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N Sawyer

Publications and source records attributed to N Sawyer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

Aequorin↗
PubMed1995

Molecular cloning and characterization of the human prostanoid DP receptor.

A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3'-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristics of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP2 receptors, followed by the hEP4 receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [3H]prostaglandin D2 (PGD2) specific binding sites, was as predicted for the DP receptor, with PGD2 >> PGE2 > PGF2 alpha = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector in HEK 293(EBNA) cells. Activation of the hDP receptor with PGD2 resulted in an elevation of intracellular cAMP and in mobilization of Ca2+, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissue showed that the hDP receptor was a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.

Amino Acid Sequence↗
PubMed1995

Ultrastructural characteristics and morphogenesis of porcine reproductive and respiratory syndrome virus propagated in the highly permissive MARC-145 cell clone.

A Québec reference strain of PRRSV (IAF-KLOP) was successfully propagated in MARC-145 cells, a highly permissive cell clone to PRRSV derived from the MA-104 cell line. Purified extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter, with an isometric core about 25-30 nm. By indirect immunofluorescence, detection of viral antigens within the cytoplasm was possible as soon as 6 h.p.i. Nucleocapsids, budding at smooth endoplasmic reticulum (ER), and enveloped viral particles that tended to accumulate in the lumen of ER or Golgi vesicles, were the main features of the viral morphogenesis. The virus apparently was released by exocytosis.

Animals↗
PubMed1994

Microsomal glutathione S-transferase is the predominant leukotriene C4 binding site in cellular membranes.

Dimethyl sulfoxide-differentiated U937 (dU937) cells express high affinity G-protein-coupled receptors for leukotriene (LT)D4 and LTB4 and, as described here, specific binding sites for LTC4. The specific binding of [3H]LTC4 was of low affinity (KD = 26 nM) and high abundance (Bmax = 33 pmol/mg of protein), as compared to LTD4 and LTB4 receptors. In addition, although [3H]LTC4 specific binding was enhanced by divalent cations, it was not inhibited by nonhydrolyzable GTP analogs. [3H]LTC4 specific binding to dU937 cell membranes does not have, therefore, the characteristics of binding to a G-protein-coupled receptor. Competition for [3H]LTC4 specific binding to dU937 cell membranes by leukotrienes and related analogs, including N-methylated LTC4, as well as glutathione, suggested a dependence on the presence of an arachidonic acid backbone, although varying degrees of saturation were well tolerated, and that the glutathione moiety of LTC4 in particular was important in determining affinity. The possibility that [3H]LTC4 specific binding was to a member of the glutathione S-transferase (GST) family of enzymes, such as LTC4 synthase, cytosolic GST, or microsomal GST, was therefore investigated. [3H]LTC4 specific binding sites could be separated from LTC4 synthase and cytosolic GSTs by differential detergent solubilization, but cofractionated with microsomal GST during solubilization and subsequent anion exchange chromatography. In membranes that were depleted of LTC4 synthase and cytosolic GSTs, 125I-azido-LTC4 (a photoaffinity probe based on LTC4) specifically photolabeled in a cation-dependent manner a 17-kDa polypeptide that was comparable in mass to the microsomal GST polypeptide. Furthermore, [3H]LTC4 bound specifically to purified human microsomal GST with the same characteristics as to the endogenous dU937-cell membrane specific binding sites. The principal [3H]LTC4 specific binding site present in dU937 cells, therefore, is not a G-protein-coupled receptor, LTC4 synthase, or cytosolic GSTs, but is microsomal GST. Finally, the 1:3 stoichiometry of [3H]LTC4 specific binding to purified microsomal GST is consistent with the enzyme functioning as a homotrimer.

Affinity Labels↗
PubMed1994

Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype.

A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 >> iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

Amino Acid Sequence↗
PubMed1994

Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung.

Specific binding sites for [3H]leukotriene (LT)D4 and [3H]LTC4 have been identified in sheep lung parenchymal membranes. [3H] LTD4 specific binding was of high affinity (KD = 0.56 nM), saturable (Bmax = 43 fmol/mg of protein), stimulated by divalent cations and inhibited by nonhydrolyzable GTP analogs. LTs and LTD4-receptor antagonists competed for [3H]LTD4 specific binding with the rank order of potency predicted for the LTD4 receptor: LTD4 > ONO-1078 > ICI 204,219 > MK-571 > LTE4 > LTC4 > BAY u9773 >> LTB4. In contrast, [3H]LTC4 specific binding was of lower affinity (KD = 27 nM), abundant (Bmax = 87 pmol/mg of protein) and although stimulated by divalent cations was unaffected by GTP analogs. LTs and LTC4 analogs competed for [3H]LTC4 specific binding with the following rank order of potency: LTC2 > LTC3 > LTC4 > LTC5 >> N-methyl-LTC4 >> LTD4 approximately LTB4 approximately LTB4. [3H]LTD4 specific binding to sheep lung membranes has, therefore, the characteristics of being to a G-protein-coupled LTD4 receptor, whereas the profile of [3H]LTC4 specific binding strongly suggests that these sites are not LT-receptor related. Photolabeling of sheep lung membranes using [125I]azido-LTC4, a photoactivable LTC4 analog, resulted in the selective photolabeling of two polypeptides migrating at 30 kDa and 19 kDa. The selective photolabeling of the 19 kDa polypeptide could be modulated in an identical manner to [3H]LTC4 specific binding. This protein is, therefore, a candidate for being the principal [3H]LTC4 specific site in sheep lung membranes and has a comparable molecular mass to microsomal glutathione S-transferase, recently shown to be the predominant LTC4 binding protein in cellular membranes.

Animals↗
PubMed1992

Dialysate calcium reduction in CAPD patients treated with calcium carbonate and alfacalcidol.

The use of oral calcium carbonate as a phosphate binder is often complicated by hypercalcaemia, particularly with concomitant use of vitamin D analogues. We previously found that stepwise reduction of dialysate calcium effectively countered this complication in haemodialysis patients, and have now assessed the strategy in CAPD patients. Seventeen patients underwent conversion from aluminium hydroxide to calcium carbonate and were followed for 5 months, with subsequent addition of alfacalcidol for a further 5 months. Standard CAPD dialysate (1.75 mM calcium) was used, reducing to 1.45 mM and, if necessary, to 1.00 mM in patients who became hypercalcaemic. While receiving calcium carbonate alone, 12 of the 17 patients became hypercalcaemic, this responding in four to dialysate calcium reduction to 1.45 mM. In the remaining eight patients, further reduction to 1.00 mM was required and in two patients even this failed to control hypercalcaemia adequately, necessitating reversion to aluminium hydroxide. Phosphate control remained unchanged, as did calcium x phosphorus product. There were transient increases of blood ionised calcium, and decreases of parathyroid hormone, with progressive reduction of serum aluminium and alkaline phosphatase. The addition of alfacalcidol (0.25 microgram/day) led to hypercalcaemia in six subjects, successfully countered by dialysate calcium reduction in four. The results show that standard CAPD dialysate calcium at 1.75 mM is too high for the majority of calcium carbonate treated patients and that substantial reductions of the dialysate calcium concentration are required if calcium carbonate is to be used effectively.

Aluminum Hydroxide↗
PubMed1991

Expression and sorting of rat plasma kallikrein in POMC-producing AtT-20 cells.

A vaccinia virus (VV) vector was used to express rat plasma kallikrein (rPK) in the constitutively secreting cells, BSC-40, and in the endocrine regulated cells, AtT-20. Using a specific rPK antibody and a fluorogenic substrate, Phe-Phe-Arg-AMC, we demonstrated that in both cell lines VV infections resulted in the synthesis of an immunoreactive enzyme predominantly present as a zymogen which can be activated with trypsin. Stimulation of VV:rPK-infected AtT-20 cells with either 5mM 8-bromo-cAMP or 56 mM KCl resulted in a different pattern of rPK and ACTH secretion, strongly suggesting that rPK follows the constitutive secretory pathway. Finally, the 10% rPK activity found within AtT-20 cell extracts had no effect on pro-opiomelanocortin (POMC) processing either intracellularly or extracellularly. The above data show that the biosynthetic machinery of both cell lines analyzed does not allow the efficient activation of plasma prekallikrein. Finally, despite the PK's demonstrated ability to cleave various hormone precursors in vitro at pairs of basic residues, in vivo, we did not obtain evidence that this hepatic enzyme can also act as an intracellular pro-protein processing enzyme.

Adrenocorticotropic Hormone↗
PubMed1990

Fast (4-h) or slow (24-h) infusions of pamidronate disodium (aminohydroxypropylidene diphosphonate (APD)) as single shot treatment of hypercalcaemia.

Twenty-five patients with hypercalcaemia (ionised calcium, 1.57 +/- 0.04 mM; total calcium, 3.33 +/- 0.06 mM) due to malignancy were randomised to receive pamidronate (1 mg/kg body wt.) by infusion over 4 h (12 patients) or 24 h (13 patients). Forced saline diuresis was not utilised. Two patients died within 24 h and were not included in the analysis. Both groups combined (n = 23) showed a decrease in ionised calcium to 1.44 +/- 0.04 mM by 2 days (P less than 0.05 vs. day 0). By 4 days there had been a further fall to 1.27 +/- 0.05 mM (P less than 0.05 vs. day 2) and 7 days after treatment ionised calcium was 1.19 +/- 0.05 mM (P less than 0.05 vs. day 4). Total calcium declined from 3.33 +/- 0.06 to 2.65 +/- 0.11 mM over 7 days (P less than 0.001). Separate analyses of the two groups showed that the fast (4-h) infusion led to an earlier reduction in calcium than did the 24-h infusion (P less than 0.02), although after 7 days there was little difference between the two groups (4 h, 1.18 +/- 0.09 mM; 24 h, 1.23 +/- 0.06 mM, n.s.). Hypercalcaemic symptoms (analogue rating scale) improved between each of the time intervals 0, 4 and 7 days (P less than 0.05) and did not differ between the two groups. We recommend a single 4-h infusion of pamidronate (1 mg/kg) as primary treatment for malignancy-induced hypercalcaemia.

Calcium↗
PubMed1990

Mouse plasma kallikrein: cDNA structure, enzyme characterization, and comparison of protein and mRNA levels among species.

There is differential regulation of liver mRNA levels of rat (r) and mouse (m) plasma kallikrein (PK), as observed on Northern blots. Affinity purification of mPK and rPK, microsequencing, and radioimmunoassay in either rat or mouse showed that the difference in mRNA levels does not appreciably affect the circulating PK concentration. Nuclear run-off assays demonstrated that the regulation of the mRNA level of PK is post-transcriptionally controlled. Complete cDNA sequence determination of mPK was achieved using a combination of polymerase chain reaction and lambda gt11 library screening procedures. Within the coding region, the overall sequence homology between mPK and rPK is about 91-92% in amino acid and nucleotide sequence. Although the 3' noncoding segment of mPK is shorter than that of rPK, we calculate a 53% homology with a 5% higher A/T content for mPK. The largest difference is found at the 5' end of the mRNAs: whereas rPK is predicted from its gene structure to have a 167-nucleotide leader sequence, mPK is expected to have more than 605 nucleotides, of which the last 291 are very similar to those found in the rPK gene. The regulation of the mRNA stability and/or turnover rate of PK may possibly be affected by its 5' end in a species-dependent manner.

Amino Acid Sequence↗
PubMed1989

Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay.

Rat plasma kallikrein (rPK) was purified to homogeneity form plasma using affinity and high-performance liquid chromatography techniques, and subjected to NH2-terminal sequencing. The data showed that the sequenced segments of the regulatory (heavy) and catalytic (light) chains of the proteinase, respectively, display 73 and 91% sequence similarity with their counterpart in human plasma kallikrein. This sequence homology in conjunction with the determined molecular structure and inhibitor sensitivity support the identity of the isolated enzyme as plasma kallikrein. A polyclonal antiserum against rPK was obtained after immunization of rabbits with the purified enzyme, and a specific radioimmunoassay was developed. Since Tyr-iodinated rPK was not recognized by the antiserum, two alternative approaches were found to be successful. These included the use of a tracer consisting of rPK modified with either the affinity reagent 125I-labeled DTyr-Glu-Phe-Lys-Arg chloromethyl ketone or with the Bolton Hunter reagent. The usable range of the assay is between 15-150 fmol per tube. The antibody was shown to bind both monomeric and dimeric forms of rPK. Denaturation of the enzyme in sodium dodecyl sulfate does not abolish immune recognition only as long as the regulatory subunit is attached to the catalytic chain. Oxidation or reduction of rPK results in complete loss of immunoreactivity. This observation suggests that perhaps the disulfide linkage of the catalytic and regulatory polypeptides somehow helps to protect the antigenic epitope from denaturation. Alternatively, the epitope(s) recognized by the antibody spans a domain which includes both Tyr and Cys residues necessary for immune recognition.

Amino Acid Sequence↗
PubMed1989

One-shot high-dose pamidronate disodium (APD): effective, simple treatment for hypercalcaemia in haematological malignancy.

Fifteen patients with haematological malignancy and hypercalcaemia (mean +/- SEM calcium 3.44 +/- 0.14 mmol/l) received pamidronate (1 mg/kg) by infusion on 17 occasions (two patients were retreated 2 and 6 months after the first dose). After 4 days the plasma calcium had fallen to 2.84 +/- 0.14 mmol/l (P less than 0.001; n = 17), and in 10/17 episodes was less than 2.6 mmol/l. The mean fall was 0.61 mmol/l (95% confidence intervals 0.49-0.72 mmol/l). By 7 days a further decrease to 2.53 +/- 0.10 mmol/l had occurred, continuing to 2.38 +/- 0.11 mmol/l after 14 days. Plasma phosphate fell from 1.18 +/- 0.16 to 0.74 +/- 0.07 mmol/l at 7 days (P less than 0.001; n = 12). In 9 of the 17 episodes plasma creatinine initially exceeded 120 mumol/l (four of these greater than 300 mumol/l). This did not impair the response to pamidronate and there was no significant change in plasma creatinine following treatment. A single large dose of pamidronate was a simple, well-tolerated and very reliable treatment for hypercalcaemia complicating haematological malignancy, irrespective of renal function.

Adult↗
PubMed1989

High-dose calcium carbonate with stepwise reduction in dialysate calcium concentration: effective phosphate control and aluminium avoidance in haemodialysis patients.

To reduce potentially toxic aluminium exposure, the phosphate binding agent aluminium hydroxide was replaced by high-dose oral calcium carbonate in 15 haemodialysis patients. Stepwise reduction in dialysate calcium concentration (from 1.75 to 1.35 mmol/l and then to 1.05 mmol/l) was made when necessitated by hypercalcaemia. After 6 months, the mean daily dose of calcium carbonate was 62 mmol (range 25-150 mmol). This dose maintained good control of plasma phosphate (baseline, 1.34 +/- 0.32 mmol/l (mean +/- SD); 12 weeks, 1.30 +/- 0.22 mmol/l; 24 weeks, 1.51 +/- 0.31 mmol/l). Calcium x phosphate product did not rise significantly (baseline, 3.41; 12 weeks, 3.44; 24 weeks, 4.02). Apart from a transient early increase, ionised calcium did not change significantly (baseline, 1.23 +/- 0.10 mmol/l; 12 weeks, 1.24 +/- 0.10 mmol/l). Intact (1-84) parathyroid hormone concentration decreased from 241 pg/ml to 116 pg/ml (median values, P less than 0.05) after 12 weeks. This simple and well-tolerated regimen almost completely eliminated oral aluminium exposure, effectively controlled plasma phosphate and calcium concentrations, and reduced hyperparathyroidism.

Administration, Oral↗
PubMed1988

Prevalence, concentration, and prognostic importance of proteinuria in patients with malignancies.

Protein was found significantly more frequently in single urine samples from 504 patients with malignancy (290; 58%) than in 529 controls (119; 22%) (p less than 0.01). Median protein concentration was greater (p less than 0.001) in patients with neoplasia (0.14 g/l) than in controls (0.07 g/l). Actuarial analysis showed a median survival of 4.5 months in patients with proteinuria compared with 10 months in those without (p less than 0.001). The association between proteinuria and shorter survival was statistically significant for patients with gut tumours, lung tumours, and tumours at other sites analysed as a group. Patients with myeloma or urinary tract tumours were not studied. In many patients with malignancy the presence of proteinuria may be associated with a substantially reduced survival time.

Adult↗
PubMed1988

Progressive hypokalaemia in elderly patients taking three thiazide potassium-sparing diuretic combinations for thirty-six months.

Three different thiazide potassium-sparing diuretic combinations were given to elderly patients for heart failure. Eighty patients received their allocated combinations for 3 years and had 6-monthly measurements of plasma potassium. A further 84 were recruited for study but 29 died within 6 months and 55 had to be withdrawn from the trial. The triamterene-containing preparation was discontinued most frequently (6/44) because of hypokalaemia (plasma potassium less than 3.0 mmol/l); amiloride (5/44) and spironolactone (1/47). The median fall in plasma potassium over 3 years in those patients not withdrawn because of hypokalaemia was similar in each case (P greater than 0.05) and possibly failed to reach significance because of the withdrawal rate (9%). The trend was for a greater fall in those patients taking triamterene. The spironolactone-containing preparation may be the least unsatisfactory of the three.

Aged↗
PubMed1984

Clinical aspects of the acquired immune deficiency syndrome in the United Kingdom.

Between August and November 1983, seven new patients with AIDS were seen at this hospital; two with disseminated Kaposi's sarcoma, and five with opportunist infections, of whom three have died. We present the case histories of the five patients with opportunist infections, which show the wide clinical range of AIDS and suggest that the pattern of infection in the UK may differ from that reported in the USA.

Acquired Immunodeficiency Syndrome↗