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R A Allen

Publications and source records attributed to R A Allen.

At least 19 recordsLinked to original sources
PubMed2004

4-thiophenoxy-N-(3,4,5-trialkoxyphenyl)pyrimidine-2-amines as potent and selective inhibitors of the T-cell tyrosine kinase p56lck.

We have developed a series of 4-thiophenoxy-N-(3,4,5-trialkoxyphenyl) pyrimidine-2-amines as potent and selective inhibitors of p56lck tyrosine kinase activity. In particular, the most potent inhibitor shows cellular activity in T-cell receptor (TCR) stimulated models of cytokine release, which suggests an immunomodulatory role for this class of inhibitor.

Adjuvants, Immunologic↗
PubMed2002

CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors.

The discovery, synthesis and biological activity of a series of triarylethane phosphodiesterase 4 inhibitors is described. Structure-activity relationship studies are presented for CDP840 (29), a potent, chiral, selective inhibitor of PDE 4 (IC(50) 4nM). CDP840 is non-emetic in the ferret at 30mgkg(-1) (po), active in models of inflammation and reverses ozone-induced bronchial hyperreactivity in the guinea pig.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Polymorphisms in the TNF-alpha and TNF-receptor genes in patients with coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is both multifactorial and polygenic in nature. Atheroma formation, the pathological hallmark of CAD, is an inflammatory process, with pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha), having a major role in its pathogenesis. We have therefore investigated whether polymorphisms in the TNF-alpha (- 238 and - 308), TNF receptor 1 (position - 609 and + 10, intron 6) and TNF receptor 2 (position + 422, codon 198) genes show an association with CAD. MATERIALS AND METHODS: Patients with angiographically proven single vessel (n = 58) and multivessel (n = 122) CAD were compared to patients with angiographically proven normal coronary arteries (n = 79) and volunteers without clinical evidence of CAD (n = 250). Genotyping was performed by PCR-RFLP analysis. For the TNF-alpha polymorphisms, a meta-analysis of all published studies was also undertaken. RESULTS: No significant differences in allele or genotype frequencies were found between the normal coronary artery group or healthy volunteers and patients with CAD for any of the polymorphisms. There was also no difference in allele frequency between patients with single- and multivessel disease. For the - 308 and - 238 TNRalpha gene polymorphisms, a meta-analysis of our data and previously published studies failed to demonstrate any significant association with CAD. CONCLUSIONS: Polymorphisms in the TNF-alpha promoter region and TNF-receptor genes are not associated with the development of CAD.

Adult↗
PubMed1999

Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors.

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Characterization of CHO-K1 cells stably expressing PDE-IV enzymes. Whole-cell cAMP determinations vs broken-cell enzymatic assays.

A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Primary adenocarcinoma, signet-ring, and transitional cell carcinoma of the bladder with penile metastasis.

This article reports a rare case of primary signet-ring cell carcinoma of the bladder with transitional cell and adenocarcinoma variants and metachronous metastases to the penis and lungs. This combination of lesions has not previously been reported. Together, they portend widespread dissemination and an early demise as is frequently the case with signet-ring cell carcinomas arising in other organs such as the breast and gastrointestinal tract. The optimal therapeutic intervention for this myriad of neoplasms with metastasis to the penis has yet to be ascertained because of the rarity of the lesions. Early diagnosis and an aggressive surgical approach appear to offer the best chance for quality survival and possible cure.

Adenocarcinoma↗
PubMed1995

Differential coupling of the formyl peptide receptor to adenylate cyclase and phospholipase C by the pertussis toxin-insensitive Gz protein.

In neutrophils, activation of receptors for the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) leads to changes in intracellular events such as phosphoinositide turnover and Ca2+ mobilization. Studies have shown that activation of the cloned fMLP receptor can also lead to inhibition of cyclic AMP (cAMP) accumulation [Lang, Boulay, Li and Wollheim (1993) EMBO J. 12, 2671-2679; Uhing, Gettys, Tomhave, Snyderman and Didsbury (1992) Biochem. Biophys. Res. Commun. 183, 1033-1039]. These responses are apparently mediated through pertussis toxin-sensitive Gi proteins. Since other chemotactic factor receptors can couple to multiple G proteins, we examined the ability of the fMLP receptor to utilize a pertussis toxin-insensitive G protein, Gz, in its signal transduction pathways. The human fMLP receptor was transiently expressed in 293 and Ltk- cells, and subsequently assayed for receptor-mediated inhibition of cAMP accumulation and stimulation of phosphoinositide-specific phospholipase C. In transfected 293 cells, fMLP inhibited choriogonadotropin-stimulated cAMP accumulation by 50% and the response could be abolished by pertussis toxin. Co-expression of the fMLP receptor with the alpha subunit of Gz rendered the fMLP response pertussis toxin-insensitive, indicating that the endogenous Gi proteins can be substituted efficiently by Gz. In contrast, Ltk- cells expressing the fMLP receptor were able to respond to fMLP with an increase in the production of inositol phosphates, but this response was completely abolished by pertussis toxin even in cells co-expressing the alpha subunit of Gz. Thus, although both signalling pathways appeared to utilize Gi-like proteins, Gz can only replace Gi in mediating inhibition of cAMP accumulation, and not in the stimulation of phospholipase C. Differential interaction with Gz might represent a novel mechanism by which fMLP receptors regulate intracellular events.

Adenylate Cyclase Toxin↗
PubMed1995

The human chemoattractant complement C5a receptor inhibits cyclic AMP accumulation through Gi and Gz proteins.

The human C5a receptor is known to signal through Gi proteins. The ability of the cloned C5a receptor to inhibit adenylyl cyclase or to stimulate phospholipase C through Gi proteins was examined in transfected cells. Activation of recombinant C5a receptors resulted in the stimulation of phospholipase C in Ltk- cells and inhibition of adenylyl cyclase in 293 cells. Pertussis toxin potently abolished both responses indicating the involvement of Gi proteins. Previous studies have shown that Gi-mediated inhibition of adenylyl cyclase can be similarly regulated by the pertussis toxin-insensitive GZ. In 293 cells co-transfected with the alpha subunit of GZ, the C5a-mediated inhibition of cAMP accumulation became pertussis toxin-resistant, signifying functional coupling between the C5a receptor and GZ. However, GZ cannot substitute for Gi in the C5a-induced stimulation of phospholipase C or inhibition of adenylyl cyclase in Ltk- cells.

Adenylyl Cyclases↗
PubMed1995

Stimulation of type II adenylyl cyclase by chemoattractant formyl peptide and C5a receptors.

The capacity of N-formylmethionyl-leucyl-phenylalanine (fMLP) and C5a receptors to regulate type II adenylyl cyclase was examined in transient transfection studies. Coexpression of either one of the chemoattractant receptors with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the corresponding chemotactic factor to stimulate cAMP accumulation in a dose-dependent manner. The chemoattractant-induced stimulation of type II adenylyl cyclase was absolutely dependent on the presence of GTP-bound alpha subunit of GS, as revealed by the coexpression of alpha s-Q227L, a constitutively activated mutant of alpha s. Stimulation of type II adenylyl cyclase by either fMLP or C5a was mediated via pertussis toxin-sensitive Gi-like proteins, because the response was abrogated by the toxin. The ability of Gz (a pertussis toxin-insensitive G protein that can couple to a number of Gi-linked receptors) to replace Gi in chemoattractant-induced stimulation of type II adenylyl cyclase was examined. The chemoattractant-induced response became insensitive to pertussis toxin upon coexpression of the alpha subunit of Gz. Interestingly, coexpression of alpha z significantly enhanced the chemotactic factor-stimulated type II adenylyl cyclase activities. When other G protein alpha subunits were tested under similar experimental conditions, all three forms of alpha 1 and alpha o1 were able to potentiate the fMLP response to various extents, whereas alpha q and alpha t slightly inhibited the fMLP response. The alpha subunit-mediated potentiation of the type II adenylyl cyclase response appears to reflect a productive coupling between alpha subunits and the fMLP receptor, because such enhancements were not seen with the constitutively activated alpha subunit mutants. Coexpression of the constitutively activated mutants of alpha z, alpha q, alpha 01, and alpha i1-3 neither enhanced nor inhibited the fMLP-stimulated cAMP accumulation. These results indicated that the observed enhancement of type II adenylyl cyclase responses was dependent on the ability of the wild-type alpha subunits to functionally interact with the fMLP receptor and that the fMLP receptor can couple to Gi1-3, Gz, and Go1 but not to Gs, Gq, or Gt.

Adenylyl Cyclases↗
PubMed1995

Pityriasis rosea.

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Adolescent↗
PubMed1993

Early treatment of Class III incisor relationship using the chincap appliance.

Twenty-three patients (10 male, 13 female), with Class III relationships of the incisor teeth at the mixed dentition stage, were treated with the chincap appliance associated, where appropriate, with an upper removable appliance to procline upper incisors and to free the occlusion. Cephalometric analyses at the beginning and at the end of active treatment were compared with those in a carefully matched untreated control group selected from the Belfast Growth Study. At the beginning of treatment patients differed significantly from the controls in the SNB was larger, ANB was negative, Wits' analysis was negative, and the intermaxillary angle significantly reduced. The overjet was reversed, the lower incisors were ahead of A-Po, and the upper lip was far behind the E line. Significant changes brought about by the treatment were improvement in the Wits' relationship (but there was no significant change in the ANB angle), increases in the intermaxillary angle and lower facial height, improvement in the overjet, proclination of upper incisors and retroclination of lower incisors, backward movement of the lower incisors in relation to A-Po, and backward movement of the lower lip. The efficacy of the chincap appliance can be attributed to retroclination of lower incisors and downward movement of the mandible which may improve the jaw relationship without affecting the ANB angle. There were marked changes in the lip posture.

Case-Control Studies↗
PubMed1991

Identification and partial characterization of a retinal pigment epithelial membrane receptor for plasma retinol-binding protein.

We have developed a membrane binding assay by which we have been able to characterize the interaction between 125I-labeled retinol-binding protein and its receptor in microsome fractions derived from retinal pigment epithelial cells. The binding of retinol-binding protein to the membranes was fast, with a dissociation constant in the range of 31-72 nM, and maximum binding occurred at neutral pH. Receptor binding sites were also found in microsome fractions of liver and kidney, whereas lung and muscle contained few, if any. Chemical cross-linking of retinol-binding protein to the microsomal membranes yielded a major molecular complex of Mr 86,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein responsible for binding of retinol-binding protein was identified as a Mr 63,000 protein using a label transfer cross-linking technique. Further characterization demonstrated that the receptor for retinol-binding protein is a terminally glycosylated membrane protein noncovalently associated with a high molecular weight complex.

Binding Sites↗
PubMed1991

Na+/Ca2+ exchange activity is increased in Alzheimer's disease brain tissues.

These studies were performed to determine the changes that occur in Na+/Ca2+ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 degrees C) in buffer containing 132 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2+ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaCl or 132 mM choline chloride and 45CaCl2 then terminated by addition of 200 microM LaCl3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2+ ionophore A23187 (10 microM) with 100 microM EGTA was added after 4 min, Ca2+ content was reduced to 10% of its original value. Ruthenium red (10 microM) had no effect on Ca2+ content. Na(+)-dependent Ca2+ uptake (Ca2+ content measured in choline chloride minus that measured in NaCl) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2+ content and in elevated values of peak plateau Ca2+ content.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism.

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.

Adenosine Triphosphate↗
PubMed1991

Calcium inhibition of cardiac adenylyl cyclase. Evidence for two distinct sites of inhibition.

Increasing the free calcium concentration from 10(-8) M to 10(-4) M inhibited cardiac sarcolemmal adenylyl cyclase activated by the addition of 5 X 10(-4) M forskolin or 1 X 10(-4) M GTP or Gpp(NH)p. The calcium inhibition curve in the presence of all three activators was shallow and best fit by a two site model of high affinity (less than 1.0 microM) and low affinity (greater than 0.1 mM). Gpp(NH)p appeared to decrease the sensitivity of adenylyl cyclase to inhibition by calcium at the high affinity site. Similar inhibition constants were obtained with each of the activators. Calmodulin content of native freeze-thaw vesicles was 76.2 +/- 14.2 ng/mg. Treatment of the vesicles with 1 mM EGTA to remove calmodulin significantly reduced calmodulin content to 19.7 +/- 1.35 ng/mg. This treatment had no significant effect on the calcium inhibition profile. Increasing free calcium to 3 X 10(-6) M was shown to have no effect on the EC50 estimated for either Gpp(NH)p or forskolin but did slightly increase the EC50 estimated for Mg2+ in the presence of maximal concentrations of either activator. Nevertheless, maximally stimulating concentrations of Mg2+ were unable to overcome calcium inhibition. Pretreatment of sarcolemmal membranes with pertussis toxin was shown to have no significant effect on calcium inhibition of adenylyl cyclase. The results suggest that the overall inhibitory action of calcium was most likely calmodulin independent and involved a direct interaction with the catalytic subunit at two distinct sites of high and low affinity. At the low affinity site calcium most likely competes with Mg2+ for an allosteric divalent cation binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1990

Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein.

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.

Acute-Phase Proteins↗