduced in immune deficient hosts, we proposed that lymphocytes might enhance tumor growth. To demonstrate this, splenic lymphocytes from tumor-bearing (B16 or 3LL) or control mice were injected with B16 cells into young, immune competent hosts. Tumors (B16) developed earlier and growth was more rapid in mice that received spleen cells from tumor-bearing (B16) mice. Subsequent cell depletion experiments to determine the mediator of tumor enhancement implicated a T-cell fraction that was neither of T-helper nor T-suppressor cell type phenotypically. Immune deficiency states that are associated with dysfunction of those cells that account for tumor enhancement might explain the reduced tumor aggressiveness that is observed frequently in these conditions.

Animals↗

PubMed1988

Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism.

The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin-resistant rate of O(2) uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone-resistant O(2) uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O(2) uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge.

Journal Article↗

PubMed1988

Effect of commercial peritoneal dialysis fluids on the lytic function of lymphokine-activated killer cells.

Intraperitoneal (ip) immunotherapies are often administered in saline or peritoneal dialysis solutions. In preparation for a Phase I i.p. interleukin-2/lymphokine-activated killer cell (IL-2/LAK) trial in patients with recurrent ovarian cancer, we evaluated the effect of six solutions, including two 1.5% dextrose peritoneal dialysis solutions (PDS), on the lytic function of LAK. In vitro IL-2-activated LAK cells were exposed to solutions, washed with Hanks' balanced salt solution, and lytic activity was measured in a standard 4-h chromium release assay using Daudi as a tumor target. LAK function was abrogated after 2 h of exposure to Inpersol and after only 20 min of exposure to Dianeal PD-2. Five percent dextrose also significantly decreased lytic capabilities of LAK, whereas 0.9% sodium chloride or lactated Ringers had no deleterious effect on function. Adjustment of PDS to pH greater than 6.5 decreased the damaging effect on LAK function, suggesting that the low pH of PDS and 5% dextrose results in a loss of LAK cell viability and therefore lytic function. Based on these data, we have chosen to administer IL-2/LAK i.p. in lactated Ringers.

Cytotoxicity Tests, Immunologic↗

PubMed1987

Pyruvate transport by thermogenic-tissue mitochondria.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

Acrylates↗

PubMed1987

Inhibition of lymphocyte nucleic acid metabolism and antibody production by trimetrexate.

Trimetrexate is a lipid soluble dihydrofolate reductase inhibitor which, unlike methotrexate, does not depend upon the membrane folate transport system for cell entry. We investigated the possibility that trimetrexate (but not methotrexate) might permeate intermitotic lymphocytes and, following stimulation, impair only the responding cells, rather than all dividing cells, as is the case with methotrexate. Peripheral blood mononuclear cells from normal individuals were incubated for 1 hr in three moderate to high concentrations (1, 10 and 100 microM) of methotrexate or trimetrexate, washed, and incubated with phytohemagglutinin. Intracellular folate activity, as assessed by the deoxyuridine suppression test, was abnormal at all three concentrations of trimetrexate but only at the highest concentration of methotrexate. Similarly, incorporation of [3H]deoxyuridine was depressed profoundly in trimetrexate-treated cells (2% of control) but unaffected by methotrexate. Analysis of cell cycle distribution by flow cytometry confirmed G0 + G1 arrest in trimetrexate but not methotrexate-treated cells. Neither drug altered morphologic transformation, Tac antigen expression, or incorporation of [3H]thymidine by the "salvage" pathway. Therefore, brief exposure to methotrexate has little effect on intermitotic lymphocytes, whereas trimetrexate very specifically inhibits the conversion of deoxyuridine to thymidine in these cells and leads to the arrest of DNA synthesis in the G0 + G1 phase. This metabolic abnormality markedly reduces in vitro antibody synthesis: a 1-hr treatment of lymphocytes with 10 or 100 microM trimetrexate prior to incubation with pokeweed mitogen on four occasions completely inhibited both IgG and IgM secretion. Similar treatment with methotrexate had no effect until the highest concentration (100 microM) was used. We conclude that brief exposure of peripheral blood mononuclear cells to the nonclassical dihydrofolate reductase inhibitor, trimetrexate, results in inhibition of nucleic acid synthesis and impairment of antibody production. This drug effect may permit more incisive modulation of immune responses.

Antibody Formation↗

PubMed1987

Tumor vasculature in young and old hosts: scanning electron microscopy of microcorrosion casts with microangiography, light microscopy and transmission electron microscopy.

Tumor growth in vivo is dependent upon new blood vessel formation. When B16-F10 melanoma cells are implanted subcutaneously in young (3 mo) and old (24 mo) C57BL/6 mice the rate of growth is dependent on the age of the mice. This study involved a wide range of histological and microscopic techniques but was limited primarily to the initial phase of tumor growth. Stereological point counting from light microscopy (LM) of standard histological sections has been used to yield data regarding blood content. Tumor-bearing mice were perfused through the aorta with a fixation solution and were infused with a low-viscosity radiopaque gel (Microfil) or resin (Mercox). Soft x-rays of the whole animal were used for identifying the feeding vessels to the tumor. Tumors with Microfil were sliced and used for microangiography and light-microscopic observation while those with resin were used to make corrosion casts for scanning electron microscopy (SEM). The different characteristics of the tumor blood vessels in different aged mice were most obvious through SEM of vascular corrosion casts. In comparison with tumors in young mice those of similar size in old hosts had more necrosis, reduced presence of angiogenic features, decreased vessel density, reduced penetration into the tumor, and enhanced tortuosity of the vessel lumen. Transmission electron microscopy (TEM) revealed incompletely developed wall structure of the vessels regardless of host. The above results are consistent with the hypothesis that retarded angiogenesis may be responsible in part for the limited growth of tumors in old hosts.

Animals↗

PubMed1986

Immunological analysis of plant mitochondrial NADH dehydrogenases.

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.

Chemical Precipitation↗

PubMed1986

Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.

Adenosine Triphosphatases↗

PubMed1986

The age-related decline in antibody response is transferred by old to young bone marrow transplantation.

The immune response declines with age. This decline correlates with thymic involution and involves primarily a loss in T-cell function, whereas humoral immunity is more variably affected. In the current experiments we have measured immunoglobulin synthesis in vitro after mitogen stimulation, and specific antibody response after vaccination. We found that the response to pokeweed mitogen by non-specific immunoglobulin production, and the response to vaccine was shown to be transferred to lethally irradiated young mice by old to young bone marrow transplantation. Both pokeweed mitogen and tetanus toxoid require T-cell help for optimal response, and, therefore, our observations are in accordance with the age-associated decline in T-cell immunity. The finding that young hosts transplanted with old bone marrow produce less antibody than young hosts transplanted with young bone marrow highlights the importance of the decline in cellular function with age.

Aging↗

PubMed1986

Slower B16 melanoma growth but greater pulmonary colonization in calorie-restricted mice.

Recently, it has been demonstrated that the immune function is preserved longer and spontaneous tumors occur less frequently in calorie-restricted (but not malnourished) mice. In the present report the effect of similar dietary manipulation on tumor growth, metastases, and survival was studied. C57BL/6 mice were fed regular laboratory diets, either restricted in calories or not restricted, and later inoculated with B16 melanoma sc, iv, or ip. Local tumor growth was found to be slower; however, survival after ip injection was no different, and the number of pulmonary metastatic colonies after iv injection was greater for the underfed mice. In this weakly immunogenic tumor model, factors in addition to immunity influenced tumor growth and were altered by calorie restriction.

Animals↗

PubMed1985

Purification of F1-ATPase from cuckoo-pint (Arum maculatum) mitochondria. A comparison of subunit composition with that of rat liver F1-ATPase.

Plant mitochondrial ATPase has been chloroform-solubilized and purified by gel filtration from spadices of cuckoo-pint (Arum maculatum). The subunit composition of purified plant and rat liver ATPase were compared by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The delta- and epsilon-subunits of the plant enzyme are larger than their supposed rat liver counterparts and, as such, A. maculatum mitochondrial ATPase shows structural homologies with the enzyme from Escherichia coli [Futai, Sternweis & Heppel (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2725-2729] rather than with the rat liver enzyme.

Animals↗

PubMed1985

Interleukin-2 and aging: decreased interleukin-2 production in healthy older people does not correlate with reduced helper cell numbers or antibody response to influenza vaccine and is not corrected in vitro by thymosin alpha 1.

The capacity of lymphocytes obtained from healthy young or old volunteers to produce interleukin-2 was measured and the results were compared with other measures of immune function. The in vitro effect of thymosin alpha 1 on interleukin-2 production was also measured. Interleukin-2 was lower in lymphocytes from the elderly, and individuals with low production also had lower proliferative responses in vitro to phytohemagglutinin. These individuals did not have a reduced helper T-cell number, abnormal ratio of helper to suppressor T-cells or reduced antibody production in response to vaccine. Thymosin alpha 1 did not have a consistent effect on interleukin-2 production.

Adult↗

PubMed1985

Specific antibody synthesis in vitro. III. Correlation of in vivo and in vitro antibody response to influenza immunization in young and old subjects.

Young and elderly volunteers received a trivalent influenza vaccine and blood samples were obtained prior to, 1 week and 3 weeks after immunization. Plasma antibody levels and the capacity for in vitro lymphocyte synthesis of specific anti-influenza antibody were assessed. Additionally, prior to immunization, peripheral blood mononuclear cells were cultured in the presence of antigen, and the capacity for in vitro immunization was determined. Antibody response was therefore measured after in vivo and in vitro immunization. After in vivo immunization, plasma antibody level and in vitro synthesis capabilities were significantly greater in the young subjects, and these assays correlated significantly. The capacity for in vitro immunization was also greater in the young, but the difference did not reach statistical significance and there was no correlation with in vitro immunization potential and the response after in vivo vaccination. Assessment of in vitro antibody synthesis after vaccination however, may prove a useful tool for probing immune deficient states and testing the success, in vitro of various immune reconstitution measures.

Adult↗

PubMed1984

Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria.

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

Chromatography, Gel↗