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PubMed1991

In vivo utilization of N-(phosphonomethyl)-anilines and related substances by Pseudomonas spec. GS.

Utilization of various phosphonates as source for phosphorus by the glyphosate degrading strain Pseudomonas spec. GS was investigated. Metabolites of phosphonate degradation were characterized indicating the cleavage of the C-P-bound as primary step of breakdown. The phosphonate N-(phosphonomethyl)-4'-nitroazobenzene-4-amine (azophon) was characterized as a suitable substrate for detection of C-P-bond splitting activity in vivo. Pseudomonas cells permeabilized by toluene treatment were also capable of phosphonate degradation whereas no in vitro activity of a putative C-P-bond cleaving enzyme was detectable after cell disruption.

Aniline Compounds↗

PubMed2005

Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities.

The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.

Animals↗

PubMed1989

Hydrolysis of GTP by the alpha-chain of Gs and other GTP binding proteins.

The functions of G proteins--like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)--depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein alpha-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the alpha-chain of Gs (alpha s) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of alpha s in human pituitary tumors inhibit the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in alpha s that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of GTPase inhibiting mutations in alpha s occurs in the codon for an Arg residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by alpha s. This Arg residue is located in a domain of alpha s not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2000

Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5.

We have characterized the promoter regions of the gtfB and gtfC genes from Streptococcus mutans GS-5. Using a plasmid-based reporter system, we discovered that the gtfC promoter is an order of magnitude stronger than the gtfB promoter. In addition, we found that a variety of growth conditions failed to affect or discriminate between the expression of the two promoters. Only during the various phases of growth could we demonstrate significant changes in expression. We conclude that gtfB and gtfC promoters are coordinately expressed.

Base Sequence↗

PubMed1993

Imbalance of the Gs and Gi/o function in post-mortem human brain of depressed patients.

The amounts of various G protein subunits in postmortem brain samples from the parietal and temporal cortices were the same in controls and depressive patients as demonstrated by immunoblotting. However, photoaffinity GTP labeling (AAGTP) of Gi/o alpha, but not Gs alpha, was significantly increased in depressives in both cortex regions. Furthermore, the ratio of Gs/Gi/o AAGTP incorporation revealed a significant reduction in depressives in these regions. The present findings suggest that an imbalance of second messengers via G protein function may be involved in the pathophysiology of depression.

Affinity Labels↗

PubMed1996

Modulatory effects of Gs-coupled excitatory opioid receptor functions on opioid analgesia, tolerance, and dependence.

Electrophysiologic studies of opioid effects on nociceptive types of dorsal root ganglion (DRG) neurons in organotypic cultures have shown that morphine and most mu, delta, and kappa opioid agonists can elicit bimodal excitatory as well as inhibitory modulation of the action potential duration (APD) of these cells. Excitatory opioid effects have been shown to be mediated by opioid receptors that are coupled via Gs to cyclic AMP-dependent ionic conductances that prolong the APD, whereas inhibitory opioid effects are mediated by opioid receptors coupled via Gi/Go to ionic conductances that shorten the APD. Selective blockade of excitatory opioid receptor functions by low (ca. pM) concentrations of naloxone, naltrexone, etorphine and other specific agents markedly increases the inhibitory potency of morphine or other bimodally acting agonists and attenuates development of tolerance/dependence. These in vitro studies have been confirmed by tail-flick assays showing that acute co-treatment of mice with morphine plus ultra-low-dose naltrexone or etorphine remarkably enhances the antinociceptive potency of morphine whereas chronic co-treatment attenuates development of tolerance and naloxone-precipitated withdrawal-jumping symptoms.

Analgesics, Opioid↗

PubMed2004

Identification, molecular cloning, and sequence analysis of a deoxyribose aldolase in Streptococcus mutans GS-5.

Bacterial fitness in the environment, where nutrients are limited and competition is intense, plays a central role in survival and virulence of the organisms. Deoxyribose aldolase, found in several species of bacteria, is known to be involved in the catabolism of deoxynucleosides arising from dead cells, thereby giving a selective advantage to the microorganisms with a capability to consume DNA as an alternative carbon and energy source. A gene encoding a deoxyribose aldolase gene ( deoC) was identified in the cariogenic Streptococcus mutans strain GS-5 by comparative sequence analysis and gene cloning. The gene encodes a protein of 220 amino acids, having a predicted molecular weight of 23.3 kDa with a p I of 5.44. The gene was cloned into the expression vector pFLAG-1, and the biological function of the gene product was analyzed by a complementation assay with a deoC(-) Escherichia coli mutant SPhi063. Transformation of the E. coli SPhi063 with the plasmid construct allowed this organism to grow on glucose minimal medium supplemented with 2 mM deoxyadenosine or deoxythymidine. These results showed activity of deoxyribose aldolase, confirming the identity of the gene. Utilization of exogenous deoxynucleotides as a carbon and energy source may confer a survival and growth advantage to S. mutans over other bacteria in dental plaque, suggesting that deoxyribose aldolase may be a contributing factor to virulence.

Aldehyde-Lyases↗

PubMed1990

Increased mitogenic responsiveness of Swiss 3T3 cells expressing constitutively active Gs alpha.

Mutational replacement of glutamine-227 with a leucine residue in the GTP-binding domain of the alpha subunit of GS (Q227L alpha S) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L alpha S caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (cAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for cAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors.

Adenylyl Cyclases↗

PubMed1990

Cholera toxin-A subunit blocks opioid excitatory effects on sensory neuron action potentials indicating mediation by Gs-linked opioid receptors.

Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1996

Hippocampal and cortical G protein (Gs alpha, G(o) alpha and Gi2 alpha) mRNA expression after electroconvulsive shock or lithium carbonate treatment.

GTP-binding proteins (G proteins) are heteromers composed of alpha, beta and gamma subunits. The expression of some G protein subunits is altered both by affective disorders and by antidepressant treatments. Here we have studied three G protein alpha subunit mRNAs in the hippocampus and frontoparietal cortex of rats treated with lithium for 14 days or with repeated electroconvulsive shock (five shocks over 10 days). After electroconvulsive shock, the three mRNAs changed differentially in the hippocampus. Specifically, Gs alpha mRNA was decreased in CA3 and CA1, whilst G(o) alpha mRNA was increased in dentate gyrus and Gi2 alpha mRNA was reduced in dentate gyrus and CA3. Lithium carbonate treatment produced a modest, uniform increase in the three mRNAs in dentate gyrus and CA3, and a selective elevation of G(o) alpha mRNA in CA1. Neither treatment altered the G protein mRNAs in the cortex nor cyclophilin mRNA in any region. These data extend the evidence that altered G protein expression is a part of the biochemical response to antidepressant treatments. Differences in the molecular and anatomical pattern of the alterations induced by electroconvulsive shock compared to lithium may contribute to their different therapeutic profiles.

Animals↗

PubMed1989

Receptor-regulated formation of GTP[gamma S] with subsequent persistent Gs-protein activation in membranes of human platelets.

Preincubation of human platelet membranes with the ATP analog ATP[gamma S] led to persistent adenylate cyclase activation. This stimulation was increased by copreincubation with PGE1 and obliterated by removing endogenous GDP by the NTP-regenerating system, creatine phosphate plus creatine kinase. PGE1 partially reversed the action of the regenerating system. Control formation of GTP[gamma S] from ATP[gamma S] and GDP in platelet membranes was apparently not stimulated by PGE1. In contrast, in the presence of creatine phosphate plus creatine kinase, which prevented formation of GTP[gamma S], PGE1 stimulated formation of this GTP analog, by partially reversing the action of the NTP-regenerating system. The data indicate that GTP[gamma S] can be formed by a membrane-associated nucleoside diphosphokinase from ATP[gamma S] and GDP, resulting in persistent Gs-protein activation, and that this process can be stimulated by an agonist-activated receptor.

Adenosine Triphosphate↗

PubMed1992

A substantial proportion of cardiac Gs is not associated with the plasma membrane.

The precise interactions between the subunits of Gs (alpha s, beta, gamma) and the plasma membrane remain to be established. If alpha s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant alpha s (52-66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of alpha s (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac alpha s with cyc- S49 membranes revealed that the supernatant fraction contained approximately 16% of total cellular alpha s activity. The alpha s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying alpha s which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular alpha s, but that the majority of this population of cardiac alpha s may not be functional, at least with respect to adenylyl cyclase activation.

Adenosine Diphosphate Ribose↗

PubMed1994

GTP-dependent binding of Gi, G(o) and Gs to the gamma-subunit of the effector of Gt.

The gamma-subunit of the cGMP-phosphodiesterase (PDE gamma) of retinal rods forms a tight complex with the activated alpha-subunit of transducin (Gt alpha GTP gamma S). We observe that while PDE gamma is not the physiological effector of other G alpha subtypes, it can still detectably interact with them. This interaction is strong with Gi1 alpha and Gi3 alpha (Kd approximately 10 nM) and weaker with Go alpha and Gs alpha (Kd approximately 1 microM). For all these G alpha subtypes, similar intrinsic fluorescence changes are observed upon PDE gamma binding. Moreover, similar relative decreases in affinity are obtained when the GDP forms of Gi1 alpha, Gi3 alpha or Gt alpha are used in lieu of the GTP forms. This points to a conserved GTP-dependent effector-interaction domain.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1996

Exclusive expression of the Gs-linked prostaglandin E2 receptor subtype 4 mRNA in mononuclear Jurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937 cells.

Prostaglandin E2 (PGE2) is regarded as a potent regulator of the immune system. It can regulate apoptosis in mononuclear cells and modulate the cytokine secretion pattern from T-helper cell subpopulations via an increase in cyclic AMP (cAMP). Of the 4 PGE2 receptor subtypes (EP1-EP4) that are defined pharmacologically by their affinity to subtype-specific ligands and their coupling to G proteins, EP2 and EP4 receptors couple to Gs. It is as yet unknown which of these two receptor subtypes mediates the immunomodulatory effects. By quantitative RT-PCR, the mRNA for EP4 receptors was demonstrated and quantified in the human mononuclear cell lines Jurkat, KM-3 and U-937. However, EP2 receptor mRNA was only present in U-937 cells and was 100-fold less abundant than EP4 receptor mRNA. PGE2 increased cAMP formation with an ED50 of 50-100 nM in all cell lines. cAMP formation was inhibited by the EP4R-specific antagonist AH23848. Since AH23848 inhibited PGE2-induced cAMP formation in U-937 cells to a similar extent as in Jurkat and KM-3, EP2 receptors seem to play, if any, only a secondary role for the PGE2-mediated cAMP formation in U-937 cells.

Biphenyl Compounds↗

PubMed1992

Identification of effector-activating residues of Gs alpha.

The heterotrimeric G proteins transduce extracellular signals by interacting with specific intracellular effectors. We have used a scanning mutagenesis approach to identify amino acids of alpha S, the alpha subunit of Gs, that determine the specificity of its interaction with its effector, adenylyl cyclase. In alpha subunit chimeras, residues 236-356 of alpha S comprise the shortest linear stretch that is required for activation of adenylyl cyclase. Within these 121 residues, we identified four clusters of residues in which substitutions prevented effector activation. Mutations in three of these regions did not affect alpha subunit expression or the GTP-induced conformational change. The identified alpha S residues in the NH2-terminal half of the 121-residue region endowed the cognate alpha i2 segment with the ability to activate effector, while those in the COOH-terminal half did not. In a three-dimensional G alpha model, based on the structure of p21ras, the effector-activating residues of alpha S form a surface on the membrane-facing side of the molecule; this surface includes a region that changes conformation upon binding GTP.

Amino Acid Sequence↗

PubMed1996

Evaluation of Cidofovir (HPMPC, GS-504) against adenovirus type 5 infection in vitro and in a New Zealand rabbit ocular model.

The antiviral inhibitory activity of Cidofovir [1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate, HPMPC, GS-504] against adenovirus type 5 (Ad5) in the New Zealand rabbit ocular replication model was evaluated. The 50% inhibitory dose (ID50) of Cidofovir was determined to be 4.7-9.5 micrograms/ml against four adenoviruses (two Ad5, Ad8 and Ad14) by plaque reduction assay in A549 cells. Twenty-four New Zealand rabbits received intrastromal inoculation and topical application of 2 x 10(6) plaque-forming units (PFU) per eye of Ad5 McEwen, a clinical isolate. Cidofovir was administered topically at three different concentrations twice per day, beginning 16 h postinoculation and continuing for 20 consecutive days. The inhibitory effects were determined by measuring suppression of virus replication and by observation of the clinical effects. Compared to the placebo group, the 1% and 0.5% Cidofovir-treated groups showed significantly reduced Ad5 ocular titers, fewer days of viral shedding and less severe subepithelial opacities (P = 0.0001). The 1% Cidofovir group had the lowest humoral antibody titer against adenovirus antigens, but the difference was not significant (P = 0.24). Cidofovir proved to have potent antiviral activity against adenovirus replication and may have great promise for the treatment of adenovirus infection. Further investigation is recommended.

Adenoviridae Infections↗

PubMed1992

Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function.

Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Stimulation of glycogenolysis by three locust adipokinetic hormones involves Gs and cAMP.

Insect adipokinetic hormones (AKHs) have been shown to mobilize fat body carbohydrate by glycogen phosphorylase activation. In this study, the signal transduction pathways of AKH-I, -II and -III from the migratory locust are further elucidated. We show that the AKHs enhance fat body cAMP levels in vitro. For all hormones, maximal levels are reached after 1 min and correspond to a 200% increase compared to resting levels. Although cAMP levels induced by massive doses of AKH-I, -II and -III are equal, AKH-III is the most potent when applied in a physiological dose. This difference in potency also applies to glycogen phosphorylase activation. Cholera toxin (CTX) likewise ennhaces cAMP levels and phosphorylase activity, however pertussis toxin (PTX) has no effect. Increases induced by CTX and AKH are not additive, suggesting that they share the same pathway. Phosphorylase activation by the AKHs is strongly attenuated by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). These results demonstrate a role for cAMP in AKH signal transduction and indicate that the AKH receptor(s) are coupled to cAMP formation and glycogen phosphorylase activation via the stimulatory guanine nucleotide-binding protein (Gs).

Animals↗