Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.     

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.     

Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism

Purine and pyrimidine deoxyribonucleoside metabolism was studied in G1 and S phase human thymocytes and compared with that of the more mature T lymphocytes from peripheral blood. Both thymocyte populations have much higher intracellular deoxyribonucleoside triphosphate (dNTP) pools than peripheral blood T lymphocytes. The smallest dNTP pool in S phase thymocytes is dCTP (5.7 pmol/10(6) cells) and the largest is dTTP (48 pmol/10(6) cells), whereas in G1 thymocytes, dATP and dGTP comprise the smallest pools. While both G1 and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. We have studied ribonucleotide reduction and deoxyribonucleoside salvage in S phase thymocytes in the presence of extracellular deoxyribonucleosides. Based on these studies, we propose a model for the interaction of deoxyribonucleoside salvage and ribonucleotide reduction in S phase thymocytes. According to this model, extracellular deoxycytidine at micromolar concentrations is efficiently salvaged by deoxycytidine kinase. However, due to feedback inhibition of deoxycytidine kinase by dCTP, the maximal level of dCTP which can be achieved is limited. The salvage of both deoxyadenosine and deoxyguanosine (up to 10(-4) M) is completely inhibited in the presence of micromolar concentrations of deoxycytidine, whereas the salvage of thymidine is unregulated resulting in large increases in dTTP levels. Moreover, significant amounts of the salvaged deoxycytidine is used for dTTP synthesis resulting in further increase of dTTP pools. The accumulated dTTP inhibits the reduction of UDP and CDP while stimulating GDP reduction and subsequently also ADP reduction. The end result of the proposed model is that S phase thymocytes in the presence of a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthesized primarily by ribonucleotide reduction. Using the proposed model, it is possible to predict the relative intracellular dNTP pools found in fresh S phase thymocytes.     

Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA.

Breathing 100% O2 at 1 atmosphere absolute (ATA) is known to be associated with a decrease in cerebral blood flow (CBF). It is also accompanied by a fall in arterial Pco2 leading to uncertainty as to whether the cerebral vasoconstriction is totally or only in part caused by arterial hypocapnia. We tested the hypothesis that the increase in arterial Po2 while O2 was breathed at 1.0 ATA decreases CBF independently of a concurrent fall in arterial Pco2. CBF was measured in seven healthy men aged 21-62 yr by using noninvasive continuous arterial spin-labeled-perfusion MRI. The tracer in this technique, magnetically labeled protons in blood, has a half-life of seconds, allowing repetitive measurements over short time frames without contamination. CBF and arterial blood gases were measured while breathing air, 100% O2, and 4 and 6% CO2 in air and O2 backgrounds. Arterial Po2 increased from 91.7 +/- 6.8 Torr in air to 576.7 +/- 18.9 Torr in O2. Arterial Pco2 fell from 43.3 +/- 1.8 Torr in air to 40.2 +/- 3.3 Torr in O2. CBF-arterial Pco2 response curves for the air and hyperoxic runs were nearly parallel and separated by a distance representing a 28.7-32.6% decrement in CBF. Regression analysis confirmed the independent cerebral vasoconstrictive effect of increased arterial Po2. The present results also demonstrate that the magnitude of this effect at 1.0 ATA is greater than previously measured.     

Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of platinum replicas

We have studied the distribution of cytoskeletal elements in detergent-extracted mouse embryo fibroblasts using the platinum replica technique. It was shown that lamelloplasm can be subdivided into three zones: 1) the ruffle edge with dense microfilament meshwork; 2) the sparse zone adjacent to the ruffle edge and containing relatively few cytoskeletal elements; 3) the lamella proper occupied with a three-dimensional network of microfilaments, microtubules, intermediate filaments; this zone contained adhesion plaques corresponding to cell-substrate focal contacts and associated with the microfilament bundle ends. The cytoskeleton structure of the central (endoplasm) region of the cell was markedly different from that of the lamelloplasm. Its main feature was a dense microfilament sheath at the dorsal cell surface. Sites of microfilament bundle convergence can be visualized near the nucleus after partial removal of the sheath by more complete detergent extraction.     

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.     

A behavioral summary for completely random nets

This paper characterizes the cycle structure of a completely random net. Variables such as number of cycles of a specified length, number of cycles, number of cyclic states and length of cycle are studied. A square array of indicator variables enables conveninent study of moment structure. Additionally, exact and asymptotic distributional results are presented.