SEARCH · PubMed Health

Results for “GS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources

PubMed2001

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.

Amino Acid Sequence↗

PubMedDate not supplied

Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMPA, in blood.

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[(R)-2-[[[[(S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.

Adenine↗

PubMed1991

Stoichiometry of receptor-Gs-adenylate cyclase interactions.

Little is known about the relative stoichiometry of guanine nucleotide-binding (G) proteins relative to the effector systems to which they link. We addressed this question for the stimulatory G protein (Gs) linked to adenylate cyclase. Forskolin stimulates the catalytic subunit of adenylate cyclase (C), but it has a higher efficacy and potency when C also interacts with the G protein Gs. Accordingly, we measured high-affinity [3H]forskolin binding to intact cells to assay alpha s-C complexes. No high-affinity specific binding occurred with unstimulated cells. The beta-adrenergic agonist isoproterenol promoted the binding of [3H]forskolin to about 3000 sites per cell, suggesting that each receptor on average activates at least several Gs molecules. Activating Gs directly with cholera toxin maximally promoted [3H]forskolin binding to a similar number of sites, suggesting that this is the maximal number of alpha s-C complexes formed per cell. We conclude that each cell likely contains only a few thousand functional copies of C, and that the availability of C (rather than Gs, which exists in more than 100,000 copies per cell) is likely to be limiting for agonist stimulation of adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed2001

Corneal temperature reversal after storage in Chen medium compared with Optisol GS.

PURPOSE: To compare corneal endothelial cell function by measuring corneal thickness during temperature reversal between corneas stored in two different storage media, Optisol GS and Chen Medium (CM). METHODS: Twenty paired corneas from 10 human donors were randomly assigned for storage at 4 degrees C in Optisol GS (10 corneas) or CM (10 corneas). The storage media were masked, and measurements were done in a masked fashion. After storage for 48 hours, corneal thickness was measured by ultrasonic pachymetry at 2-hour intervals for 12 hours, during which time the corneas were perfused with BSS (balanced salt solution) Plus at 37 degrees C. Scanning electron microscopy of two pairs of corneas from two donors was performed to assess ultrastructural change after 12 hours of warming. RESULTS: Corneal thickness decreased during the first 4 hours of the warming period and then increased during the 6-to 12-hour warming period. These changes in corneal thickness over time were similar for the two storage media (p = 0.212). Scanning electron microscopy showed greater amounts of endothelial cell disruption in Optisol GS-stored corneas than those stored in CM after 12 hours of warming and perfusion. CONCLUSIONS: The endothelial pump of corneas stored in CM appear to be as well-preserved as those stored in Optisol GS, although greater endothelial disruption may be present with Optisol GS by scanning electron microscopy. Further studies are required to compare the clinical effectiveness of these two media.

Aged↗

PubMed2006

The effect of temperature on the antimicrobial activity of Optisol-GS.

PURPOSE: To determine the survival of different bacteria inoculated in Optisol-GS at refrigerated storage temperature (6 degrees C) and after subsequent warming to room temperature (19-22 degrees C). METHODS: Staphylococcus aureus, Enterococcus faecium, Streptococcus pneumoniae, and Pseudomonas aeruginosa were chosen from stock clinical isolates for inclusion in the study. The first group consisted of 12 Optisol-GS vials. The second group consisted of 12 Optisol-GS vials containing corneas inappropriate for transplantation according to the Eye Bank Association of America (EBAA) protocols. Each group was inoculated with 3 concentrations of approximately 10, 10, and 10 colony-forming units (CFU)/mL of each bacterial species and then refrigerated per EBAA protocol. After 48 hours of refrigeration, all vials were placed at room temperature (RT) and counts were performed at 48, 50 (2 hour RT), 54 (6 hour RT), 60 (12 hour RT), 72 (24 hour RT), and 96 (48 hour RT) hours. At 96 hours, the corneal tissue from 10 and 10 inocula were cultured. All samples underwent serial dilution, spiral plating on blood agar plates, and incubation at 35 degrees C. Viable colony counts were determined at 24 hours. RESULTS: Except for the 10 CFU/mL inocula of P. aeruginosa, all isolates were viable after 48 hours of refrigeration. Rapid bactericidal activity was observed against P. aeruginosa after 2 hours at RT, with complete sterilization by 6 hours. The rate and extent of killing against S. aureus were influenced by the initial inoculum. Bactericidal activity was achieved after 2 hours at RT with 10 CFU/mL of S. aureus versus 24 hours with the 10 inoculum. Of note, bactericidal activity was not observed against S. pneumoniae and E. faecium following 24 hours of storage at RT. The presence of corneal tissue did not affect viable counts, with counts from corneal tissue cultures reflecting the counts seen from Optisol-GS after 48 hours at RT. CONCLUSIONS: The antimicrobial activity of Optisol-GS was reduced at refrigerated temperature and enhanced at RT. Bactericidal activity was not observed against E. faecium at either refrigerated temperature or RT.

Anti-Bacterial Agents↗

PubMed1998

Characterization of human influenza virus variants selected in vitro in the presence of the neuraminidase inhibitor GS 4071.

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

Acetamides↗

PubMed2000

Characterization of transcripts expressed from human herpesvirus 6A strain GS immediate-early region B U16-U17 open reading frames.

Several gene fragments of human herpesvirus 6 (HHV-6) have been shown to activate the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR). An open reading frame (ORF) designated B701 (Y. Geng, B. Chandran, S. F. Josephs, and C. Wood, J. Virol. 66:1564-1570, 1992), found within a 22-kb HHV-6A strain GS [HHV-6A(GS)] genomic fragment and a 3.8-kb SalI subfragment, was shown to activate the HIV LTR. B701, also known as HHV-6 U16, is located in the immediate-early B (IE-B) region of the genome. The sequence of the 3.8-kb genomic fragment of HHV-6A(GS) is nearly identical to the published sequence of HHV-6A strain U1102, with minor differences. The HHV-6A(GS) B701 ORF (U16) was used to screen an HHV-6A(GS) cDNA library, and two different but overlapping cDNAs were identified. These cDNAs represent differently spliced transcripts ending at different polyadenylation signals. The ORFs included in the cDNAs are positionally homologous to the human cytomegalovirus (HCMV) UL36 ORF. The ORF in one cDNA was generated by splicing together in frame ORFs U17 and U16, and the second cDNA included ORFs U16 and U15. A third differentially spliced cDNA (U16+), was identified by 5' rapid amplification of cDNA ends. The predicted protein was identical to the U16 portion of the U17/U16 spliced gene product but did not include the U17 portion. 5'-extension analyses of the mRNAs demonstrated that at least two potential transcription initiation sites were used to express the transcripts encoding U17 and U16 gene products. Single-stranded U16 and U17 gene-specific RNA probes hybridized with at least five RNA species from infected cells and demonstrated that the expression of these transcripts was differentially regulated. The U17/U16 spliced gene products were expressed at IE times after infection, but a multiply spliced gene product encoded by U16 was expressed as a late gene. The U17/U16 and the U16+ gene products transactivated the HIV LTR. Thus, while there are similarities to the HCMV UL36-UL38 gene family, some of the IE-B U17/U16 transcripts are unique to HHV-6.

Base Sequence↗

PubMed1996

Developmental differences in the expression of the cholera toxin sensitive subunit (Gs alpha) of adenylate cyclase in the rat small intestine.

BACKGROUND: The stimulatory guanosine triphosphate (GTP) binding protein alpha subunit (Gs alpha) of adenylate cyclase is the target protein for cholera toxin. AIMS/METHODS: The expression of this signal transducer was analysed in the small intestine of developing rats by RNA transfer (northern blot) analysis by immunoblotting, and by ADP-ribosylation of membrane proteins. RESULTS: Intestinal Gs alpha mRNA (about 1.9 kb) was increased in the neonate compared with the adult rat. Two isoforms of Gs alpha proteins, a 45,000 and a 52,000 form, were expressed in the small intestinal epithelial cell and both were ADP-ribosylated by cholera toxin. A significant increase in the larger isoform (52,000) and in its ribosylation was noted in the 2 week old suckling compared with post-weaned older animals. The protein content or ribosylation of the smaller form (45,000) did not significantly change with age. CONCLUSION: These data show that a developmental decline of intestinal Gs alpha expression seems to be, in part, regulated at the mRNA level. An increased Gs alpha expression in the immature intestine may help to explain a previously reported, dose dependent increased adenylate cyclase response and an increase in fluid secretion to cholera toxin in neonates compared with adults.

Adenylyl Cyclases↗

PubMed1994

Different pharmacological characteristics of structurally similar benzylisoquinoline analogs, papaverine, higenamine, and GS 389, on isolated rat aorta and heart.

In the present study, we compared the pharmacological properties of structurally similar benzylisoquinoline compounds, papaverine, higenamine, and GS 389, using isolated rat aorta and atrial preparations. The three benzylisoquinoline compounds, concentration dependently, relaxed phenylephrine (3 microM) induced contraction of rat aortic rings, with the rank order of potency being higenamine > papaverine > GS 389. They also relaxed high K+ (60 mM) induced contraction, with the rank order of potency being papaverine > GS 389 >> higenamine. The relaxation was not modified by the presence of endothelium. To assess whether these compounds directly interfere with Ca2+ influx, the effects of these compounds on Ca(2+)-induced contraction in Ca(2+)-free media were examined. Among the three compounds, papaverine most strongly inhibited Ca(2+)-induced contraction of both K+ stimulated and phenylephrine-stimulated aorta. Higenamine was least potent in inhibition of Ca(2+)-induced contraction in high K+ depolarized aorta. In atrial tissues, lower concentrations of papaverine increased spontaneous beats and isometric tension, whereas above 30 microM its action was reversed. GS 389 decreased heart rate without affecting the contractility. On the other hand, higenamine concentration dependently increased both heart rate and isometric tension, as well as cyclic AMP levels in atrial tissues as a result of beta-receptor activation. Cyclic AMP and cyclic GMP dependent phosphodiesterases from rat atrial and ventricular tissue homogenates were inhibited by papaverine and GS 389, but not by higenamine. These results suggest that calcium antagonistic action of these compounds is at least in part responsible for vasodilation action, but not for cardiac action.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗

PubMed1990

Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells.

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗

PubMed1994

Stimulation of rat parietal cell function by histamine and GLP-1-(7-36) amide is mediated by Gs alpha.

It was the aim of the present study to determine in rat parietal cells whether Gs alpha, the stimulatory subunit of adenylate cyclase, mediates adenosine 3',5'-cyclic monophosphate (cAMP)-dependent parietal cell function in response to histamine and glucagon-like peptide 1 (GLP-1)-(7-36) amide. Cytoplasmic membrane from enriched (83 +/- 5%) rat parietal cells were incubated for 30 min with 30 microCi/ml [32P]NAD+ and 40 micrograms/ml preactivated cholera toxin (CT), a pharmacological tool for activation of Gs alpha. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed [32P]ADP ribosylation of Gs alpha represented by three proteins with molecular masses ranging from 42 to 45 kDa. In intact parietal cells, CT (10(-12)-10(-8) M) caused marked stimulation of [14C]aminopyrine accumulation and cAMP production confirming the functional importance of Gs alpha in regulation of H+ production. Identical membrane preparations were preincubated (2 h, 4 degrees C) in parallel with and without RM/1, a rabbit polyclonal anti-Gs alpha-antibody. Subsequently, adenylate cyclase was stimulated by histamine, GLP-1-(7-36) amide, CT, or forskolin. At a 1:10 dilution, the antiserum completely abolished adenylate cyclase activity in response to maximal concentrations of histamine, GLP-1-(7-36) amide, and CT while reducing forskolin stimulation by only 22.0 +/- 4.9%. At 1:50, RM/1 reduced responses to histamine, GLP-1-(7-36) amide and CT by 20-30% but failed to inhibit forskolin-stimulated enzyme activity. At 1:100, the antiserum was ineffective versus all stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1997

Cardiomyopathy induced by cardiac Gs alpha overexpression.

The goal of this study was to determine whether chronic endogenous sympathetic stimulation resulting from the overexpression of cardiac stimulatory G protein alpha subunit (Gs alpha) in transgenic mice (15.3 +/- 0.1 mo old) resulted in a clinical picture of cardiomyopathy. The left ventricular ejection fraction, measured by echocardiography, was reduced in older mice with Gs alpha overexpression (50.4 +/- 5.4%) compared with age-matched control mice (70.9 +/- 1.6%; P < 0.05). When ejection fractions were compared at similar heart rates, the Gs alpha mice exhibited a greater left ventricular end-diastolic dimension than control mice (4.3 +/- 0.2 vs. 3.7 +/- 0.1 mm; P < 0.05). Baseline heart rates were elevated in conscious Gs alpha mice (722 +/- 27 beats/min; n = 5) compared with control mice (656 +/- 28 beats/min; n = 5). Moreover, electrocardiographic monitoring demonstrated a high incidence of arrhythmias. Increased mortality compared with control mice (31.6 vs. 3.0%; P < 0.01) was also observed. Thus older mice with Gs alpha overexpression exhibit many of the features of dilated cardiomyopathy. This study supports the concept that chronic sympathetic stimulation over an extended period of time, i.e., over the life of an animal, is deleterious and actually may result in cardiomyopathy.

Animals↗

PubMed1997

Expression of the Gs protein alpha-subunit disrupts the normal program of differentiation in cultured murine myogenic cells.

The manner in which growth factors acting at the cell surface regulate activity of myogenic basic-helix-loop-helix proteins in the nucleus and thus control the fate of committed skeletal myoblasts remains poorly understood. In this study, we report that immunoreactive Gs protein alpha-subunits (Gs alpha) localize to nuclei of proliferating C2C12 myoblasts but not to nuclei of differentiated postmitotic C2C12 myotubes. To explore the biological significance of this observation, we placed a cDNA encoding Gs alpha in an expression vector under the control of a steroid-inducible promoter and isolated colonies of stably transfected C2C12 myoblasts. Dexamethasone-induced expression of activated Gs alpha markedly delayed differentiation in comparison with uninduced stably transfected cells, which differentiated normally in mitogen-depleted media. Northern blot analysis showed that impaired differentiation was associated with delayed up-regulation of MyoD and myogenin and delayed down-regulation of Id, a dominant negative inhibitor of differentiation. Similar impairment of differentiation could not be reproduced in wild-type C2C12 cells by increasing intracellular cAMP either with forskolin or treatment with a cell-permeable cAMP analog. However, treatment of myoblasts with cholera toxin markedly inhibited myogenic differentiation. Taken together, these findings suggest a novel role for Gs alpha in modulating myogenic differentiation.

Animals↗

PubMed1980

Comparison of the glucosyltransferases of Streptococcus mutans gs-5 to a non-cariogenic mutant.

The extracellular glucosyltransferase (GTF) enzymes of Streptococcus mutans GS-5 and avirulent mutant GS-511 were fractionated using agarose and DEAE-cellulose columns. GTF of GS-5 produced both water-soluble and -insoluble glucans, while those of GS-511 made soluble products almost exclusively. Nearly all (98%) of the small amount of insoluble GTF enzyme made by GS-511 was bound to the cell wall.

Carbohydrates↗

PubMed1986

Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5.

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Bacillus subtilis↗

PubMed1992

Lowered responsiveness of the catalyst of adenylyl cyclase to stimulation by GS in heterologous desensitization: a role for adenosine 3',5'-monophosphate-dependent phosphorylation.

Treatment of chick hepatocytes with glucagon results in homologous and heterologous desensitization of the receptor-stimulated adenylyl cyclase. The loci of postreceptor heterologous desensitization was studied. The addition of excess purified GS to glucagon-desensitized hepatocyte membranes did not fully restore fluoride stimulation of adenylyl cyclase, even though the absolute activity was increased at least 2-fold. Treatment of chick hepatocytes with 8-bromo-cAMP resulted in a similar reduction of fluoride stimulation that could not be restored by the addition of purified GS. When membranes from control and glucagon-treated hepatocytes were treated with purified catalytic subunit of protein kinase-A (PKA), fluoride stimulation was lowered in control, but not glucagon-treated, membranes. Treatment of membranes from S49 kin- lymphoma cells with PKA also resulted in decreased fluoride- and forskolin-stimulated adenylyl cyclase activity, but activity stimulated by Mn2+ was not altered. Since previous studies from our laboratory had shown that GS and G(i) are not substrates for protein kinase-A, it appears that the catalyst of adenylyl cyclase is the likely locus of modulation. To determine if both chick hepatocytes and S49 cells contain similar types of adenylyl cyclase that could account for the similar PKA regulatory properties, we used polymerase chain reaction-based techniques to identify GS-stimulated adenylyl cyclases present in these systems. The chick liver contains both type 5 and type 6 adenylyl cyclases, while S49 cells contain the type 6 enzyme. Type 5 and 6 adenylyl cyclases are members of one widely expressed subfamily of mammalian GS-responsive adenylyl cyclases and share a predicted PKA phosphorylation site in the central cytoplasmic loop. This site is not found in other known adenylyl cyclases (types 1-4), although the olfactory-specific type 3 enzyme has a predicted site nearby. These data indicate that one component of hormone-induced desensitization of the adenylyl cyclase system can be at the level of the catalyst, where PKA-mediated phosphorylation could result in lowered responsiveness. The types 5 and 6 adenylyl cyclases are likely candidates for such regulation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Familial Albright's hereditary osteodystrophy with hypoparathyroidism: normal structural Gs alpha gene.

Albright's hereditary osteodystrophy (AHO) is a characteristic skeletal phenotype, including short stature, obesity, round face, and brachydactyly. AHO appears in patients with pseudohypoparathyroidism (PHP) who have resistance to PTH and in their eumetabolic family members who have pseudopseudohypoparathyroidism (PPHP). The differential diagnosis of AHO in families without PHP includes brachydactyly E, whose existence as a distinct entity has been questioned. We studied a patient with familial AHO who presented with hypocalcemia. To our surprise, PTH levels were low, and the response to PTH administration was normal. This is the first case of familial AHO with hypoparathyroidism. The proband's family included 22 affected subjects spanning 3 generations, who had variable degrees of AHO manifestations, with an autosomal dominant inheritance trait. The metacarpophalangeal pattern profile was typical of that of PHP-PPHP. As deficient activity and inactivating mutations of Gs alpha were described in PHP as well as in PPHP, we measured the biological activity of Gs in family members, which was normal. To exclude subtle abnormalities in the Gs alpha gene, we sequenced the entire coding region of Gs alpha in the propositus, which was normal. We conclude that hypocalcemia should be adequately evaluated even in the presence of familial AHO, and that familial AHO can occur with a normal coding structural Gs alpha gene. Identification of the molecular defect in familial AHO without PHP will shed light on the pathogenesis of AHO in general.

Adult↗

PubMed1997

Olfactory dysfunction in type I pseudohypoparathyroidism: dissociation from Gs alpha protein deficiency.

The discovery of variably decreased olfactory ability in Type Ia pseudohypoparathyroidism (PHP), a syndrome in which generalized hormone resistance is associated with deficiency of the alpha chain of the stimulatory guanine nucleotide-binding protein (Gs alpha) of adenylyl cyclase, has been used to support the hypothesis that Gs alpha plays a major role in human olfactory transduction. However, only a limited number of olfactory tests have been administered to such patients, and these patients have other problems that might cause or contribute to their olfactory dysfunction, including an unusual constellation of skeletal and developmental deficits termed Albright hereditary osteodystrophy (AHO). In this study, we administered tests of odor detection, identification, and memory to (i) 13 patients with Type Ia PHP; (ii) 8 patients with Type Ib PHP; (iii) 7 patients with pseudopseudohypoparathyroidism (PPHP); and (iv) 3 sets of normal controls matched to these groups on the basis of age, gender, and smoking history. Although we confirm that PHP Type Ia patients evidence olfactory dysfunction, we also demonstrate that (i) patients with Type Ib PHP, who have no AHO, no generalized hormone resistance, and normal Gs alpha activity, also evidence olfactory dysfunction relative to matched controls; and (ii) patients with PPHP, who have AHO, no generalized hormone resistance, and deficient Gs alpha protein activity, have relatively normal olfactory function. These observations do not support the hypothesis that the olfactory dysfunction associated with PHP is the result of generalized Gs alpha protein deficiency and imply that other mechanisms (e.g. ones associated with PTH or PTHrP resistance) are responsible for the olfactory deficits of this disorder.

Adult↗