Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.

The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea.

Background

G protein-coupled receptors (GPCRs) constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum.

Results

Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification.

Conclusions

Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

A 330-360 GHz spectral survey of G 34.3+0.15 .1. Data and physical analysis

A 330--360 GHz spectral survey of the hot molecular core associated with the 'cometary' ultracompact HII region G 34.3+/-0.15 observed with the James Clerk Maxwell Telescope has detected 338 spectral lines from at least 35 distinct chemical species plus 19 isotopomers. 70 lines remain unidentified. Chemical abundance and rotation temperature have been determined by rotation diagram analysis for 12 species, and lower limits to abundance found for 38 others.

Late glacial interhemispheric climate dynamics revealed in South African hyrax middens

Our ability to identify the timing and extent of past major climate fluctuations is central to understanding changes in the global climate system. Of the events that have occurred in recent geological time, the Younger Dryas (YD, 13-11.5 ka), an abrupt return to near-glacial conditions during the last glacial-interglacial transition (ca. 18-11.5 ka), is one of the most widely reported. While this event is apparent throughout the Northern Hemisphere (Peteet, 1995), evidence for its occurrence in the Southern Hemisphere remains equivocal due to a lack of well-dated terrestrial records. Here we report high-resolution stable carbon and nitrogen isotope records obtained from a rock hyrax midden, revealing the first unequivocal terrestrial manifestation of the YD from the southern African subtropics. These results provide key evidence for the relative influence of the YD, and suggest that a subtropical-temperate transition zone existed along the oceanic Subtropical Front (similar to 41 degrees S) across the Southern Hemisphere, with the Northern Hemisphere exerting a strong influence on all but the higher latitudes of the Southern Hemisphere after the Heinrich Stadial 1 (15 ka).

WASP-39b: a highly inflated Saturn-mass planet orbiting a late G-type star

We present the discovery of WASP-39b, a highly inflated transiting Saturn-mass planet orbiting a late G-type dwarf star with a period of 4.055259 +/- 0.000008 d, Transit Epoch T-0 = 2 455 342.9688 +/- 0.0002 (HJD), of duration 0.1168 +/- 0.0008 d. A combined analysis of the WASP photometry, high-precision follow-up transit photometry, and radial velocities yield a planetary mass of M-pl = 0.28 +/- 0.03 M-J and a radius of R-pl = 1.27 +/- 0.04 R-J, resulting in a mean density of 0.14 +/- 0.02 rho(J). The stellar parameters are mass M-star = 0.93 +/- 0.03 M-circle dot, radius R-star = 0.895 +/- 0.23 R-circle dot, and age 9(-4)(+3) Gyr. Only WASP-17b and WASP-31b have lower densities than WASP-39b, although they are slightly more massive and highly irradiated planets. From our spectral analysis, the metallicity of WASP-39 is measured to be [Fe/H] = -0.12 +/- 0.1 dex, and we find the planet to have an equilibrium temperature of 1116(-32)(+33) K. Both values strengthen the observed empirical correlation between these parameters and the planetary radius for the known transiting Saturn-mass planets.

Christian Gottlob Neefe and the Bonn National Theatre, with New Light on the Beethoven Family

When the Bonn stage was closed after the death of Elector Max Friedrich in 1784, the director of its theatre company, Gustav Friedrich Wilhelm Großmann, decided to leave the city to further his career elsewhere in the Rhinelands. During the next few years, he was kept informed about developments in Bonn by two of his erstwhile colleagues, Christian Gottlob Neefe and Nikolaus Simrock, whose correspondence paints a vivid picture of musical life in the city during the later 1780s. The new Elector, Maximilian Franz, permitted a visiting troupe to perform during Carnival each year, but repeatedly delayed the decision to re-establish a resident troupe. In 1787 Christoph Brandt, a singer in the Bonn Hofkapelle, attempted a home-grown initiative, perhaps to test the market for a new permanent company. Although this failed almost immediately, a single, well-attended public rehearsal of Monsigny’s Le Déserteur was given, in which Johann van Beethoven made what was probably his last stage appearance. In a letter dated 14 May 1787, Simrock rated his performance ‘zimlich gut’. In the event, a new Bonn troupe was not recruited until 1789, when it featured the young singer Magdalena Willmann. Neefe and his musical colleagues were relieved finally to be able to resume their theatrical careers.

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.