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P D Reynolds

Publications and source records attributed to P D Reynolds.

17 recordsLinked to original sources
PubMed2000

Squirrel monkey immunophilin FKBP51 is a potent inhibitor of glucocorticoid receptor binding.

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Amino Acid Sequence↗
PubMed1999

Glucocorticoid resistance in the squirrel monkey is associated with overexpression of the immunophilin FKBP51.

Squirrel monkeys are neotropical primates that have high circulating cortisol to compensate for expression of glucocorticoid receptors (GRs) with reduced affinity. The low binding affinity of squirrel monkey GR does not result from substitutions in the receptor, because squirrel monkey GR expressed in vitro exhibits high affinity. Rather, squirrel monkeys express a soluble factor that, in mixing studies of cytosol from squirrel monkey lymphocytes (SML) and mouse L929 cells, reduced GR binding affinity by 11-fold. In an effort to identify this factor, the cellular levels of components of the GR heterocomplex in SML and human lymphocytes (HL) were compared. The immunophilin FKBP51 was 13-fold higher in SML than in HL cytosol; FKBP52 in SML was 42% of that in HL cytosol. A role for changes in immunophilins, causing glucocorticoid resistance in neotropical primates, is supported by the following: the changes in FKBP51 and FKBP52 were observed in cells from other neotropical primates with glucocorticoid resistance; the elevated level of FKBP51 was reflected in an abundance of FKBP51 in heat shock protein 90 complexes in SML; when cytosols of SML and L929 cells were mixed, the decrease in GR binding was associated with incorporation of FKBP51 into GR heterocomplexes; the effect of SML cytosol on GR binding was reproduced with cytosol from COS cells expressing squirrel monkey FKBP51; and both the effect of SML cytosol on GR binding and the incorporation of FKBP51 into GR heterocomplexes were blocked by FK506. Regulation of GR binding by FKBP51 represents a previously unrecognized mechanism for regulating glucocorticoid sensitivity.

Animals↗
PubMed1998

S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells.

As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 +/- 0.16 versus 2.93 +/- 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total alpha-tubulin levels in untreated (4.0 +/- 0.6 versus 1.76 +/- 0.4 ng of alpha-tubulin/mg of total protein) and nerve growth factor-treated pAntisense clones (4.15 +/- 0.4 versus 2. 04 +/- 0.5 ng of alpha-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 +/- 8%) than control cells (116 +/- 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 +/- 3.0 versus 59.5 +/- 1.2 micron). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.

Animals↗
PubMed1998

Glucocorticoid-resistant B-lymphoblast cell line derived from the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

The goal of the study reported here was to develop a continuous cell line from the squirrel monkey that expresses the species-specific phenotype of impaired sensitivity to glucocorticoids. Thirty milliliters of blood from a male Bolivian squirrel monkey (Saimiri boliviensis boliviensis) was fractionated, and the buffy coat was obtained and incubated in the presence of B95-8 cell-conditioned medium, an abundant source of Epstein-Barr virus (EBV), and 2 micrograms of cyclosporin A/ml. Cell growth was detected within 8 weeks, after which the cells were cloned by use of the limiting dilution method. One clone (4D8) was characterized in detail. The chromosomal count and G-banding pattern confirmed that the cells were of Bolivian squirrel monkey origin. The B-cell origin of these cells was indicated by electron microscopic analysis and was confirmed by expression of CD20. The cells stained strongly for LMP1, a marker of latent EBV infection, and occasionally for the lytic infection marker ZEBRA (BZLF1). The responsiveness of clone 4D8 cells to glucocorticoids was determined by comparing the effects of dexamethasone on cell growth and the induction of a glucocorticoid-inducible mRNA in 4D8 cells with the effects on a human EBV-transformed B-lymphoblast cell line (HL). Dexamethasone inhibited the growth of HL cells, with IC50 of approximately 9 nM, but had no effect on the growth of 4D8 cells. The induction of FK506-binding protein FKBP51 mRNA by dexamethasone was also significantly blunted in 4D8 cells. Thus, we have developed and characterized a squirrel monkey lymphoblastic cell line derived by transformation of B-lymphocytes with EBV; the cell line has diminished growth and transcriptional responses to glucocorticoids.

Animals↗
PubMed1997

Cyclic GMP accumulation in pulmonary microvascular endothelial cells measured by intact cell prelabeling.

Cyclic GMP accumulation in cultured rat pulmonary microvascular endothelial cells (RPMVEC) was studied with a new prelabeling method developed using intact platelets and smooth muscle cells (1). [3H]-hypoxanthine was used to radiolabel the cellular guanine nucleotide pool. Neutral alumina and Dowex-50 double column chromatography was used to purify and quantitate the levels of [3H]-cyclic GMP. Changes in cyclic GMP metabolism in short and long term RPMVEC cultures were studied using rat atrial naturetic factor 8-33 (ANF) and sodium nitroprusside (SNP) in the presence and absence of cyclic nucleotide (CN) phosphodiesterase (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a time-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tritium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF treatment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [3H]-cyclic GMP in the absence of CN PDE inhibitors (EC50 = 5.4 nM). However, incubation with SNP showed no changes in cellular cyclic GMP accumulation. Several relatively selective CN PDE inhibitors had no effect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF induced cGMP accumulation was verified by radioimmunoassay. These studies confirm the utility of the hypoxanthine prelabeling technique to monitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.

Animals↗
PubMed1997

Confirmation of nitric oxide synthesis in active ulcerative colitis by infra-red diode laser spectroscopy.

BACKGROUND: The synthesis of nitric oxide (NO) in the colonic mucosa of patients affected by ulcerative colitis (UC) has been previously investigated using indirect measurements of enzyme activity and non-specific measurements of luminal NO gas. The aim of this study was to determine unequivocally if luminal NO gas in active UC is present in greater concentrations than in healthy control subjects. METHODS: Luminal gas was collected from eight patients with active UC and eight healthy volunteers. NO gas concentration was measured using diode laser spectroscopy. RESULTS: NO gas was detected in four of eight patients with active UC (range 0.13-1.1 nmol) and none of the healthy control subjects. CONCLUSION: NO gas is present in greater concentrations in patients with active UC than in control subjects. This supports the view that mucosal NO synthesis is increased in ulcerative colitis.

Colitis, Ulcerative↗
PubMed1997

Cloning and expression of the glucocorticoid receptor from the squirrel monkey (Saimiri boliviensis boliviensis), a glucocorticoid-resistant primate.

New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent Kd, 20.9 +/- 1.8 and 4.3 +/- 0.2 nmol/L, respectively; n = 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528-777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421-486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the human sequence were found in the N-terminal region (amino acids 1-421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 +/- 1.2 and 4.3 +/- 0.5 nmol/L, respectively; n = 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 +/- 2.0 nmol/L; n = 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function.

Amino Acid Sequence↗
PubMed1996

Xanthine oxidase activity is not increased in the colonic mucosa of ulcerative colitis.

AIM: To measure urate and xanthine by high-performance liquid chromatography in mucosal biopsies from patients with ulcerative colitis and controls. RESULTS: Reactive oxygen metabolite activity was demonstrated by a cystine to cysteine ratio of 0.24 +/- 0.08 in the control biopsies compared with 1.79 +/- 0.25 in ulcerative colitis. The product to substrate ratio (urate to xanthine) for xanthine oxidase and mucosal urate concentrations were not different between ulcerative colitis patients and controls. Xanthine oxidase activity after incubation of homogenized biopsies was 0.451 (+/- 0.175) nmol.min/mg protein in ulcerative colitis patients compared with 2.585 (+/- 0.823) nmol.min/mg protein in controls (P < 0.01). CONCLUSION: These results suggest that xanthine oxidase may not be the major source of superoxide formation in ulcerative colitis.

Biopsy↗
PubMed1995

The effects of aminosalicylic acid derivatives on nitric oxide in a cell-free system.

AIMS AND METHODS: To determine the effect of aminosalicylic acid derivatives on the concentration of nitric oxide produced in a cell-free system, by the use of a sensitive and specific polarographic meter. RESULTS: The aminosalicylic acid derivatives 3-ASA (IC50 100 microM), 4-ASA (IC50 350 microM) and 5-ASA (IC50 5 microM) all decreased the nitric oxide signal. These drugs had a similar inhibitory effect on the formation in vitro of nitrite from sodium nitroprusside (IC50: 200 microM, 500 microM and 100 microM, respectively). Sulphasalazine (31.1 +/- 5% decrease in signal at 1 mM) was less effective than 5-ASA, but sulphapyridine, N-acetyl 5-ASA, indomethacin and hydrocortisone produced no decrease in nitric oxide signal at all. CONCLUSIONS: Nitric oxide binding may be part of the mechanism by which ASA derivatives exert their therapeutic effect, and this work suggests that it may be an important factor in the pathogenesis of ulcerative colitis.

Aminosalicylic Acids↗
PubMed1995

Validation of 99Tcm-HMPAO leucocyte scintigraphy in ulcerative colitis by comparison with histology.

Leucocyte scintigraphy offers an alternative to more invasive techniques in the investigation of inflammatory bowel disease. The accuracy of 99Tcm-HMPAO leucocyte scintigraphy has not been assessed by comparison with colonic histology, which was the aim of this study. 15 patients with ulcerative colitis underwent 99Tcm-HMPAO leucocyte scintigraphy (TLS) less than 5 days before colonoscopy. Histological features of mucosal biopsies were compared with total and segmental colonic TLS scores. Segmental and total scintigraphy scores correlated most strongly with histological grades for acute inflammation (r = 0.75, p < 0.001 and r = 0.9, p < 0.001, respectively) and chronic inflammatory cell infiltration in the lamina propria (r = 0.76, p < 0.001 and r = 0.86, p < 0.001, respectively). 99Tcm-HMPAO leucocyte scintigraphy detected acute inflammation in the colon of patients with ulcerative colitis with a sensitivity of 91% and negative predictive value of 80% and localized acute inflammation to a particular colonic segment with a sensitivity of 82%, specificity of 94%, accuracy of 88%, positive predictive value of 94% and negative predictive value of 91%. 99Tcm-HMPAO leucocyte scintigraphy positivity predicts and localizes colonic acute inflammation with a high degree of confidence, but negative scintigraphy does not exclude acute inflammation.

Adult↗
PubMed1985

Effects of glucose and D-3-hydroxybutyrate on human pancreatic islet cell function.

Beta-Cell function in human islets derived from a number of kidney donors was investigated by using various types of islet preparations. With fresh islets, both insulin release and biosynthesis were increased by raising glucose concentrations, although the response was a variable one. In fresh islets, the effects of 5 mmol of glucose/l on release were potentiated by 10 mmol of D-3-hydroxybutyrate/l. Insulin release at 20 mmol of glucose/l was inhibited by adrenaline (0.1 mmol/l), and potentiated by theophylline (10 mmol/l) in the presence of 5 mmol of glucose/l, in islets cultured for 4 days. After culture for 8 days, islets still showed an increase in insulin release and biosynthesis in response to glucose. Pancreas slices derived from fresh human tissue also responded to increasing concentrations of glucose with a sigmoidal curve for insulin release.

3-Hydroxybutyric Acid↗
PubMedDate not supplied

Pharmacotherapy of inflammatory bowel disease.

The standard treatments for inflammatory bowel disease have been aminosalicylates and corticosteroids, administered both systemically and topically. They are frequently extremely effective, especially at higher doses. Unfortunately steroid side effects are too frequent and agents with low systemic bioavailability (budesonide, beclamethasone and tixocortol) are being investigated. Azathioprine, although a useful adjunct to steroids, has occasional and unpredictable severe side effects. Cyclosporin is an important new therapy in severe refractory disease. Several new phospholipid mediator inhibitors, mepacrine, zileuton, and ridogrel, may be useful in moderate colitis. Other topical treatments, butyrate, acetarsol and bismuth subsalicylate, can be beneficial in refractory distal disease. Quadruple antimycobacterials, antioxidants and antimicrobials warrant further study, while newer immunosuppressives such as methotrexate, FK 506 and monoclonal antibodies against helper T lymphocytes show some early promise.

Colitis, Ulcerative↗
PubMedDate not supplied

An open-label phase I/II dose escalation study of the treatment of pancreatic cancer using lithium gammalinolenate.

There are currently no satisfactory treatments for inoperable pancreatic cancer. Median survivals for untreated patients are of the order of 100 days and, with one exception, no chemotherapy or radiotherapy regime has been found to produce a worthwhile extension of life with reasonably tolerable side effects. Gamma-linolenic acid (GLA) has been found to kill about 40 different human cancer cell lines in vitro without harming normal cells. The lithium salt of GLA (LiGLA) can be administered intravenously and a dose escalation study of a 10 day infusion followed by oral therapy in patients with inoperable pancreatic cancer was carried out in 48 patients in two centres. Peripheral venous infusion caused thrombophlebitis but this could be avoided by infusing via a central vein with appropriate heparinisation. Too rapid infusion caused haemolysis which could be avoided by slow dose escalation in the first few days and maintenance of plasma lithium below 0.8 mmol/l. Doses ranged from 7 to 77g/patient cumulatively delivered over 2-12 days. Other than the above described events there were no important side effects and patients felt well during the infusions. A Kaplan-Meier analysis showed that survival was not significantly influenced by which centre the patients were treated in, the sex of the patients or the presence or absence of histological confirmation. The presence or absence of liver metastases, the patients' Karnofsky scores and the-dose of LiGLA had significant effects on survival from treatment. A Cox proportional hazards model revealed similar results: in both centres, in both sexes, and in patients with and without liver metastases according to the model the highest doses of LiGLA were associated with longer survival times as compared with the lowest doses. LiGLA deserves investigation in a randomised prospective study.

Aged↗