Biomedical subjects

F Antoni

Publications and source records attributed to F Antoni.

At least 73 records · Page 4Linked to original sources

PubMed1986

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine.

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.

Cell Membrane Permeability↗

PubMed1986

Interaction of casein with human polymorphonuclear cells.

Attachment of 125I-casein to PMN cells was investigated. Iodination did not decrease the chemotactic effect of casein. 125I-casein binding was increasing toward a maximum reached at about 45 min at 24, and 37 degrees C. At 4 degrees C the binding was proportional to time for 45 min. No saturation was achieved even at 15 mg/ml casein. About 40% of casein remained attached to PMN in a casein-free medium after 60 min, at 37 degrees C. Pretreatment of the cells with trypsin or butanol, or the presence of indomethacin, azide, and PMSF did not affect the binding of casein. The hydrophobic amino acid, leucin counteracted the attachment of casein. Our data show that at chemotactic doses casein is bound specifically to cell membranes by hydrophobic forces. The induction of chemotaxis may be due to micellar casein-membrane lipid complexes.

Adult↗

PubMed1986

Poly(ADP-ribose) and replicative DNA synthesis studied in permeable mouse thymocytes.

Poly(ADP-ribose) and DNA synthesis were followed in a random population of freshly isolated and permeabilized mouse thymocytes. Different conditions were found to be optimal for the synthesis of poly(ADP-ribose) and DNA, respectively. The suspension of one of the processes did not influence significantly the synthesis of the other macromolecule. Our results suggest that there is no direct relationship between the two processes.

Animals↗

PubMed1985

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes.

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.

Animals↗

PubMed1985

Specific substrate for histone kinase II: a synthetic nonapeptide.

Based on the previously determined intrinsic substrate specificity of histone kinase II, a nonapeptide was synthesized which was a specific substrate for this enzyme. The Vmax value of phosphorylation of the peptide (Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide) was about the same as that for H1 histone and the apparent Km for the phosphorylation of the peptide was 0.2 mM, an order of magnitude higher than that for H1 histone. H1 histone inhibited the phosphorylation of the peptide, while the peptide did not inhibit the phosphorylation of H1 histone. In the crude extracts of calf thymus, spleen and liver, histone kinase II was the only enzyme which phosphorylated the synthetic peptide. The rate of phosphorylation of this peptide was used to determine the activity of histone kinase II in the crude extracts of several tissues obtained from different species.

Animals↗

PubMed1985

Characteristic biochemical differences in human T and B lymphocytes separated on nylon wool.

T and B lymphocytes from human tonsils were separated on a nylon wool column. T. cells are enriched in the nonadherent, B cells in the adherent fraction. Several enzymes and other markers were tested in separated and non separated lymphocyte populations. Certain enzymes and other properties can be used as T or B lymphocyte markers because of their preferential occurrence and because of the advantages of their estimation (simple, quick methods, objective evaluation). The following characteristics were considered as markers on the basis of our results: (I) acid phosphatase, Na+-K+-activated ATPase, BAEE-peptidase and chromium labeling in T lymphocytes; (II) 5'-nucleotidase, FITC-IgG binding, N-acetyl-D-glucosaminidase, thymidine and valine incorporation in B lymphocytes.

B-Lymphocytes↗

PubMed1985

Preparation and characterization of myosin from cultured cells of Escherichia coli (02:K1 30156 strain).

In this study the myosin preparations isolated from E. Coli cell cultures were analysed. The isolation of myosin from fresh cultures resulted in a substantially higher (approximately 10-fold) yield than from stored cells. The coli myosin, despite the two DEAE-cellulose treatments, contained more RNA and P-lipid than the myosin prepared from skeletal muscle. The RNA content can be removed gradually by acetone denaturation and lipid removal followed by subsequent washings. The fresh preparations contained protein-bound alkali-stable P; part of this was released by Cu ions. The coli myosin can be phosphorylated. The phosphate uptake depended on the concentrations of ATP and Mg2+ and on the time of incubation. The alkaline hydrolysate of the phosphorylated, washed lipid-free preparation was resolved into 8 P-containing peaks on ion-exchange chromatography. Of fractions P-Arg, Pi, P-Lys and 2 P-His conformer were identified by means of synthetic compounds, elution pattern and specific reactions. The remaining compounds could not be identified. The most abundant component was P-Arg suggesting that this compound might play an important role in the cytokinetic movements of E. coli.

Adenosine Triphosphate↗

PubMed1985

Pyrimidine salvage enzymes in human tonsil lymphocytes: II. Purification and properties of deoxycytidine kinase.

Human tonsillar lymphocytes incorporate about 7-10 times more [3H]-deoxy-thymidine than [3H]-deoxycytidine at the same extracellular nucleoside concentration, and both incorporations could be inhibited by 10(-5) M arabinosyl-cytosine to 99%. On the other hand, the crude extract of these cells had about 10 times more deoxycytidine kinase than deoxythymidine kinase specific activity, as it was reported previously as well. Therefore, the differences in the labelling of these cells by [3H]-deoxycytidine and [3H]-thymidine cannot be simple due to the different levels of phosphorylating enzymes in the cells. The deoxycytidine kinase of human tonsillar lymphocytes was purified 38.6-fold by DEAE-Sephadex chromatography, and some kinetic and regulatory properties of the purified enzyme were investigated. Deoxycytidine kinase was eluted as a single peak from DEAE-Sephadex column and also from Sephadex G-100 column indicating a molecular weight of about 60 000; no isoenzymes could be detected. During the purification procedure an increase of the enzyme activity was observed suggesting the inhibition of the enzyme in the cells. An apparent Km of 13 microM for deoxycytidine and Ki of 55 microM for arabinosyl-cytosine were found for the tonsillar deoxycytidine kinase. The enzyme was strongly inhibited by dCTP, but not by the other deoxyribonucleoside triphosphates.

Cell-Free System↗

PubMed1985

Pyrimidine salvage enzymes in human tonsil lymphocytes. I. Separation and properties of thymidine kinase isoenzymes.

Two forms of deoxythymidine kinase were separated from human tonsillar lymphocytes on DEAE-Sephadex column (peak 1 and peak 2 isoenzymes) and identified by gel electrophoresis. Both isoenzymes were found in freshly prepared lymphocytes as well as in lymphocytes cultured in the presence or absence of phytohaemagglutinin, but freshly prepared and phytohaemagglutinin-stimulated cells contained higher activity of peak 1 isoenzyme than the cultured, non-stimulated ones. Kinetic properties and feed-back inhibition of the two separated isoenzymes were compared. The apparent Km values of isoenzyme 1 and 2 were 4 microM and 5 microM for thymidine and were 0.15 mM and 0.12 mM for ATP, respectively. Isoenzyme 1 was found to be more sensitive to heat denaturation at 55 degrees C, and to feedback inhibition by dTTP than isoenzyme 2. On the contrary, isoenzyme 1 was more resistant to dCTP inhibition than the other one. It is concluded that the isoenzyme 1 activity correlates with the synthesis of DNA, while the activity of isoenzyme 2 seems to be constant. The high activity of isoenzyme 1 in freshly isolated tonsil lymphocytes supports their active proliferation in the organ.

Adenosine Triphosphate↗

PubMed1984

Cultured tonsillar lymphocytes excrete [3H]thymidine labeled DNA and revert to resting state.

Large tonsillar lymphocytes labeled with [3H]thymidine reverted to small lymphocytes with concomitant loss of [3H]DNA upon culturing. The decrease of labeled DNA content and size of large lymphocytes was demonstrated by flow cytometry and cell sorting. These observations suggest that stimulated lymphocytes may revert from their proliferative phase to resting phase by shedding 'extra'DNA under cell culture conditions. This released DNA is not due to cell damage and can be hybridized to chromosomal DNA.

Basal Metabolism↗

PubMed1984

Uptake of arachidonic acid, arachidic acid, oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex.

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time- and concentration- dependent. 94-98% of the arachidonic acid was incorporated into the phospholipid and triacylglycerol fractions following a 60 min incubation period at 37 degrees C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.

Animals↗

PubMed1984

The effect of emetine on the immune response of mice.

Emetine (33 mg/kg body weight) administered intraperitoneally blocked the immune response of mice to 10(9) sheep red blood cells (SRBC). The inhibition was almost complete when the drug was administered simultaneously or 24 hr after immunization, while partial inhibition was caused by treatment at 48 and 72 hr. Incorporation of 14C-leucine and 3H-thymidine by spleen cells isolated 4 hr after emetine injection of the mice was strongly decreased. Incorporation was approaching the control level in cells isolated 72-96 hr after emetine administration. However, the incorporation of labeled precursors was less than after SRBC treatment only, even after 72-96 hr. Emetine apparently blocked the development of immune response at an early stage and, in contrast to macromolecule synthesis, the inhibition of the antibody response was irreversible.

Animals↗

PubMed1984

Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes.

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T-150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours. 2'-Deoxycytidine 5'-triphosphate is effectively substituted by 5-mercuri-2'-deoxycytidine 5'-triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol-agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (aCTP), nalidixic acid and novobiocin and by density shift experiments. More than 80% of the newly synthesized low-molecular-mass DNA, 8-60 nucleotides in length, consists of RNA-linked DNA. This conclusion is based on phosphorylation with [gamma-32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5' end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.

Animals↗

PubMed1984

Analysis of 5'-ends of short DNA fragments excreted by phytohemagglutinin stimulated lymphocytes.

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and the excreted DNA was isolated from the medium after four days of incubation of cells. The excreted DNA was labeled at the 5'-end with [gamma-32P]ATP and polynucleotide kinase. Analysis of the end-labeled material revealed a size distribution with a chain length of 6 - 60 nucleotides. These short DNA fragments did not contain ribo-nucleotides at their 5'-termini. P1 nuclease digestion did not release specific deoxyribonucleoside monophosphates from the 5'-end of the excreted DNA fragments. These results point to the non-specific degradation of DNA excreted by stimulated lymphocytes.

DNA↗

PubMed1984

Prostaglandin and thromboxane synthesizing activity in isolated murine hepatocytes and nonparenchymal liver cells.

Prostanoid synthesis from 3H-arachidonic acid was compared in isolated parenchymal and nonparenchymal murine liver cells. The cells incorporated arachidonic acid into phospholipids but no prostanoid synthesis could be measured during 30 min incubation. Conditions necessary for prostanoid synthesis were different in parenchymal and nonparenchymal cells and the products were also different. Prostanoid synthesis could be induced by in vitro partial hepatectomy: parenchymal cells synthesized thromboxane A2 whereas nonparenchymal cells produced prostaglandin E2 and F2 alpha. Prostaglandin E2 and F2 alpha synthesis could be provoked also by homogenization of the nonparenchymal cells prepared from normal liver, while the homogenates of parenchymal cells prepared from normal liver did not synthesize thromboxane. Imidazole and indomethacin inhibited the production of thromboxane and prostaglandins, respectively. Our results suggest that the various cell types of the liver respond by the synthesis of different and specific prostanoids after the same injury.

Animals↗

PubMed1984

Kinetics of thymidine entry into tonsillar lymphocytes and its alteration in the presence of a lymphokine.

Thymidine uptake into the acid soluble cell fraction of human tonsillar lymphocytes was studied in vitro. Uptake was linear for 15-20 minutes at low concentrations (less than 1.2 microM) of thymidine. The plot of uptake versus time could be extrapolated to the origin. Value for KM (0.5-0.6 microM) and values for Vmax were determined. In the presence of a lymphokine which inhibited thymidine incorporation into DNA the uptake of thymidine into the acid soluble cell fraction was also inhibited. The decreased uptake could be characterized by an increase in the apparent KM, without the alteration of Vmax. Lymphokines which inhibit thymidine incorporation may influence and regulate in vivo the entry of the exogenous thymidine into the cells.

DNA↗

PubMed1984

Ethanol-soluble substances isolated from Escherichia coli culture filtrate induce polymorphonuclear chemotaxis.

Substances chemotactic (CT) for human PMN were isolated from the culture medium of Escherichia coli O2 : Kl grown to stationary phase. The ethanol soluble fraction of the lyophilized culture filtrate, which contained the bulk of CT activity, was gel filtrated on Sephadex G-10. The 5 X 10(3) molecular weight fraction showed the highest CT activity. On the basis of partial chemical characterization the active CT substances probably consist of glycopeptides and lipids originating from the cell envelope.

Amino Acids↗

PubMed1983

Resolution and reconstitution of the rec BC deoxyribonuclease of Escherichia coli.

The inactivation of rec BC nuclease activity and simultaneously the separation of 3 DNA-dependent ATPases and an ATP-independent DNases specific for single-stranded DNA have been observed after DEAE-cellulose chromatography of cell extracts from Escherichia coli. Two of the ATPases catalyze the strand separation of duplex DNA. Reconstitution of ATP-dependent DNase activity has been carried out by the combination of the separated enzymes.

Adenosine Triphosphatases↗