The Drosophila cation channel trpl expressed in insect Sf9 cells is stimulated by agonists of G-protein-coupled receptors.

Structures and regulations of vertebrate channels responsible for sustained calcium elevations after hormone stimulation are largely unknown. Therefore, the Drosophila photoreceptor channels, trp and trpl, which are assumed to be involved in calcium influx, serve as model system, trpl expressed in Sf9 cells showed spontaneous activity. Hormonal stimulations of calcium influx (detected by fura-2) and of an outwardly rectifying current were observed in Sf9 cells coinfected with baculoviruses encoding trpl and various heptahelical receptors for histamine, thrombin, and thromboxane A2, all known to cause phospholipase C-beta activation in mammalian cells.

Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells.

We sought to determine the effects of exogenous epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF-alpha), single-chain precursor hepatocyte growth factor (SC-HGF), double-chain mature HGF (DC-HGF), and keratinocyte growth factor (KGF) on proliferation, motility, and differentiation of first passage cultures of human corneal epithelial cells in serum-free chemically defined medium. The effect of EGF, HB-EGF, TGF-alpha, SC-HGF, DC-HGF, KGF or combinations of the growth factors on proliferation was measured by counting cells present after 3 weeks of culture and by immunostaining for the cell-cycle-specific nuclear proliferation antigen Ki-67. The effect of the factors on epithelial cell motility was assessed by morphometric analysis of photographs of cells migrating from confluent islands of cells.

Rapid isolation of G alpha 13 from bovine brain membranes: supportive effect of ethylene glycol.

G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated G alpha 13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of G alpha 13 markedly.

Purification of the G-protein G13 from rat brain membranes.

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11.

Detection of G-protein heterotrimers on large dense core and small synaptic vesicles of neuroendocrine and neuronal cells.

Heterotrimeric G proteins, initially believed to be exclusively present in the plasma membrane, have also been found to be associated with intracellular membrane compartments. There they are involved in various membrane trafficking processes including regulated secretion (reviewed in Bomsel, M., K.

Complex information processing by the transmembrane signaling system involving G proteins.

Much of the information cells receive is transduced by a membranous signaling system that uses heterotrimeric guanine nucleotide binding proteins (G proteins) to functionally couple cell surface receptors to a variety of effectors. During recent years it has been shown that receptors, G protein alpha, beta and gamma subunits as well as effectors involved in this signaling system exhibit a remarkable structural diversity and that the interactions of these components display a bewildering complexity. Even though many questions remain to be answered, it is becoming obvious that G proteins form the basis of a complex membranous signaling network which allows the cell to coordinate and to process incoming signals already on the level of the plasma membrane.

Heterotrimeric G proteins involved in the modulation of voltage-dependent calcium channels of neuroendocrine cells.

Various mechanisms have been identified by which hormones and neurotransmitters, interacting with heptahelical receptors, modulate the intracellular Ca2+ concentration in neuronal, endocrine, and neuroendocrine cells. All of them involve heterotrimeric G proteins. Best documented are hormonal stimulations and inhibitions of voltage-dependent Ca2+ channels.

Transforming growth factor-alpha is a constant component of human tear fluid.

Growth factors are known as a family of polypeptides with powerful influences on angiogenesis, tumor growth and wound healing. Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are structurally related peptides which bind to the same receptor, EGF-R, and also exert similar effects. EGF is a natural component of human tears, and ocular disease leads to decreased concentrations in tear fluid.

Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells.

The alpha-subunits of the widely expressed G-proteins Gq and G11 indistinguishably activate beta-isoforms of phospholipase C. In this report we have tested whether differences exist in the activation of both G-proteins via phospholipase C-linked receptors. We found that bombesin and vasopressin, with very similar potencies and time dependencies, induce the activation of both Gq and G11 in Swiss 3T3 cells, suggesting that these G-proteins, at least in part, serve interchangable functions.

Temporal and spatial expression of major histocompatibility complex class I H-2K in the early mouse embryo.

The pattern of expression of major histocompatibility complex class I H-2K in early mouse embryos was determined through reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization methods. H-2K transcripts were detected in all stages of preimplantation embryos from oocyte to blastocyst. In the blastocyst, transcripts were found in both trophectoderm and inner cell mass.

Maitotoxin activates cation channels distinct from the receptor-activated non-selective cation channels of HL-60 cells.

We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493].

Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea.

The purpose of this study was to determine whether epidermal growth factor (EGF), EGF receptor, transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), acidic fibroblast growth factor (acidic-FGF), basic fibroblast growth factor (basic-FGF), and interleukin-1-alpha (IL-1-alpha) proteins were present in cultures of human corneal cells and/or in sections of human corneal tissue. Immunohistochemistry was performed on human corneal sections. Immunofluorescent cell staining was used to evaluate corneal epithelial, stromal fibroblast, and endothelial cells in primary culture.

Synthetic lipopeptide Pam3CysSer(Lys)4 is an effective activator of human platelets.

Lipopeptide analogues of the NH2-terminus of bacterial lipoprotein are known to induce activation of macrophages, neutrophils, and lymphocytes. We studied the effect of the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine [Pam3CysSer(Lys)4] on several functions of human platelets. Pam3CysSer(Lys)4 led to the aggregation of platelets and induced the secretion of serotonin with an effectiveness similar to thrombin.

Purification of a novel G-protein alpha 0-subtype from mammalian brain.

Three distinct G-protein alpha o-subtypes, i.e. alpha o1, alpha o2 and a newly observed 'alpha o3', are present in membranes of mammalian brain, each appearing as two isoforms on SDS/PAGE.

The human thyrotropin receptor activates G-proteins Gs and Gq/11.

The human thyrotropin receptor leads upon activation to the stimulation of phospholipase C and adenylyl cyclase. It is presently not known whether this bifurcating signaling occurs via two different G-proteins (Gq/11 and Gs) or via one G-protein (Gs). Receptor-activated Gs releases beta gamma subunits and alpha s, which then could regulate phospholipase C and adenylyl cyclase, respectively.

Transfected muscarinic acetylcholine receptors selectively couple to Gi-type G proteins and Gq/11.

Regulation of effector functions by muscarinic acetylcholine receptor subtypes is mediated by pertussis toxin-sensitive and -insensitive G proteins. In membranes from human embryonic kidney 293 cells transfected with m1, m2, and m3 muscarinic acetycholine receptors, we detected the pertussis toxin-sensitive G proteins Gi1, Gi2, and Gi3 and the pertussis toxin-insensitive G proteins Gq/11 and Gs. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled with [alpha-32P] GTP azidoanilide, in the absence and presence of carbachol, revealed the selective coupling of activated muscarinic receptors to G protein subtypes.

High-performance liquid chromatographic determination of 3-hydroxy-3-methylglutaryl coenzyme A as diphenacyl ester of 3-hydroxy-3-methylglutarate.

A method for derivatization of (S)-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) by phenacylation in tetrahexylammonium hydrogensulphate as phase-transfer reagent and high-performance liquid chromatography of the HMG-diphenacyl ester is described. To avoid interference with buffers and other ions used to dissolve biological material, the alkaline hydrolysate of CoA esters has to be neutralized with phosphoric rather than hydrochloric acid. The latter reacts with phenacyl bromide.

Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores.

The results of binding studies suggest the presence of histamine H1-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca2+ concentration ([Ca2+]i) and superoxide anion (O2-) formation in HL-60 cells differentiated towards monocytes with 1 alpha,25-dihydroxycholecalciferol. In HL-60 monocytes, HA increased [Ca2+]i with a half-maximal effect at 8 microM and a maximum at 30-100 microM.

A growth factor phenotype map for ovine preimplantation development.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development.

The H1 receptor agonist 2-(3-chlorophenyl)histamine activates Gi proteins in HL-60 cells through a mechanism that is independent of known histamine receptor subtypes.

In dibutyryl-cAMP-differentiated HL-60 cells, histamine H1 and formyl peptide receptors mediate increases in the cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive G proteins of the Gi family. We compared the effects of 2-(3-chlorophenyl)-histamine (CPH) [2-[2-(3-chlorophenyl)-1H-imidazol-4-yl] ethanamine], one of the most potent and selective H1 receptor agonists presently available, with those of histamine and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in these cells. CPH increased [Ca2+]i through Ca2+ mobilization and Ca2+ influx.

Insulin-like growth factor binding proteins are transcribed by preimplantation mouse embryos.

Preimplantation embryos have been reported to synthesize insulin-like growth factors (IGF) and their receptors, and reproductive tract fluid has been found to contain insulin and IGF I. In this communication, we report that all stages of preimplantation mouse embryos transcribe IGF binding proteins (IGF-BP) 2, 3 and 4, and that blastocysts also transcribe IGF-BP6. IGF-BP5 was not detected at any preimplantation stage.

Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.