Nucleoside transport and metabolism in lymphocytes, polymorphonuclear cells and cerebral synaptosomes.

The salvage of nucleosides dominates over de novo biosynthesis in lymphocytes, polymorphonuclear cells (PMN) and in central neutral nervous system (CNS) in higher organisms. Earlier works in our laboratory have shown that the salvage of deoxycytidine (dCyd) did not correlate with DNA synthesis. The uptake and metabolism of dCyd was higher in undifferentiated germinal center lymphocytes and in follicles comparing to more differentiated cells. Recently we have compared the transport of thymidine (dThd), dCyd, uridine (Urd) and adenosine (Ado) in the three cell systems in which the salvage of nucleosides is dominating. It was found that dCyd was transported 30 times more effectively into lymphocytes than into PMN and synaptosomes, while Urd was transported about the same rate into the two cells and into synaptosomes. All transport processes could be inhibited by dipyridamole, NBRPR, papaverine and dilazep. The dCyd and dThd was phosphorylated even at 0 degrees C up to TTP and dCTP without incorporation into DNA and into liponucleotides. Our results show that the processes of transport-phosphorylation, as well as the processes of DNA-CDP-phospholipid synthesis are tightly coupled to each other in intact cells and organelles.

Formation of leucyl-leucine-O-methylester in leucine-O-methylester treated cells.

Porcine polymorphonuclear cells (PMN) and murine macrophages (M phi) were treated in vitro with Leu-OMe or Leu-Leu-OMe (1.5-5.0 mM) for various periods of time. It was found that the Leu-OMe and Leu-Leu-OMe entered cells rapidly, concomitantly the intracellular leucine accumulated. The methyl derivative diffused faster than Leucine due to its lipophylic character. The Leucine-O-methylesters hydrolysed rapidly as a consequence of the esterase and peptidase activities. The cells treated with Leu-OMe accumulated a high amount of Leucine and some Leu-Leu-OMe too. It was found that the formation of the didpeptide-methylester is not a spontaneous process, rather an enzymatic one. The Leu-OMe treated cells serve as a model which can be used to investigate the effect of the amino acid metabolism and the formation of dipeptides intracellularly and extracellularly.

In vitro effect of leucine-O-methylester on polymorphonuclear and mononuclear cells.

The uptake of Leu-OMe and Leu-Leu-OMe was studied in vitro in porcine PMN cells. Both methylesters are metabolized leading to the intracellular accumulation of leucine. Part of the hydrolyzed leucine gradually filtrates back into the culture medium in a time-, temperature- and methylester substrate concentration-dependent manner. Another portion of Leu-OMe is converted to Leu-Leu dipeptide. With respect to the cellular effects of Leu-OMe treatment ultrastructural studies showed the presence of large vacuoles without significant alteration of cell viability. Increased exocytosis of lysosomal enzymes did not lead to lytic events. Changes in the plasma membrane are indicated by the observation that Leu-OMe treatment causes the loss of the chemotactic activity to formyl-Met-Leu-Phe.

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.

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.

Cytotoxic material released from Staphylococcus epidermidis. I. Effects on [3H]thymidine incorporation of human lymphocytes.

Incorporation of [3H]thymidine ([3H]TdR) into tonsil lymphocytes was inhibited by native Staphylococcus epidermidis while Staphylococcus aureus Cowan I caused stimulation. The inhibitory effect of S. epidermidis was abolished by formalin treatment but not by heat killing. A toxic agent was released from S. epidermidis on gentle water extraction without lysing the bacteria. The extract contained protein and other UV-absorbing material, but did not exhibit haemolytic, lysozyme, catalase or protease activity. The heat-resistant, formalin-sensitive inhibitor present in the aqueous extract of S. epidermidis inhibited [3H]TdR incorporation of lymphocytes in a dose-dependent manner and decreased the viability of lymphocytes.

Cytotoxic material released from Staphylococcus epidermidis. II. Fractionation and some effects of the fractions on lymphocytes and hepatocytes.

Cytotoxic substance(s) of about 4 X 10(3) molecular weight, containing 9.5% peptide and 73% carbohydrate was released from Staphylococcus epidermidis in phosphate buffered saline. The material was soluble in ethanol and was heat-resistant. It blocked amino acid uptake and E-rosette formation of human tonsillar and blood lymphocytes. In isolated mouse hepatocytes the toxin inhibited protein synthesis, but only in the presence of calcium ions. The results suggest that eukaryotic cell membranes are damaged by the coccal agent.

The adherence of Staphylococcus epidermidis to human tonsil lymphocytes.

Attachment of various bacteria to human, peripheral blood and tonsil lymphocytes was investigated in vitro. About 20% of tonsil lymphocytes bound Staphylococcus epidermidis, whereas the binding of other strains was negligible. The influence on cytoadherence of human serum, immunoglobulins (human IgM, IgG, IgA, as well as their respective anti-Ig's), and carbohydrates (mono and polysaccharides) was measured. It was found that heterogenous surface structures of the lymphocytes participate in the attachment.

Lethla effect of nitrous acid on Escherichia coli.

The effect of nitrous acid (NA) on viability, integrity of cellular DNA and on membrane transport were studied in 5 strains of Escherichia coli. Stationary phase cells, grown on mineral salts medium, were exposed to NA. The viability of strains decreased in thefollowing order: W3110 wild-type greater than WP2 wild-type, WP2 uvrA greater than NG30 recA greater than P3478 polA. Alterations were found in the DNA sedimentation profile in alkaline sucrose gradient. Disturbance of DNA synthesis was measured by 3H-labelled thymidine ([3H]Thd) incorporation. No degradation of DNA was found after NA treatment. Low doses of NA caused significant inhibition of leucine and glucose transport into whole cells. The results are interpreted in terms of the multi-target action of NA causing the death of cells.