Solubilization of histamine H-1, GABA and benzodiazepine receptors.

Dopamine 3-0-sulfate is present in considerable amounts in mammalian plasma and peripheral tissues. Incubation of dopamine 3-0-sulfate (0.1 μmole) with purified bovine dopamine-β-hydroxylase resulted in the formation of free norepinephrine (7.3 × 10^?3 μmole). The conversion to norepinephrine was inhibited by 0.6 mM of fusaric acid, an inhibitor of dopamine-β-hydroxylase. The reaction of dopamine 3-0-sulfate with dopamine-β-hydroxylase followed Michaelis-Menten kinetics. The calculated K_m was 17 mM, different from the K_m for free dopamine (0.1 mM). The incubation medium does not contain any sulfatase activity.     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.     

A simple, sensitive and specific radioreceptor assay for beta-adrenergic antagonist drugs.

A radioreceptor assay for β-adrenergic blocking drugs described here is based on the ability of the blood content of drugs to compete with the binding of ³H-dihydroalprenolol (³H-DHA) to β-adrenergic receptors in calf cerebellar membranes. Plasma protein greatly inhibits the binding of ³H-DHA to β-receptors by binding the ³H-DHA so it is unavailable to the β-receptors. As little as 0.01 ml of human plasma in a final volume of 1 ml reduces binding 25–45%. Assays conducted on plasma dialysates can be performed without such inhibition. The radioreceptor assay is simple to perform as 100 samples can be processed in a morning. It is sensitive, detecting low nanomolar concentrations of plasma propranolol, and it is specific. No drugs clinically employed other than β-blocking agents compete for β-receptor binding. The assay detects all pharmacologically active metabolites of β-blocking drugs as well as the parent drug.     

Burn syndactyly.

When the entire digital web space has been destroyed by burn scarring and there is a contracture of the volar aspect of the web as well as the dorsum, Z-plasties and skin grafts alone seldom produce a satisfactory web space. During the past 3 years, for the release of 46 contracted web spaces in 20 burned patients, we have turned a rectangular flap from the dorsal surface of the web through into an inverted-T incision in the palm. The adjacent sides of the defects have been skin grafted. In all these patients, we obtained satisfactory release of the contracture and restoration of the web space.     

A possible role for cyclic GMP in mediating the effect of luteinizing hormone releasing hormone on gonadotropin release in dispersed pituitary cells of the female rat.

In continuing studies on cyclic nucleotide involvement in the regulation of gonadotropin release, we have measured the cyclic nucleotide content and rate of LH and FSH release during stimulation by LHRH of dispersed overnight cultured cells from the pituitaries of adult female rats. The minimal effective concentration of LHRH was 0.1 nM and half maximal stimulation of gonadotropin release was observed in the presence of 1.0 nM LHRH. Significant release of both LH and FSH was detectable after only 10 min in the presence of 5 nM LHRH. The presence of fetal calf serum (FCS) in the overnight culture medium increased basal cGMP levels significantly, whereas horse serum (HS) had no effect, therefore all experiments were conducted on cells cultured in the presence of HS. Treatment of the cultured cells with the phosphodiesterase inhibitors theophylline (TH) or isobutyl-methyl-xanthine (MIX) revealed a preferential stimulatory effect of TH on basal cAMP levels and of MIX on cGMP levels. Throughout these experiments, LHRH had no effect on cAMP levels. In the presence of MIX, concentrations of the releasing hormone as low as 1 nM induced a significant rise in the level of cGMP whereas in its absence, cGMP levels appeared to be unchanged by LHRH. The increase was detectable after 10 min of incubation. MIX alone slightly increased LH and FSH release and significantly potentiated the response of the cells to increasing doses of LHRH up to, but not beyond, 10 nM. The data support the possibility that cGMP may be involved in the mechanism of action of LHRH.     

Catecholamine binding to CNS adrenergic receptors

The properties of ³H-catecholamine binding to α- and β-adrenergic receptors in CNS are reviewed. ³H-epinephrine and ³H-norepinephrine label one class of α-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. ³H-epinephrine labels β₂, but not β₁, receptors in CNS, especially in bovine cerebellum. The same β-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na⁺-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

A coenzyme A-independent transacylase is linked to the formation of platelet-activating factor (PAF) by generating the lyso-PAF intermediate in the remodeling pathway

The remodeling pathway for the biosynthesis of platelet-activating factor (PAF) consists of the following reaction sequence: alkylacylglycerophosphocholine----lyso-PAF----PAF. Results presented in this article describe a novel transacylase activity that generates the lyso-PAF intermediate, which can then be acetylated to form PAF. Ethanolamine-containing lysoplasmalogens, 1-acyl-2-lyso-sn-glycero-3-phosphoethanolamine, alkyllysophosphoethanolamine, unlabeled lyso-PAF, 1-acyl-2-lyso-GPC, where GPC is sn-glycero-3-phosphocholine, and choline-containing lysoplasmalogens were all able to stimulate the formation of [3H]lyso-PAF from a [3H]alkylacyl-GPC precursor pool associated with HL-60 cell (granulocytic type) membranes. Other glycerolipids containing free hydroxyl groups (3-alkyl-2-lyso-sn-glycero-1-phosphocholine, lysophosphatidylserine, lysophosphatidylinositol, diacylglycerols, alkylglycerols, and monoacylglycerols), cholesterol, phosphatidylcholine, and phosphatidylethanolamine had no stimulatory effect on the release of [3H]lyso-PAF from the prelabeled membranes under identical incubation conditions. The observed transacylase reaction is directly coupled to PAF production, since the addition of a lysoethanolamine plasmalogen preparation to HL-60 membranes in the presence of [14C]acetyl-CoA stimulated PAF formation; under these conditions the lysoethanolamine plasmalogen was acylated. The transacylase responsible for the release of lyso-PAF from the membrane-associated alkylacyl-GPC was not affected by Ca2+, EGTA, or a known phospholipase A2 inhibitor, p-bromophenacyl bromide. The fact that the unnatural analog of lyso-PAF, lysophosphatidylserine, and lysophosphatidylinositol did not influence transacylase activity, whereas detergents such as deoxycholate and Triton X-100 inhibited the activity, demonstrated the observed stimulatory effects of the choline- and ethanolamine-containing lysophospholipids on the formation of [3H]lyso-PAF from [3H]alkylacyl-GPC were not due to any detergent property of these lysophospholipids. Thus, we conclude a CoA-independent transacylase (possessing phospholipase A2/acyltransferase activities) can be responsible for the formation of the lyso-PAF intermediate in the remodeling route of PAF biosynthesis.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Chemoattraction of a bactivorous ciliate to bacteria surface compounds

The locomotory response to cell surface compounds extracted from two prey species,Vibrio natriegens andVibrio neries, was tested for a bacterivorous ciliate,Pseudochnilembus marinus Thompson 1966. Chemoattraction of the ciliate to the surface compounds stabilized in agarose baits was not equal for the two prey species. Fractionation of the extracts suggested the attractive substance was a high molecular weight compound. The expression of the differential response was dependant on the physiological condition and prior prey species exposure of the ciliate test population. The recognition and response to material normally found on the surface of prey cells supports evidence for the involvement of chemical sensing of gradients of prey particles and dissolved compounds of prey origin in the natural swimming behavior of bacterivorous ciliates. The prey species-specific reactions and influence of ciliate physiological state on chemosensory response suggest ciliate-bacteria interactions may be more complex than preciously assumed.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.