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PubMed2004

Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila.

Although synapses are assembled in a highly regulated fashion, synapses once formed are not static structures but continue to expand and retract throughout the life of an organism. One second messenger that has been demonstrated to play a critical role in synaptic growth and function is cAMP. Here, we have tested the idea that signaling through the heterotrimeric G protein, Gs, plays a coincident role with increases in intracellular Ca(+2) in the regulation of adenylyl cyclases (ACs) during synaptic growth and in the function of synapses. In larvae containing a hypomorphic mutation in the dgs gene encoding the Drosophila Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization, as well as defects in the facilitation of synaptic transmission. Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha either pre- or postsynaptically rescued the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. In addition, this Gs alpha mutation interacted with fasII, dnc, and hyperexcitability mutants in a manner that revealed a coincident role for Gs alpha in the regulation of cAMP and FASII levels required during growth of these synapses. Our results demonstrate that Gs alpha-dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.

Animals↗

PubMed2005

Classification of wheat low-molecular-weight glutenin subunit genes and its chromosome assignment by developing LMW-GS group-specific primers.

On the basis of sequence analysis, 69 known low-molecular-weight glutenin subunit (LMW-GS) genes were experimentally classified into nine groups by the deduced amino acid sequence of the highly conserved N-terminal domain. To clarify the chromosomal locations of these groups, 11 specific primer sets were designed to carry out polymerase chain reactions (PCR) with the genomic DNA of group 1 ditelosomic lines of Chinese Spring, among which nine primer sets proved to be LMW-GS group-specific. Each group of LMW-GS genes was specifically assigned on a single chromosome arm and hence to a specific locus. Therefore, these results provided the possibility to predict the chromosome location of a new LMW-GS gene based on its deduced N-terminal sequence. The validity of the classification was confirmed by the amplifications in 27 diploid wheat and Aegilops accessions. The length polymorphisms of LMW-GS genes of groups 1 and 2, and groups 3 and 4.1 were detected in diploid A-genome and S-genome accessions, respectively. The diploid wheat and Aegilops species could be used as valuable resources of novel allele variations of LMW-GS gene in the improvement of wheat quality. The nine LMW-GS group-specific primer sets could be utilized to select specific allele variations of LMW-GS genes in the marker-assisted breeding.

Breeding↗

PubMed2001

The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast.

The chloroplastic isoform of glutamine synthetase (GS-2, EC 6.3.1.2) from Nicotiana tabacum L. is phosphorylated at the serine residues. At least three of the six GS-2 subunits separated by two-dimensional polyacrylamide gel electrophoresis cross-reacted with an antibody raised against phosphoserine. This provoked the question as to whether 14-3-3 proteins might be present in the chloroplast and bind to chloroplastic GS-2. Although two different 14-3-3 proteins of 32 and 30 kDa were present in total leaf extracts, in the soluble fraction of chloroplasts, only the 32-kDa 14-3-3 protein was immunodetected with an antibody raised against a conserved region of 14-3-3 protein from corn. This demonstrates the presence of a chloroplast-located isoform of 14-3-3 proteins in tobacco. To examine a putative binding of GS-2 to these 14-3-3 proteins in vivo, the native GS-2 holoenzyme was probed with a 14-3-3 antibody. The strong cross-reaction between GS-2 and the 14-3-3 antibody clearly points to a binding of GS-2 and 14-3-3 in tobacco chloroplasts. Only those oligomers of GS-2 that were strongly associated with 14-3-3 proteins were catalytically active.

14-3-3 Proteins↗

PubMed1994

Human-Xenopus chimeras of Gs alpha reveal a new region important for its activation of adenylyl cyclase.

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The alpha subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gs alpha subunits are 92% identical to those found in the short form of human Gs alpha. Despite this high homology, the X. laevis Gs alpha clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc- membranes in contrast with human Gs alpha similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gs alpha. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gs alpha. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTP gamma and isoproterenol. The results obtained here clearly indicate that the G alpha sequence that extends from amino acid 70 to 140, is important for the functionality of human Gs alpha in activating adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1994

Exclusion of close linkage of bipolar disorder to the Gs-alpha subunit gene in nine Australian pedigrees.

Growing evidence suggests that guanine nucleotide binding proteins (G proteins) may be involved in both the pathogenesis and treatment of bipolar affective disorder. Both overactive G proteins and increased levels of the alpha subunit of the stimulatory form (Gs-alpha) have been demonstrated in peripheral leucocytes of manic patients while an increase of Gs-alpha subunit levels has also been found in a postmortem study of bipolar disorder. The function of Gs and Gi alpha subunits has now been shown to be affected by lithium. The present study aimed to determine whether bipolar affective disorder was linked to the Gs-alpha subunit gene which has been mapped to chromosomal region 20q13.2. Linkage analysis utilized the PCR amplification of a portion of the Gs-alpha gene that contains a dinucleotide repeat (CA repeat) polymorphism. Linkage of bipolar disorder and recurrent depression to the Gs-alpha subunit gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness. Close linkage to the Gs-alpha subunit gene was strongly excluded using each model and definition. Thus, our study indicates that a genetic defect in the Gs-alpha subunit gene is unlikely to be the cause of bipolar disorder.

Adolescent↗

PubMed1993

A mutant substituting valine for glycine at position 49 of Gs alpha induces neuronal differentiation of PC12 cells without activation of adenylate cyclase.

A GTPase-deficient mutant of the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylate cyclase, substituting valine for glycine 49 (G49V) was transiently expressed in COS7 cells. The basal level of cAMP as well as an agonist-dependent accumulation of cAMP was two-fold higher in transfectants of Gs alpha (G49V) than in those of the normal counterpart. A stable transformant of PC12 cells expressing Gs alpha (G49V) under the control of a metallothionein promoter was then established. Two independent clones showed neurite outgrowth when the mutated Gs alpha was expressed by adding Cd2+ to culture medium. However, the level of basal cAMP of the transformant of PC12 cells with Gs alpha (G49V) was lower than that of the parental cells. The response to an agonist of the adenosine A2-receptor was suppressed in transformants. Although a cAMP-responsive element (CRE) was slightly activated by transfection and transient expression of Gs alpha in PC12 cells, no activation but a suppression of CRE was observed with PC12 cells transiently expressing Gs alpha (G49V). These results suggest that the mutant Gs alpha (G49V) couples adenylate cyclase in a different way in PC12 cells and may transduce differentiation signals through a cAMP-independent pathway.

Adenylyl Cyclases↗

PubMed1994

Gs alpha-dependent and -independent desensitisation of prostanoid IP receptor-activated adenylyl cyclase in NG108-15 cells.

NG108-15 mouse neuroblastoma x rat glioma cells were treated with the prostanoid IP receptor agonist iloprost (1 microM) and the time course of changes in the levels of prostanoid IP receptors, adenylyl cyclase activity, and the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein, Gs, were measured. Incubation of cells with iloprost produced a biphasic time course of desensitisation of prostanoid IP receptor-activated adenylyl cyclase. Parallel analysis of iloprost-induced loss of membrane Gs alpha, NaF-stimulated adenylyl cyclase and [3H]iloprost binding suggested only monophasic curves, with t0.5 values similar to the initial phase of desensitisation of iloprost-stimulated adenylyl cyclase activity. This suggests that the loss of receptor and Gs alpha occur at the same time and account for the initial period of desensitisation due to iloprost pretreatment. Pretreatment of NG108-15 cells with cholera toxin produced a near complete loss of membrane-associated Gs alpha, but the loss of [3H]iloprost binding due to iloprost treatment was not affected by pretreatment with cholera toxin, suggesting that prostanoid IP receptors can be down-regulated in the absence of any coupling to Gs. The second phase of desensitisation of iloprost-stimulated adenylyl cyclase activity, during which there was no further change in NaF-stimulated adenylyl cyclase or in the membrane levels of Gs alpha, was not due to protein kinase A activation, since elevating intracellular cyclic AMP levels with forskolin did not subsequently decrease iloprost-stimulated adenylyl cyclase activity or [3H]iloprost binding. These results demonstrate that iloprost pretreatment of NG108-15 cells induces two distinct phases of desensitisation; an initial desensitisation due to concurrent loss of prostanoid IP receptors and Gs alpha, and then a further desensitisation by an as yet uncharacterized mechanism during which there is no further loss of Gs alpha.

Adenylyl Cyclases↗

PubMed2006

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro.

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.

Animals↗

PubMed1997

Solution structure of the sodium channel antagonist conotoxin GS: a new molecular caliper for probing sodium channel geometry.

BACKGROUND: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. RESULTS: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. CONCLUSIONS: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

Amino Acid Sequence↗

PubMed1998

Biexponential decomposition of a neuraminidase inhibitor prodrug (GS-4104) in aqueous solution.

PURPOSE: To examine the degradation kinetics and identify the degradation products of a neuraminidase inhibitor prodrug. GS-4104. METHODS: Degradation was studied as a function of pH and temperature using a stability-indicating RP-HPLC assay. Degradation products were isolated by RP-HPLC and identified by NMR. Specific rate constants were calculated based on a scheme defined by products(s) analysis. RESULTS: Three distinct degradation products were observed in the pH region studied (pH 2-8): isomer I, GS-4071, and isomer II. Isomer I resulted from the N, N-migration of the acetyl group. Gs-4071 was formed by the hydrolysis of the ethyl ester. Both GS-4071 and isomer I degraded further to isomer II by N, N-acyl migration and ester hydrolysis, respectively. The N, N-acyl migration reaction was characterized using two dimensional heteronuclear multiple bond correlation (HMBC) NMR. The decomposition kinetics of GS-4104 follow a biexponential decay at pH 2-7. The degradation kinetics of Gs-4104 at pH 4.0, 70 degree C were independent of the initial GS-4104 concentration. CONCLUSIONS: The degradation profile indicates that development of solution or solid dosage from of GS-4104 with adequate shelf-life stability at room temperature is feasible.

Amines↗

PubMed1989

Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs).

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.

Animals↗

PubMed1992

Beta-adrenergic-receptor-mediated dissociation and membrane release of the Gs protein in S49 lymphoma-cell membranes. Dependence on Mg2+ and GTP.

We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Animals↗

PubMed1993

Attenuation of Gs alpha coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters.

In order to localize site(s) of beta-adrenergic desensitization found in brown adipocytes from cold-acclimated animals, total brown-adipose-tissue homogenates (postnuclear supernatant) were obtained from control or cold-acclimated hamsters and were fractionated on discontinuous sucrose gradients. A low-density band (cytosolic proteins) and a high-density band (mitochondria) were obtained; in the middle fractions only low levels of protein were recovered. However, these fractions displayed a high level of specific [3H]ouabain binding, indicating that they represented fractions enriched in plasma membranes. The level of [3H]ouabain binding was significantly higher in plasma membranes from cold-acclimated animals, indicating an increased density of Na,K-ATPase units. The maximal activity of adenylate cyclase, as estimated with forskolin, was not changed by cold acclimation. However, the levels of cyclase activity observed after Gs-protein-mediated activation (with guanosine 5'-[gamma-thio]triphosphate, isoprenaline, both of these, or fluoride) were decreased, indicating a decreased coupling efficiency. Notably, a significant decrease was observed in the functional activity of the Gs protein, as directly measured by estimation of the ability of cholate extracts of brown-fat plasma membranes to reconstitute Gs-protein-mediated stimulation of adenylate cyclase in cyc- membranes. Further, a functionally significant decrease (to 72%) was observed in the ratio between the amount of functional Gs proteins and adenylate cyclase units. The total content of Gs alpha protein was decreased to the same extent as the coupling efficiency of the membranes, indicating that a lower content of functionally equivalent Gs alpha molecules could explain the decreased coupling. It could therefore be concluded that a decrease in Gs-protein-mediated coupling efficiency, owing to a decrease in the amount of Gs alpha, is at least one site of beta-adrenergic desensitization in cold-acclimated animals. This may, at least in part, explain that desensitization takes place despite the fact that the beta 3-adrenoceptor itself apparently lacks some of the sites known to be involved in the desensitization process in other beta-adrenergic receptors.

Acclimatization↗

PubMed1988

Genetic deficiency of the alpha subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy.

Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the alpha subunit of the G protein that stimulates adenylyl cyclase (alpha Gs) (ATP pyrophosphate-lyase, EC 4.6.1.1). To discover the molecular mechanism that causes alpha Gs deficiency in these patients, we examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and alpha Gs deficiency. In these families, alpha Gs deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern. Using a cDNA hybridization probe for alpha Gs, restriction analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed approximately equal to 50% reduced mRNA levels for alpha Gs in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the alpha subunit of the G protein that inhibits adenylyl cyclase were not altered. Our findings suggest that several molecular mechanisms produce alpha Gs deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for alpha Gs deficiency in pseudohypoparathyroidism type I.

Cell Membrane↗

PubMed1999

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.

Animals↗

PubMed1992

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.

Amino Acid Sequence↗

PubMed1992

Stimulus deprivation increases pineal Gs alpha and G beta.

Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters alpha and beta subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the alpha and beta subunits of Gs (Gs alpha and G beta) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light-induced stimulus deprivation were also apparent by measuring cholera toxin-dependent ADP-ribosylation of Gs alpha, which revealed a four-fold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gs alpha and G beta during 72 h of organ culture. The constant light-induced increases in both Gs alpha and G beta were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gs alpha and G beta. These studies indicate that stimulus deprivation increases both Gs alpha and G beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1998

Oral administration of a prodrug of the influenza virus neuraminidase inhibitor GS 4071 protects mice and ferrets against influenza infection.

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.

Acetamides↗