Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus

The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action.     

Action of novel eicosanoids lipoxin A and B on human natural killer cell cytotoxicity: effects on intracellular cAMP and target cell binding

Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells.     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

Determinants in the perceptions of visual blight

Research on visual blight has been predominantly qualitative and has pointed out salient attributes of blight: that it is durable, visually demeaning, and somehow aesthetically depressing. Data from a national sample (n=3005) are used in this study to measure the effect of demographic variation on the perception of visual blight. The perception of blight shows marked and regular variation with socioeconomic status, marital status, and religiosity.Harris data for this study was purchased with funds from the Shell Foundation. The author wishes to acknowledge Robert Q. Hanham, whose assistance proved invaluable.