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Biomedical subjects

M L Eboli

Publications and source records attributed to M L Eboli.

At least 19 recordsLinked to original sources
PubMed1996

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells.

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.

Animals↗
PubMed1994

Transformation linked decrease of pyruvate dehydrogenase complex in human epidermis.

Epidermis exhibits glycolytic features peculiar to cancer cells. The activity of pyruvate dehydrogenase complex, both active (PDHa) and total (PDHt) forms, has been investigated and compared in epidermis and epidermal carcinomas from human source. Low or undetectable PDHa is found in either normal and neoplastic tissue. PDHt is unchanged in human epidermis between the second and seventh decades of life but is dramatically decreased following neoplastic transformation (0.107 and 0.026 units/g fresh tissue for epidermis and epidermal carcinoma, respectively). As PDH plays a key role in mitochondrial carbohydrate metabolism, the decrease of total enzymic capacity found in tumors suggest that different mechanisms regulate PDH expression and, in turn, glycolytic mechanisms of epidermis and cancer cells.

Adult↗
PubMed1994

Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.

Animals↗
PubMed1993

Differential involvement of protein kinase C in transmitter release and response to excitatory amino acids in cultured cerebellar neurons.

Cerebellar granule cells cultured in the presence of a differentiating factor isolated from rabbit serum exhibit, at variance with those cultured in fetal calf serum, an almost complete resistance to excitatory aminoacid (EAA)-induced cytotoxicity. We investigated the behaviour of protein kinase C (PKC), strongly implicated in EAA cytotoxicity, in the two types of culture. Phorbol esters, used to monitor the enzyme, enhanced the depolarization-evoked release of D-[3H]aspartate, but less effectively in factor-conditioned cells. EAAs increased phorbol esters binding in both cultures, but the effect was briefly lasting in factor-conditioned cells. The different behaviour of PKC is postulated to be causally related to different response to EAA of the cultures.

Amino Acids↗
PubMed1993

Cerebellar neurones: differentiation and modulation of sensitivity to excitotoxic treatment.

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C18 column. SDS-PAGE analysis of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.

Animals↗
PubMed1989

Chronic myeloid leukemia with monoclonal gammopathy terminating in myeloid crisis and immunoblastic lymphoma.

A patient, with chronic myeloid leukemia and IgA monoclonal gammopathy, who contemporaneously developed myeloid blast crisis and immunoblastic lymphoma is reported. Cytogenetic studies showed complex chromosome abnormalities concerning chromosomes 8, 14 and 22, other than the Ph chromosome. A possible relationship between the emergence of immunoblasts from slow proliferating lymphoplasmacytoid cells, myeloid blasts crisis and chromosomal changes is discussed.

Blast Crisis↗
PubMed1986

Oxidation of reduced cytosolic nicotinamide adenine dinucleotide by the malate-aspartate shuttle in the K-562 human leukemia cell line.

The activity of the malate-aspartate shuttle for the reoxidation of reduced cytosolic nicotinamide adenine dinucleotide (NADH) by mitochondria was studied in a line of human myeloid leukemia cells (K-562). The tumor cells showed mitochondrial reoxidation of cytosolic NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain in the reoxidation of cytosolic NADH was demonstrated by the action of rotenone, antimycin A, and oligomycin which strongly inhibited the formation of pyruvate from added L-lactate. Moreover, pyruvate production was greatly inhibited by the transaminase inhibitor, aminooxyacetate. Under glycolytic conditions, in the presence of aminooxyacetate, the rate of pyruvate production was also markedly inhibited, the rate of lactate accumulation was stimulated, and at 60 min the cytosolic NADH/nicotinamide adenine dinucleotide (NAD) ratio had increased progressively about 5-fold with respect to untreated cells. The maximal rate of the malate-aspartate shuttle has also been established by addition of arsenite to inhibit mitochondrial oxidation of the pyruvate formed from added L-lactate.

Aminooxyacetic Acid↗
PubMed1985

Pyruvate dehydrogenase levels in Morris hepatomas with different growth rate.

Pyruvate dehydrogenase (PDH) activity has been evaluated with respect to normal liver in 3 lines of Morris hepatomas (H), i.e. the highly differentiated H 9618A, the well differentiated H 44 and the poorly differentiated H 3924A. Assays of both total (PDHt) and active (PDHa) form show a progressive decrease of enzyme activity going from liver to the H 3924A. PDHa better correlates with the degree of hepatoma differentiation than does PDHt. Further enzyme analysis has been achieved in partially purified extracts from liver and H 3924A. The possible implications of such an enzymatic variation are discussed.

Animals↗
PubMed1983

Inhibition of mitochondrial oxidative metabolism by SKF-525A in intact cells and isolated mitochondria.

We have examined the effects of various concentrations of SKF-525A (beta-diethylaminoethyldiphenylpropyl acetate X HCl) on the energy metabolism of liver slices, isolated liver mitochondria, and two types of ascites tumor cells, as well as on ion transport in liver slices. In liver slices, 0.2 to 1.0 mM SKF-525A caused an initial stimulation of O2 uptake which was followed, at 0.5 to 1.0 mM, by a progressive inhibition of O2 consumption, a fall of slice ATP content, and a reduced transport of K+, Na+ and Ca2+. In isolated mitochondria, we studied the effects of SKF-525A on the rate of respiration and on the oxidation-reduction responses of NAD(P)+ and cytochrome b in the presence of various substrates. The results suggest that SKF-525A had three distinct actions on liver mitochondria, viz. an uncoupling action at low concentrations (0.02 to 0.17 mM); at higher concentrations (0.2 to 0.5 mM) an inhibition of the oxidation of NAD(P)+-linked substrates, exerted close to the substrate level; also at 0.2 to 0.5 mM, a less effective inhibition of electron transfer at a point between cytochrome b and O2 in the electron-transfer chain. Experiments on O2 consumption and cytochrome b oxidation-reduction changes in ascites cells showed only the first two of these effects in the intact tumor cells. We conclude that inhibition of mitochondrial energy-conserving reactions by SKF-525A can have a marked influence on energy-requiring aspects of liver-cell metabolism, one example of which is inhibition of cation active transport.

Adenosine Triphosphate↗
PubMed1981

Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells.

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (-)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as two inhibitors glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricarboxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.

Adenosine Triphosphate↗
PubMed1979

Calcium transport and translocation of adenine nucleotides in mitochondria from Morris hepatoma 3924A.

The interaction of Ca2+ with Morris hepatoma 3924A mitochondria and its effect on the adenine nucleotide translocation have been studied. The characteristics of the Ca2+ transport process in mitochondria from Morris hepatoma are not significantly different from those of normal liver mitochondria. The Km for Ca2+ is 2 to 3 microM, and the rate versus concentration curve exhibits hyperbolic kinetics. A lower activity of the adenine nucleotide translocation was found, probably due to the high endogenous Ca2+ content of Morris hepatoma mitochondria (123 +/- 15 nmol Ca2+ per mg protein). No further inhibition of the translocase activity was observed after isolated mitochondria had accumulated extra amounts of Ca2+. The total amount of adenine nucleotides in tumor mitochondria is one-half those present in control liver, and a significantly lower percentage of the pool is present as adenosine 5'-monophosphate.

Adenine Nucleotides↗
PubMed1977

Pyruvate transport in tumour-cell mitochondria.

Tumour-cell mitochondria contain a pyruvate-transporting system exhibiting the same general properties as those described in rat liver mitochondria. The Km for net pyruvate uptake in tumour-cell mitochondria is practically similar to that measured in rat liver mitochondria but the V is lower. This difference is also shown by swelling experiments. The possible implication of these observations in the context of lactate accumulation in tumour-cell is discussed.

Animals↗
PubMed1977

NAD(P)H utilization in the reduction of pyruvate to lactate in a glycogen-containing subline of Ehrlich ascites tumour cells.

The possible pathways of utilization of glucose-6-phosphate (G-6-P) produced from glycogen breakdown have been investigated in a glycogen-containing subline of Ehrlich ascites tumour cells. Addition of either mitochondrial inhibitors or pyruvate to ascites cells metabolizing endogenous substrates enhances the rate of lactate production. However, only in the former condition such effect is abolished by iodoacetate (IAA). In pyruvate-supplemented cells mitochondrial inhibitors cause a further increase in lactate production which becomes insensitive to IAA when the cells are depleted of endogenous substrates. Measurements of the glycogen content show that either in the presence of mitochondrial inhibitors or pyruvate there is a stimulation of glycogenolysis. Significant changes (about 10--20 fold increase) of the G-6-P level are observed only in the presence of both mitochondrial inhibitors and IAA, irrespective of pyruvate addition. However, with pyruvate the accumulation of G-6-P becomes lower if the cells are starved. The results obtained indicate that in our conditions G-6-P which is produced during glycogenolysis may be oxidized either through the Embden-Meyerhof pathway or the phosphogluconate pathway. Indeed, whereas mitochondrial inhibitors promote the utilization of this metabolite through the first route by enhancing the activity of phosphofructokinase, added pyruvate favours the other route by lowering the cytosolic NADPH/NADP+ ratio.

Anaerobiosis↗
PubMed1976

Transport of anionic substrates and glutamate metabolism in mitochondria from ascites tumor cells.

A study is presented of alpha-oxoglutarate and glutamate transport and of glutamate oxidation in ascites tumor cell mitochondria. Kinetics analysis of alpha-oxoglutarate transport in mitochondria from two strains of Ehrlich ascites tumor cells, the hyperdiploid and the hyperdiploid Lettré mutant, shows that the activity of the alpha-oxoglutarate carrier and its affinityfor substrates are higher in the mutant than in the wild strain. Evidence is presented showing the occurrence of carrier mediated glutamate-OH-exchange-diffusion in mitochondria from both strains. The activity of the glutamate carrier is apparently higher in the hyperdiploid Lettré mutant. Glutamate oxidation occurs mainly through transamination to asparatate in both tumor stains. The rate of deamination in the two strains correlates directly with the level of glutamide dehydrogenase (EC 1.4.1.3.), which is higher in the wild than in the mutant strain. Thus glutamate dehydrogenase per se, and not glutamate penetration, constitutes the control step for gluttochondria of glutamate with externally added oxaloacetate (arsenite present) that exclude an obligatory transport of oxaloacetate on the alpha-oxoglutarate carrier.

Animals↗