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Biomedical subjects

F Antoni

Publications and source records attributed to F Antoni.

At least 55 records · Page 3Linked to original sources
PubMed1988

cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2 alpha synthesis in mouse hepatocytes.

The role of cAMP dependent regulation in thromboxane A2, prostacyclin and PGF2 alpha synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N6,O2-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA2, PGI2 and PGF2 alpha by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchymal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.

Animals↗
PubMed1988

Structural changes in the mouse thymus and lymph node after emetine treatment.

The effect of emetine which is a potent immunosuppresant was studied on the thymus and lymph node. The subcapsular zone of the thymus was depleted and large number of adherent cells accumulated in this thymic region. The medulla enlarged but the cortico-medullary border remained distinct. In the paracortex (T dependent area) of the lymph node many non-lymphoid pyroninophil cells appeared which is followed by an increased cell proliferation 24 hours after emetine injection. 48-60 hours after administration many macrophage-like cells appeared in the medullary sinuses. This macrophage invasion precedes the adherent cell accumulation in the subcapsular zone of the thymus suggesting a possible non-lymphoid (adherent) cell migration from the lymph node's paracortex to the thymus.

Animals↗
PubMed1988

DNA synthesis and nucleoside metabolism in human tonsillar lymphocyte subpopulations.

High (HD) and low (LD) density cells were separated on 25% BSA gradient from tonsils of 3-6 years old children. Early B lymphocyte markers and sIg-s were found on the surface of 59-82% of the LD cells. This cell population was 5-6 times more active in DNA synthesis (3H-thymidine incorporation, DNA polymerase activity) than the HD cells. The total uptake of 3H-deoxycytidine was about the same as that of 3H-thymidine. As long as practically all thymidine taken up by the cells was immediately incorporated into DNA (90-95%), only 10-15% of deoxycytidine was incorporated into DNA under the same conditions, indicating different pool sizes for the DNA precursors. The majority of deoxycytidine (70%) was converted and incorporated as dTMP. A considerable part of labeled deoxycytidine could be detected in the soluble pool in form of nucleotides (3-8%), and in an unknown form, called substance X (8-14%). Substance X was purified by TL chromatography and identified by HPLC as deoxycytidine containing liponucleotides, probable precursors for plasmamembranes. The preferential utilisation of deoxycytidine for DNA and membrane synthesis in immature B lymphocytes draws the attention to its function in early events of lymphocyte maturation.

Antigens, Differentiation↗
PubMed1987

Proteolytic activation of protein kinase C in the extracts of cells treated for a short time with phorbol ester.

A 10 min treatment of human neutrophils with phorbol 12-myristate 13-acetate (PMA) has been reported to induce accumulation of the proteolytically activated Ca2+/phospholipid-independent catalytic fragment of protein kinase C in the cytosol of intact cells [(1986) J. Biol. Chem. 261, 4101-4105]. We investigated the proteolytic conversion of protein kinase C to Ca2+/phospholipid-independent form in the cytosol and membrane fractions of pig neutrophils. The activity of protein kinase C was measured with its specific oligopeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously. In our experiments the short-term treatment of neutrophils with PMA did not induce the accumulation of the proteolytically activated form of protein kinase C in the cytosol of intact cells. However, treatment of cells with PMA enhanced the limited proteolysis of protein kinase C during the preparation of cell extracts.

Animals↗
PubMed1987

Interrelationship between aminopyrine oxidation and gluconeogenesis in hepatocytes prepared from fructose-pretreated mice.

Aminopyrine oxidation was studied in isolated hepatocytes prepared from 24-h-starved mice (i) after induction of the NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase, but not the mixed function oxygenases by fructose, (ii) after induction of both mixed function oxygenases and NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase by phenobarbital and (iii) without any pretreatment. Phenobarbital pretreatment, as expected, increased the rate of aminopyrine oxidation of isolated hepatocytes. However, fructose pretreatment also enhanced the rate of N-demethylation of aminopyrine by more than 100% supporting the view that the availability of NADPH is rate limiting in drug oxidation under certain conditions. The role of malic enzyme and glucose-6-phosphate dehydrogenase in the NADPH supply for aminopyrine oxidation was investigated by the addition of two groups of gluconeogenic precursors: lactate or alanine and glycerol or fructose with the simultaneous measurement of glucose synthesis and aminopyrine N-demethylation. There was a clear correlation between the increased rate of aminopyrine oxidation and the decreases of glucose production caused by aminopyrine. Gluconeogenesis in the presence of 1 mM aminopyrine was decreased by 70-80% when alanine or lactate were used as precursors, it was decreased by only 35-40% when glucose production was started from glycerol or fructose; in an accordance with the facts that NADPH generation and gluconeogenesis starting from alanine or lactate share two common intermediates--malate and glucose-6 phosphate--, while there is only one common intermediate--glucose-6 phosphate--if fructose or glycerol are used. Similar results were obtained with the addition of the structurally dissimilar hexobarbital. It is concluded that besides malic enzyme, glucose-6-phosphate dehydrogenase also takes part in NADPH supply for drug oxidation in glycogen-depleted hepatocytes.

Aminopyrine↗
PubMed1987

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light.

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.

Chloramphenicol↗
PubMed1987

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells.

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.

Animals↗
PubMed1987

Preferential utilisation of deoxycytidine by undifferentiated (peanut positive) tonsillar lymphocytes.

Peanut agglutinin (PNA), a D-galactose specific lectin, agglutinated 10-18% of lymphocytes isolated from tonsils of 3- to 6-yr-old children. PNA+ cells were found to be mainly B lymphocytes showing a 9.6 times higher specific activity of DNA polymerase alpha compared to the PNA- cells. The specific activity of deoxycytidine kinase as well as the incorporation of [5-3H]deoxycytidine were also much higher in PNA+ cell fraction than in PNA- fraction (7.7-fold and 6-fold, respectively). On the other hand, thymidine kinase activity and [5-3H]deoxythymidine incorporation were only 3.6 and 3.9 times higher, respectively. The data presented here show a high degree of DNA synthesis and preferential utilisation of [5-3H]deoxycytidine for DNA synthesis in this undifferentiated B lymphocyte fraction.

B-Lymphocytes↗
PubMed1987

The immunosuppressive effect of acute doses of emetine on murine thymic cells.

Emetine--a general inhibitor of protein synthesis--was investigated for its ability to depress specific immune response in animal models. A single dose (33 mg/kg) of emetine administered subcutaneously to mice markedly decreased thymus weight and thymic cell numbers. DNA, RNA and protein synthesis of thymic cells were reduced by 90, 50 and 65%, respectively. RNA synthesis was the first process to recover followed by the reconstitution of DNA and protein synthesizing capacity. Histological evidence revealed an effect on the cortical region of thymus upon emetine administration. Results suggest that emetine exerts an immunosuppressive effect on T-cell maturation in the thymus. These findings may enhance a therapeutic interest in the drug.

Animals↗
PubMed1987

Prostanoid synthesis in isolated parenchymal and nonparenchymal mouse liver cells in the presence of arachidonic acid.

Prostanoid synthesis was investigated in suspensions of isolated mouse hepatocytes and nonparenchymal liver cells. A stable metabolite of thromboxane A2 (TXB2) of prostacyclin (6-keto PGF1 alpha) and one of the prostaglandins (PGF2 alpha) was detected by radio-immuno-assay (RIA). Hepatocytes synthesized mainly TXB2, while smaller amounts of 6-keto PGF1 alpha and PGF2 alpha were detected during 60 min incubation. Homogenization of hepatocytes caused a slight increase of TXB2 production and provoked the synthesis of PGF2 alpha and 6-keto PGF1 alpha. The addition of arachidonate to hepatocytes did not influence prostanoid production at concentrations below 10-5M. Higher concentrations further increased TXB2 production and also increased the synthesis of 6-keto PGF1 alpha and PGF2 alpha. Nonparenchymal cells synthesized all the three types of prostanoids and homogenization of these cells did not result in a marked change. The addition of 10(-7)-10(-5)M arachidonate increased the TXB2, 6-keto PGF1 alpha and PGF2 alpha synthesis in nonparenchymal cells. No further increase was found at higher concentrations.

Animals↗
PubMed1987

In vitro effect of emetine and chloroquine on the macromolecular biosynthesis of murine thymus cells.

The in vitro effect of antiparasitic agents, emetine and chloroquine on the DNA, RNA and protein synthesis was studied in isolated murine thymocytes. Cytotoxic effect was observed at 10(-4) and 10(-3) M emetine concentrations causing 50% and 70% cell death, respectively. The toxic effect of emetine could be prevented when emetine was removed within 10 min of treatment. Inhibitory concentrations for DNA, RNA and protein biosynthesis were 10(-5), 5 x 10(-5) and 10(-8) M, respectively. The number of living cells decreased by 30 and 50% at 10(-4) and 10(-3) M chloroquine concentrations, respectively. Gradually decreasing rate of DNA synthesis was measured at increasing concentration of chloroquine between 10(-8) and 10(-3) while RNA and protein synthesis were effected at 5 x 10(-5) M concentration. These results indicate that protein biosynthesis is primarily affected by in vitro emetine and chloroquine treatment of murine thymocytes.

Animals↗
PubMed1987

The effect of the cAMP-dependent protein kinase on protein phosphorylation and phosphatidylinositol 4-phosphate formation in a hepatocyte membrane preparation.

Plasma membrane preparation obtained from isolated mouse hepatocytes was phosphorylated by exogenous cyclic AMP dependent protein kinase in the presence of 32P-ATP. The phosphorylated proteins were analysed by SDS-polyacrylamide gel-electrophoresis of the membrane and the phosphorylated lipids were analysed by thin layer chromatography of the separated lipid fraction. The protein kinase stimulated the 32P incorporation into phosphatidylinositol 4-phosphate and it catalyzed the phosphorylation of several proteins. The main phosphoprotein bands were found at 51 kDa, 49 kDa, 46 kDa and 34 kDa. A part of the 51 kDa phosphoprotein accompanied the lipids in the course of the separation of the lipid fraction.

Animals↗
PubMed1987

Inhibitory effect of A 23187 on protein synthesis in isolated murine hepatocytes.

The effect of the Ca ionophore A 23187 on amino acid incorporation into protein, on gluconeogenesis and on synthesis of some eicosanoids was investigated in isolated mouse hepatocytes (i) in the presence of 2.5 mM Ca2+, (ii) in the absence of Ca2+ and in the presence of 0.2 mM EGTA, (iii) after calcium depletion. A 23187 inhibited amino acid incorporation into protein both in the presence and in the absence of Ca2+ in a concentration dependent manner, whereas in the absence of Ca2+ the known stimulating effects of A 23187 on eicosanoid synthesis could not be detected. In hepatocytes prepared from 24 h starved mice A 23187 increased the rate of gluconeogenesis and similarly to eicosanoid synthesis this stimulation in the absence of Ca2+ could not be observed. On the other hand, after calcium depletion the inhibition of protein synthesis by A 23187 was markedly moderated. It is concluded that the effect of A 23187 on protein synthesis is also related to its ionophoretic effect but calcium sensitivity in this effect is smaller than in gluconeogenesis or eicosanoid synthesis.

Animals↗
PubMed1987

Bivalent cation and ATP requirements of endonucleases from rat liver nuclei.

The distribution of different types of DNase in rat liver nuclei was determined after a purification procedure involving ion exchange chromatography and gel filtration. As major enzymes Ca2+, Mg2+-dependent endonuclease, Mn2+-dependent endonuclease and an acid endonuclease were identified, sharing 60, 20 and 10% of the total activity, respectively. Mn2+-dependent endonuclease is a novel enzyme with a molecular mass of 30 +/- 5 kilodaltons. The synergistic effect of Ca2+ and Mg2+ ions for the Mn2+-dependent enzyme was lower by an order of magnitude than that of the Ca2+ and Mg2+-dependent endonuclease. The Ca2+ and Mg2+-dependent nuclease activity represents a heterogeneous population of enzymes. One of the cation dependent enzymes (Mr 25 +/- 5 kD) is stimulated by ATP the ATP optimum being 0.1 mM and the Mg2+ requirement 1 mM. The ATP-dependent endonuclease belongs to the minor endonucleases separable from the major ones.

Adenosine Triphosphate↗
PubMed1986

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II.

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).

Animals↗
PubMed1986

Effect of emetine and chloroquine on phagocytic processes of rat macrophages.

Emetine and chloroquine caused a dose-dependent and time-dependent inhibition of bacterial phagocytosis in rat peritoneal macrophages. The effect of emetine was found to be stronger: 50% inhibition was achieved at 5 X 10(-6) M concentration. The inhibition of phagocytosis was irreversible. Neither the binding of bacteria to the surface of phagocytes nor membrane marker 5' nucleotidase was touched by this treatment. The adherence of macrophages to plastic dishes was also impaired by the drugs but only in a smaller extent. Both emetine and chloroquine blocked the amino acid incorporation into macrophage proteins. The effect of emetine was more marked; 10(-6) M decreased the incorporation to 25% of control, while 10(-4) M chloroquine produced a similar inhibition. The block of protein synthesis was also irreversible.

Animals↗