The kinetics of generation of influenza-specific cytotoxic T-lymphocyte precursor cells

A dramatic increase in the number of restimulatable (memory) cytolytic T-cell precursors (CTLps) has been demonstrated to occur 2 to 4 days postimmunization. By 4 or at the latest 6 days following in vivo priming, the frequency of restimulatable influenza-specific CTLps has reached that characteristic of a memory response. This indicates that the proliferative events which give rise to memory CTLps occur earlier than might have been expected and in fact are completed prior to the cessation of othe primary CTL response. Such information may have important significance to future immunomodulatory efforts aimed at perturbing the establishment of T-cell memory in vivo.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Metabolism of leukotriene A4 into C4 by human platelets

Tritium-labelled leukotriene A4 is converted by a suspension of human platelets into leukotriene C4. The conversion is stimulated by reduced glutathione and is dependent on the platelet concentration. Formation of leukotriene C4 is temperature and time dependent and is destroyed by heating the platelets at 100 degrees C for 5 min. Verification of leukotriene C4 formation was obtained by conversion into leukotriene D4 during reaction of the HPLC-purified platelet-derived leukotriene C4 with commercial gamma-glutamyl transpeptidase. In separate experiments we incubated authentic tritiated leukotriene C4 with human platelets and we showed the formation of tritiated leukotriene D4, demonstrating the presence of gamma-glutamyl transpeptidase activity in these cells. This activity could be blocked by the presence of reduced glutathione in the incubation mixture. In contrast, erythrocytes converted tritiated leukotriene A4 almost exclusively into leukotriene B4. Although platelets have been reported to lack 5-lipoxygenase activity, our study demonstrates that platelets possess the necessary machinery to transform leukotriene A4 into leukotrienes C4 and D4. Our results suggest that an intracellular interaction between platelets and leukotriene A4-forming cells, e.g., polymorphonuclear leukocytes, could lead to the formation of these potent peptidolipids in the circulation.     

Scanning electron microscopy of pulmonary vascular endothelium in rats with hypoxia-induced hypertension

Scanning electron microscopy was used to study the endothelial surface of the pulmonary trunk, artery, and vein in normobaric control rats as well as in rats exposed to hypobaric hypoxia for 7 and 21 days. The individual endothelial cells of the normobaric pulmonary trunk and hilar artery were flat and slightly elongated with elevated nuclear regions, and those of the intermediate-sized artery were more elongated and had more microvilli than the large arteries studied. Their endothelial cell boundaries were outlined by beaded cytoplasmic projections. The surfaces of the normobaric hilar and intermediate-sized veins were smooth and demonstrated numerous longitudinal streaks. These venous endothelial cells were elongated and their cell boundaries were outlined by low discontinuous marginal folds. Exposure to hypobaric hypoxia caused the following changes on the arterial surface: elevation of the endothelial cells; formation of microvilli-rich cell clusters; formation of hollow defects; and the attachment of leukocytes. Hypobaric hypoxia also caused the disappearance of the longitudinal streaks and the occurrence of microvilli-rich cells in the hilar veins. The endothelial surface modifications in the hypobaric rats could be related to thickening of the endothelium, intimal edema, increased intimal connective tissue, luminal invasion of leukocytes, and increased endothelial cell proliferation, known to occur in systemic arteries of hypertensive animals.     

Detection of the intracellular ras p21 oncogene product by flow cytometry

Rapid identification of the expression of oncogene products in specific cell types could potentially be useful in the diagnosis and treatment of human malignancy. We have now observed that through the use of lysolecithin permeabilization and fluorescence-activated flow cytometry, cells expressing high levels of the v-Ha-ras oncogene product, p21, can readily be distinguished from the nontransformed parent cells in a rapid and quantitative manner.     

Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation

A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction.     

Mutant allele frequencies in cats of Minneapolis-St. Paul

Coat color phenotype frequencies were determined in the cat population of Minneapolis and St. Paul. Mutant allele frequencies are estimated to be p (O) = 0.287, q(a) = 0.742, q(d) = 0.635, q(l) = 0.507, p(S) = 0.288. q(tb) = 0.472, p(W) = 0.016, and q(cs) = 0.214. A substantial number of cats displaying the Siamese coat pattern were found. These cats have a long history in the population.     

Photocontrol of Dark Circadian Rhythms in Stomata of Phaseolus vulgaris L

Stomatal diffusion resistance in primary leaves of Phaseolus vulgaris L. which had been grown in light:dark cycles followed a marked circadian rhythm when the plants were transferred to continuous darkness. Reentrainment of the rhythm required more than one inductive change in photoperiod. The phasing of the rhythm of dark stomatal opening was contolled primarily by the light-on (dawn) signal, whereas the rhythm of dark closure was related to the light-off (dusk) signal. The evidence points to a dual control of the circadian clock in which a product of photosynthesis plays a major role. No evidence for phytochrome involvement in the phasing of the rhythm was found. An influence of phytochrome on the amplitude of the stomatal rhythm was observed in which removal of phytochrome-far-red absorbing form caused rapid damping.     

Insulin activation of insulin receptor tyrosine kinase in intact rat adipocytes. An in vitro system to measure histone kinase activity of insulin receptors activated in vivo

We have studied the effect of incubation of intact cells with insulin on insulin receptor kinase activity. Following exposure of rat adipocytes to insulin, cells were solubilized and insulin receptors purified by specific immunoprecipitation or by insulin affinity chromatography. Kinase activity of the receptors, as measured by phosphorylation of histone 2B, was then determined. Insulin treatment of the cells resulted in a 10-20-fold increase in histone kinase activity of the subsequently isolated insulin receptors. The insulin effect was half-maximal at 3 s and maximal within 15 s of exposure, was dose-dependent (EC50 = 21 ng/ml), and was rapidly reversible following dissociation of insulin from the cells. The insulin effect in intact cells on insulin receptor kinase activity could be partially reversed in vitro by dephosphorylation of the isolated receptors by alkaline phosphatase. It is proposed that: in intact cells, insulin causes alterations in insulin receptors, such that their kinase activity toward non-receptor substrates increases; increased insulin receptor kinase activity following insulin stimulation in intact cells is, at least in part, the result of an increased phosphate content of the receptors; and effects of insulin on insulin receptors in intact cells can be preserved during receptor isolation and thus can be measured in a cell-free system.     

Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.