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Biomedical subjects

G Bolger

Publications and source records attributed to G Bolger.

At least 19 recordsLinked to original sources
PubMed2000

Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical.

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Biophysical Phenomena↗
PubMed1999

Acute murine cytomegalovirus infection: a model for determining antiviral activity against CMV induced hepatitis.

Acute intraperitoneal infection of weanling BALB/c mice with murine cytomegalovirus (MCMV) resulted in an inoculum titer-dependent weight loss, mortality and elevation of plasma transaminases (ALT: alanine transaminase and AST: aspartate transaminase). Three days post infection (p.i.) with 10(4.85) plaque forming units (pfu) there was 90% mortality with a mean death day p.i. of 4.1 +/- 0.2. Plasma levels of ALT and AST were elevated 24- and 15-fold, respectively. Organ titers of virus (log10 pfu/g tissue) were 6.16 in the liver, 6.05 in the spleen, 4.0-4.7 in the lung, heart, kidney and intestine and undetectable in the muscle and brain. Organ concentrations (units/g wet-weight) of ALT were highest in the liver, whilst for AST the highest levels were found in the heart. The concentrations of ALT but not AST were reduced (35-55%) in the infected liver; the concentrations of ALT and AST were not changed in other infected organs. There were excellent correlations (r > 0.95) between viral titers in the liver, increases of plasma ALT and depletion of liver ALT. HPMPC and ganciclovir administered either p.o. or s.c. reduced mortality, increases in plasma transaminases and viral burdens in the liver and prevented depletion of liver ALT. HPMPC was approximately 10-fold more potent than ganciclovir. These results strongly suggest that intraperitoneal infection of the BALB/c mouse with MCMV represents an animal model of CMV hepatitis that can be monitored by measuring plasma ALT.

Alanine Transaminase↗
PubMed1999

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy.

A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.

Administration, Oral↗
PubMed1999

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors.

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.

Administration, Oral↗
PubMed1996

The human cyclic AMP-specific phosphodiesterase PDE-46 (HSPDE4A4B) expressed in transfected COS7 cells occurs as both particulate and cytosolic species that exhibit distinct kinetics of inhibition by the antidepressant rolipram.

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed approximately 96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a approximately 125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was approximately 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an approximately 11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0. 195 microM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 microM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 microM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 microM; Ki' = 2.3 microM). Particulate PDE-46 thus showed a approximately 60-fold higher affinity for rolipram than cytosolic PDE-46.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5).

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family.

COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for the distribution of the approximately 109 kDa immunoreactive species. It is suggested that the 109 kDa species identified in brain cytosol and pellet fractions is the native form of RPDE-6. The PDE4A splice variants, RD1 and RPDE-6, were shown to have distinct patterns of expression among various brain regions. PDE4A and PDE4B activities appear to provide the major source of PDE4 activity in brain membranes, whereas the cytosolic PDE4 activity is suggested to reflect predominantly the activity of the PDE4D family. Alternative splicing of the PDE4A gene confers distinct N-terminal domains on RPDE-6 and RD1, which attenuates the Vmax. of these enzymes and defines their distinct subcellular distribution pattern.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

A novel cyclic GMP stimulated phosphodiesterase from rat brain.

A cDNA clone for cyclic GMP Stimulated Phosphodiesterase (cGSPDE; PDE2) was isolated from a rat brain cDNA library. The cDNA has an open reading frame which encodes a protein of 928 amino acids of which 829 are identical with the reported bovine adrenal gland cGSPDE cDNA (Sonnenburg, W.K., Mullaney, P.J., and Beavo, J.A. (1991) J. Biol. Chem. 266, 17655-17661). Although the overall homology of these two cDNAs is high, they are distinctly different in their 5' ends, with the N-terminal 37 amino acids of the rat brain protein showing no homology with the N-terminal end of the bovine adrenal protein. Hydrophilicity plots show that in contrast to the bovine adrenal cGSPDE, the N-terminal end of the rat brain cGSPDE is highly hydrophobic. Isolation and analysis of a genomic clone for cGSPDE from a rat genomic library shows the presence of an exon/intron junction at the Gln39 codon. The cGSPDE cDNA we have isolated and that of Sonnenburg et al. represent alternatively spliced mRNA products from the same gene, with the brain isoform designed to be targeted to membranes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse.

Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, we have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosome 19 (DPDE1 and DPDE2), 5q12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Interactions between endothelin and atrial natriuretic factor in the rat aortic ring preparation.

The potential interaction (s) between atrial natriuretic factor (ANF) and porcine--human endothelin (ET-1) was investigated in the endothelium-denuded rat aortic ring preparation. ET-1 produced a sustained contraction of aortic rings with an ED50 of 3.6 +/- 0.49 x 10(-9) M. Within the concentration range of 10(-9) to 10(-7) M, both rat ANF 103-126 and rat ANF 99-126 when preincubated with tissues reduced the contractile efficacy of ET-1 especially at low concentrations resulting in a small but significant rightward shift of the dose--response curve to ET-1. In contrast, at a concentration of 10(-10) M, rANF 99-126 but not rANF 103-126 produced a significant leftward shift of the dose--response curve to ET-1 and an increase in the maximal developed tension for the dose--response curve to ET-1. For tissues incubated in the absence of extracellular calcium or in the presence of the calcium channel blocker nifedipine (5 x 10(-7) M), both ANF derivatives produced a dose-dependent decrease in the maximum contraction, but no change in potency to ET-1. Addition of either rANF 103-126 or rANF 99-126 to tissues maximally contracted with ET-1 resulted in relaxation, reaching a maximum of 70%. The ED50 values for relaxation were 2.7 +/- 0.51 x 10(-8) and 3.5 +/- 0.60 +/- 10(-8) M for rANF 103-126 and rANF 99-126, respectively. ET-1 did not interact with biologically responsive and clearance receptors for ANF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein.

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

Adenylyl Cyclases↗
PubMed1990

A method for difference cloning: gene amplification following subtractive hybridization.

We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100- to 700-fold enrichment of target sequences.

Bacteriophage lambda↗
PubMed1985

Binding of a new opiate antagonist, nalmefene, to rat brain membranes.

Nalmefene (6-methylene-naltrexone) is a potent, orally active, opiate antagonist. IC50's were obtained for nalmefene, naloxone and naltrexone using radiolabelled prototype ligands for mu, kappa and delta receptors in homogenates of rat brain minus cerebellum. Nalmefene antagonized the bindings of [3H]-dihydromorphine, [3H]-ethylketocyclazocine and [3H]-D-ala-D-leu enkephalin with IC50's in the low nanomolar range. At the central mu receptor, nalmefene bound with an IC50 of 1.0 nM, equal to that of naltrexone and approximately four times lower than that of naloxone. At central kappa and delta sites the IC50's for nalmefene were somewhat lower than those of naltrexone and considerably lower than those of naloxone. All three antagonists had sodium indices less than 1.0. These results indicate that nalmefene is a universal opiate antagonist, has no agonist character at the central mu site and binds more effectively to central opiate receptors than either naloxone or naltrexone.

Animals↗