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PubMedDate not supplied

Purification of PMPA amidate prodrugs by SMB chromatography and x-ray crystallography of the diastereomerically pure GS-7340.

The diastereomers of GS-7171, aryl phosphoramidate derivatives of the anti-HIV nucleotide analog 9-[2-R-(phosphonomethoxy)propyl]adenine (tenofovir, PMPA), were isolated by batch elution chromatography and continuous simulated moving bed chromatography. The absolute configuration of the more pharmacologically active diastereomer, GS-7340, was determined to be (R,S,S) by single crystal x-ray crystallography.

Adenine↗

PubMed1992

A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production.

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.

Adenosine Diphosphate↗

PubMed1975

Oncogenesis by Marek's disease herpesvirus in chickens lacking expression of endogenous (gs, chick helper factor, Rous-associated virus-O) and exogenous avian RNA tumor viruses.

Chickens free of exogenous avian leukosis virus (ALV) infection, replicating endogenous ALV (Rous-associated virus-O), gs antigen, and chick helper factor were fully susceptible to induction of Marek's disease (MD) by ALV-free MD viruses. Dual infection with Rous-associated virus-2 and MD virus did not significantly alter the character of the MD lesions. Thus exogenous ALV infection was not requisite for MD virus-induced oncogenesis. Although participation of endogenous RNA tumor virus genes in MD lesion induction could not be excluded, expression of such genes in MD tumors as gs antigen was not established.

Animals↗

PubMed1993

Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases.

Receptor-regulated adenylyl cyclase in mammalian systems is among the best-studied of the cell surface signaling pathways that utilize G-proteins as transducers. In addition to the multiplicity of receptors and Gs proteins that function in this pathway, recent studies have shown that substantial molecular diversity exists in the effector as well. Full-length cDNAs encoding six different G-protein-regulated adenylyl cyclases have been isolated, and partial sequences identifying two more are known. These eight mammalian adenylyl cyclases can be grouped into five distinct families. The different types share some common properties such as stimulation by Gs and the diterpene forskolin. They show very distinct patterns of regulation by the beta gamma-subunits of G-proteins and protein kinases such as protein kinase C. The different types also appear to be localized in a tissue-specific manner. This diversity of regulatory features indicates that the effector can play an important role in determining the routing of signals to the cAMP pathway. The tissue and cell type-specific localization of the individual forms suggests that effectors such as adenylyl cyclase could be potential targets for a new generation of cell and tissue-specific drugs.

Adenylyl Cyclases↗

PubMed1993

Endothelin inhibits the calcium pump and stimulates phosphoinositide phospholipase C in liver plasma membranes via two different G proteins, Gs and Gq.

We have shown previously that in liver, endothelin (ET) binding to plasma membranes causes a rise in cytosolic calcium and activation of glycogenolysis. Here we show that the calcium extrusion pump in liver plasma membranes is inhibited by ET peptides, with ET-1 > or = ET-3 = sarafotoxin S6C-inhibition of the system being potentiated by GTP gamma S. Also, ET-1 stimulates PIP2 hydrolysis in liver plasma membranes in a guanine nucleotide-dependent manner, with ET-1 > or = ET-3 = sarafotoxin S6C. In order to determine the nature of G protein(s) coupling of the ETB receptor to both effectors, antibodies against the C-terminus of different G-protein alpha-subunits were used. Antibodies reactive with Gs alpha blocked ET-1 inhibition of the calcium pump, but they had no effect on ET-1 stimulation of PIP2 hydrolysis. Antibodies reactive with Gq alpha dose-dependently antagonized stimulation of PIP2 breakdown by ET-1 without affecting ET-1 inhibition of the calcium pump. Antibodies reactive with Gi1 alpha/Gi2 alpha had no effect on both systems. We conclude that the calcium signal induced by endothelins in hepatocytes may be consequent to both an activation of phospholipase C and inhibition of the calcium pump, both effectors being coupled to the ETB receptor by different G proteins, Gq and Gs, respectively.

Amino Acid Sequence↗

PubMed1993

Chromosome organization of Streptococcus mutans GS-5.

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5'-GGGCC/C), SmaI (5'-CCC/GGG), and NotI (5'-GC/GGCCGC).

Blotting, Southern↗

PubMed1999

Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).

Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.

Alcohol Oxidoreductases↗

PubMed2003

Fast algorithms for GS-model-based image reconstruction in data-sharing Fourier imaging.

Many imaging experiments involve acquiring a time series of images. To improve imaging speed, several "data-sharing" methods have been proposed, which collect one (or a few) high-resolution reference(s) and a sequence of reduced data sets. In image reconstruction, two methods, known as "Keyhole" and reduced-encoding imaging by generalized-series reconstruction (RIGR), have been used. Keyhole fills in the unmeasured high-frequency data simply with those from the reference data set(s), whereas RIGR recovers the unmeasured data using a generalized series (GS) model, of which the basis functions are constructed based on the reference image(s). This correspondence presents a fast algorithm (and two extensions) for GS-based image reconstruction. The proposed algorithms have the same computational complexity as the Keyhole algorithm, but are more capable of capturing high-resolution dynamic signal changes.

Algorithms↗

PubMed1994

Gs alpha and Gi2 alpha mutations in clinically non-functioning pituitary tumours.

BACKGROUND AND OBJECTIVE: Activating mutations of Gs alpha (gsp) and Gi2 alpha (gip) have been described in various endocrine neoplastic conditions. The objective of this study was to assess the prevalence of gsp and gip mutations in clinically non-functioning pituitary tumours (NFTs) and to compare the clinical phenotypic characteristics of tumours bearing G protein gene mutations with wild-type tumours. DESIGN: Twenty-two NFTs and 20 normal anterior pituitary glands screened for G protein gene mutations. PATIENTS: Twenty-two patients; 14 female (median age 59 years, range 19-76) and 8 males (median age 66.5 years, range 50-77). MEASUREMENTS: Site-directed hybridization or direct sequencing of polymerase chain reaction amplified Gs alpha and Gi2 alpha DNA. RESULTS: G protein gene mutations were identified in 3/22 (13%) of NFTs. Two tumours demonstrated gsp mutations, one at codon 201 arginine to cysteine, and the second at codon 227 glutamine to arginine. Three tumours demonstrated gip mutations at codon 205 glutamine to arginine. Two tumours with gsp mutations also harboured gip mutations. All tumours with G protein gene mutations demonstrated local bone infiltration into the surrounding structures. CONCLUSIONS: G protein gene mutations have been demonstrated in a proportion of non-functioning pituitary tumours. The presence of dual gsp and gip mutations in two tumours suggests the possibility of multiple hits in a stepwise pathogenesis of pituitary neoplasia.

Adenoma↗

PubMed1995

Characterization of lymphocyte beta-adrenoceptor activity and Gs-protein in patients with rheumatic heart valvular disease.

In order to test whether the beta-adrenoceptor activity in rheumatic heart valvular disease depends on the ventricular load conditions, we determined their density and binding affinity to [125I]-iodocyanopindolol in lymphocytes, as well as plasma catecholamine and cAMP levels in 69 patients with regurgitant and stenotic lesions of the aortic and mitral valves. The patients were classified as having left ventricular pressure overload (LVP), left ventricular volume overload (LVV), mixed lesions (MOL) or right ventricular pressure overload (RVP). The beta-adrenoceptor activity was determined by radioligand binding methods, catecholamines by high performance liquid chromatography using an electrochemical detector and cAMP by radioimmunoassay. The mean beta-adrenoceptor density (Bmax) of the control group was 60.1 +/- 9.5 fmol (n = 29) per 10(6) lymphocytes. In the study population, the density was decreased by 83% in LVP, 78% in LVV, 87% in MOL and 86% in RVP. Plasma norepinephrine was elevated by 89% in LVP and 60% in MOL, epinephrine by 43% in LVP, 50% in VOL, 115% in MOL and 20% in RVP, while dopamine was not significantly changed, and cAMP was slightly elevated in all four groups. Screening for activating mutational changes in the Gs alpha-protein gave negative results, possibly dissociating the elevation in plasma cAMP from stimulatory effects of such abnormalities in the Gs-protein signaling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1988

Role of stimulatory GTP-binding protein (Gs) in reduced beta-adrenoceptor coupling in the femoral artery of spontaneously hypertensive rats.

1. Arterial relaxant responses to beta-adrenoceptor agonists are decreased in spontaneously hypertensive rats (SHR) when compared with normotensive Wistar-Kyoto rats (WKY). To establish which component of the beta-adrenoceptor.adenylate cyclase (AC) system is impaired in the SHR arteries, effects of two activators of AC--cholera toxin (CTX) and forskolin--and of dibutyryl cyclic AMP (db cyclic AMP) were compared between strips of femoral arteries isolated from 13 week-old SHR and age-matched WKY. 2. In the absence of timolol, a beta-adrenoceptor antagonist, contractile responses of the strips to noradrenaline (NA) were significantly greater in the SHR than in the WKY. Timolol augmented the contractile responses to NA to a smaller extent in the SHR than in the WKY. 3. After blockade by timolol of beta-adrenoceptors, contractile responses of the strips to NA through the activation of alpha-adrenoceptors were not significantly different between the two strains. 4. Pre-treatment of the strips with CTX, an activator of the stimulatory GTP-binding protein (Gs), produced a slow-onset and long-lived antagonism of the alpha-adrenoceptor-mediated contractions. The antagonism was much smaller in the SHR than in the WKY. 5. The dose-response curves of the strips from both strains for alpha-adrenoceptor stimulation with NA determined after pretreatment with CTX were comparable to those determined in the absence of timolol. 6. Forskolin, an activator of the catalytic subunit of AC, and DB cyclic AMP also antagonized the alpha-adrenoceptor-mediated contractions. However, these antagonisms were not significantly different between the two strains. 7. Isobutyl methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterase, produced a similar antagonism of the alpha-adrenoceptor-mediated contractions between the two strains. 8. These results suggest that a reduced function of Gs is the main factor responsible for the decreased responsiveness to beta-adrenoceptor stimulation in the SHR femoral artery.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Hormonal tolerance to ethanol is associated with decreased expression of the GTP-binding protein, Gs alpha, and adenylyl cyclase activity in ethanol-treated LS mice.

Using the proopiomelanocortin (POMC) system as a marker, long sleep (LS) and short sleep (SS) lines of mice were investigated to explore the cellular events that occur during the acquisition of hormonal tolerance to ethanol. Four-day ethanol exposure (1.8 g/kg/24 hr) increased anterior pituitary POMC mRNA levels 4-fold in the LS line and 2-fold in the SS line. Following 7 days of ethanol exposure (1.8 g/kg/24 hr), anterior pituitary POMC levels returned to basal values in the LS line but remained elevated (2-fold) in the SS line. In this setting, the loss of ethanol's ability to sustain elevated POMC mRNA levels in the LS line is defined as hormonal tolerance. Since POMC biosynthesis is primarily regulated through adenylyl cyclase, ethanol-induced alterations in this signal transduction system were explored. Paralleling the effects of ethanol on POMC mRNA levels, ethanol exposure reduced GTP-gamma-S, AIF3-, and MnCl2-stimulated adenylyl cyclase activity by 35%, 21%, and 24%, respectively, in the LS line without effecting adenylyl cyclase activity in the SS line. To determine whether ethanol-induced changes in adenylyl cyclase activity in LS mice could result from alterations in G proteins, protein levels of G, alpha and Gi alpha were determined by western analysis before and after ethanol exposure. Paralleling the effect on POMC mRNA levels and adenylyl cyclase activity, ethanol induced a 35% reduction in Gs alpha protein levels in LS mice but did not alter Gi alpha levels. Neither Gs alpha nor Gi alpha levels were altered in the SS line.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1994

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA.

Streptococcus mutans GS-5 synthesizes an exo-beta-D-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans. These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗

PubMed1992

The role of Mg2+ on the formation of the ternary complex between agonist, beta-adrenoceptor, and GS-protein and an interpretation of high and low affinity binding of beta-adrenoceptor agonists.

In the absence of GTP or its analogues, the beta-adrenoceptor agonist (H), the receptor (R) and the stimulatory guanine nucleotide-binding regulatory protein (GS-protein) form a slowly dissociable complex (HRGSGDP). It is presumed that the agonist in this ternary complex is bound to the receptor with high affinity while its binding to the receptor in the HR-complex is of low affinity. We have studied the effect of Mg2+ on the formation of these two complexes by the displacement of (-)-[3H]CGP-12177 with (-)-isoprenaline in cell membranes prepared from guinea-pig lung parenchyma. In the absence of Mg2+, only monophasic displacement curves were obtained with a dissociation constant not differing from that obtained in the presence of Gpp(NH)p and Mg2+. In the presence of Mg2+ and absence of Gpp(NH)p, a shallow binding curve was obtained that fitted a two site model best. One of the dissociation constants agreed well with that obtained in the presence of Gpp(NH)p and Mg2+ but the other one was 27 times smaller. It was concluded that Mg2+ is necessary for the coupling between the HR-complex and the GS-protein. The two dissociation constants obtained in the absence of Gpp(NH)p represent the dissociation constants of the HR- and HRGSGDP-complexes and may correspond to those named KL and KH in the literature. According to our study, there may be no difference in affinity between the agonist and receptor in the HR- and HRGSGDP-complexes. The two dissociation constants obtained in the absence of GTP or its analogues refer to two consecutive reactions.

Adrenergic beta-Agonists↗

PubMed1994

Clinical behaviour over three years of GS-80 and Lojic+ amalgam alloys.

The clinical performance of GS-80 and Lojic+amalgam alloys was compared with that of Dispersalloy, Valiant PhD, Lojic N and Permite C amalgam alloys over periods of up to three years. The 1114 restorations were placed by one dentist in 23 canine, 418 premolar and 673 molar permanent teeth of 301 adult patients treated in a private practice. Direct and indirect clinical assessments were made of the restorations for failures (there were no true failures), and for the deterioration of four clinical factors. All restorations were rated as being either 'good' (A) or 'adequate' (B) for each of the four factors which, apart from surface texture, showed some slight but statistically-significant clinical deterioration with time. Lojic+ and Permite C showed clinically slight but statistically more surface roughness and marginal fracture, while Lojic+ and GS-80 showed more surface tarnishing, than did the other alloys. There were no statistically significant differences between the alloys for marginal staining.

Adult↗

PubMed1989

Splice variants of the alpha subunit of the G protein Gs activate both adenylyl cyclase and calcium channels.

Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.

Adenylyl Cyclases↗

PubMed1995

Construction of a soluble adenylyl cyclase activated by Gs alpha and forskolin.

A soluble adenylyl cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) Gs (Gs alpha). Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl cyclase activity. The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two sets of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases.

Adenylyl Cyclases↗

PubMed1981

Localization of Streptococcus mutans GS-5 glucosyltransferases and intracellular invertase.

Antibodies directed against Streptococcus mutans GS-5 intracellular invertase and glucosyltransferase fractions capable of synthesizing primarily water-soluble or insoluble glucans were used to ultrastructurally localize the enzymes by means of the unlabeled antibody peroxidase-antiperoxidase method. This immunocytochemical procedure revealed that the intracellular invertase was associated primarily with the cytoplasmic membrane of the cariogenic organism. The glucosyltransferase complex responsible for insoluble glucan synthesis was localized as aggregates attached to the cell surface or extracellular polysaccharides of strain GS-5. In contrast, the glucosyltransferase activity synthesizing primarily water-soluble glucans was distributed uniformly over the cell surface or in association with extracellular polysaccharides. These results are discussed relative to the sucrose-metabolizing ability of Streptococcus mutans.

Cell Membrane↗