Prostaglandin D2 in cultured capillary and microvascular endothelium of human brain.

Production of prostaglandin D2 (PGD2) was investigated in cultured endothelial cells derived from capillaries and microvessels (small and large) of human brain using radioimmunoassays. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester and calcium ionophore greatly stimulated the secretion of endothelial PGD2. Secretion of PGD2 induced by vasoconstricting peptides, angiotensin II and arginine-vasopressin, was almost completely abolished by their respective specific receptor antagonists [Sar1, Ala8]-Ang II and [1-6(beta-mercapto-beta,beta-cyclopentamethylene propionic acid) 2-O-methyltyrosine]. Thus, the augmented production of PGD2 by angiotensin II and arginine-vasopressin is a receptor-mediated event. It also indicates that the EC have specific angiotensin II and arginine-vasopressin (V1) receptors. This study represents the first demonstration of vasoactive agents modulating PGD2 production in capillary and microvascular endothelium of human brain.     

Secretion of the sweet-tasting plant protein thaumatin byBacillus subtilis

Summary With the -amylase promoter and ribosome binding site,Bacillis subtilis was used to express the sweet plant protein thaumatin II cDNA fused in the correct reading frame to the -amylase leader peptide. The r-thaumatin was purified from the medium on a S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The r-thaumatin and authentic thaumatin were the same size when reduced by 2-ME and the same size when not reduced.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Oligochaetes and water pollution in two deep Norwegian lakes

Analyses of the oligochaete fauna of two of the deepest lakes in Scandinavia — the Norwegian lakes Mjösa (450 m) and Tyrifjorden (295 m), revealed a totally different species composition in the deep profundal compared with the upper profundal - in contact with the nutrient-enriched epilimnion. In both lakes a pronounced thermal stratification develops in the summer, thus the epilimnion receiving gross organic pollution behaves differently from the profundal. The lakes are each effectively divided into two bodies of water with limited water exchange between them, i.e. one major oligotrophic body and one minor more nutrient-rich. Since the 1950s both lakes have been exposed to heavy pollution of various kinds. In Lake Mjösa in 1975 and 1976 unpleasant algal blooms of the blue-green alga Oscillatoria bornetii fa. tenuis occurred. Bottom samples obtained at the same time revealed that the deep central bottoms of the lake were totally dominated by oligotrophic oligochaete indicators, i.e. by Stylodrilus heringianus and Spirosperma ferox, while the fauna of the upper profundal in the vicinity of domestic and agricultural sewage outfalls, wood processing industries, etc. was dominated by Limnodrilus hoffmeisteri and Tubifex tubifex in great abundance, indicating enriched conditions. Several other species indicative of eutrophy, were absent, most of them belonging to the genus Potamothrix. A fairly similar situation exists in Lake Tyrifjorden, where, for instance, in the shallow bay of Steinsfjorden — heavily eutrophied by agricultural wastes — blooms of blue-green algae have caused problems from time to time. The same oligochaete communities as in Lake Mjösa distinguish the central oligotrophic bottoms from the regionally more enriched upper profundal. The likely reasons for an intact profundal oligochaete fauna are great volumes of oxygen-rich hypolimnic water of low temperature and a high bottom/lake surface area ratio.     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)