Biomedical subjects

V E Allgood

Publications and source records attributed to V E Allgood.

11 recordsLinked to original sources

PubMed2001

Ocular safety of INS365 ophthalmic solution: a P2Y(2) agonist in healthy subjects.

The purpose of this study was to evaluate the ocular safety and tolerability of the P2Y(2) receptor agonist, INS365, when applied as eye drops in normal human subjects. This study was a double-masked, placebo-controlled, randomized, within subject paired-comparison, dose-escalation study in five cohorts of ten healthy subjects. The concentrations of INS365 ophthalmic solution were 0.5, 1.0, 2.0, and 5.0% given three times over six hours. Safety was assessed by general and ophthalmic examination and symptomatology. Unanesthetized Schirmer tests were performed in the last cohort of 10 subjects to evaluate the acute effects of INS365 on tear secretion. There were no significant differences in the number of subjects with ocular events reported in placebo-treated eyes compared to INS365-treated eyes. Two adverse events were possibly related to INS365: painless blepharospasm and an increase in lacrimation after 5.0% INS365 instillation. Unanesthetized Schirmer testing showed no acute effects of INS365 on tear secretion, compared to its vehicle, in healthy subjects, in which reflex tearing often produced maximal Schirmer values. INS365 ophthalmic solution was well-tolerated when administered by ocular instillation. Stimulation of ocular surface P2Y(2) receptors was not associated with ocular tolerability issues in healthy subjects.

Administration, Topical↗

PubMed1997

Differential phosphorylation of chicken progesterone receptor in hormone-dependent and ligand-independent activation.

Many steroid receptors, including chicken progesterone receptor, have been shown to be activated in the absence of their cognate ligands by modulators of kinases and phosphatases. To investigate the molecular mechanism of ligand-independent activation, chicken progesterone receptor mutants in which either one or all four of the previously identified phosphorylation sites have been changed to nonphosphorylatable alanine were analyzed for their ability to be activated by progesterone, 8-bromoadenosine 3':5'-cyclic monophosphate, or a dopamine agonist, SKF82958. Our current study shows that the receptor is differently phosphorylated in ligand-dependent and ligand-independent activation. The transcriptional activity of the receptor in response to 8-bromoadenosine 3':5'-cyclic monophosphate is affected by mutation of either Ser211 or Ser260. In addition, our data demonstrated that none of the four sites is absolutely required for the activation of the receptor by either 8-bromoadenosine 3':5'-cyclic monophosphate or the dopamine agonist. Treatment with 8-bromoadenosine 3':5'-cyclic monophosphate did not increase the overall level of receptor phosphorylation or cause phosphorylation of the receptor at alternate sites. These data raise the possibility that ligand-independent activation of the chicken progesterone receptor may be mediated through changes in the phosphorylation of coregulators or other protein factors interacting with the receptors.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Analysis of chicken progesterone receptor function and phosphorylation using an adenovirus-mediated procedure for high-efficiency DNA transfer.

The expression of heterologous DNA in mammalian cells is crucial to understanding physiological functions or determining biological properties of encoded proteins. However, expression for biological assay or at levels sufficient for recovery and subsequent physical analysis has been limited by the poor efficiency, variability, or cost of current DNA transfer methodologies. We have modified a DNA transfer procedure which exploits the capacity of replication-deficient adenovirus to infect a wide range of cell types, carrying with it transiently associated DNA. We have established conditions for achieving 80% transfection of CV1 cells and have used this procedure for DNA transfer into several mammalian cell lines and primary cell cultures. We have shown that biologically active avian progesterone receptor may be readily detected, both immunologically and functionally, using less than 1 ng of progesterone receptor-encoding plasmid DNA per 2 x 10(5) cells. We previously reported the identification of four phosphorylation sites in chicken progesterone receptor using oviduct tissue minces labeled with [32P]PO4 under nonequilibrium conditions. We now find, using adenovirus-mediated infection and equilibrium labeling conditions, that the same sites are phosphorylated in receptor expressed in CV1 cells and report that there are no additional major phosphorylation sites in chicken progesterone receptor. The ease, efficiency, sensitivity, and wide applicability of this DNA transfer method should simplify current efforts to study heterologous protein expression in mammalian cells.

Adenoviridae↗

PubMed1997

Chimeric receptors as gene switches.

There is a recognized need for eukaryotic molecular gene switches that are tightly regulated by the administration of small molecule drugs. The modular nature of intracellular receptor proteins has allowed the recent development of chimeric receptors that fulfill this need. These switches will help dissect the roles that specific proteins play in signaling pathways, cell differentiation and development. Some of these switches will also be used in gene therapy to regulate therapeutic gene expression.

Animals↗

PubMed1994

Modulation of steroid receptor-mediated gene expression by vitamin B6.

Gene transcription mediated by steroid hormones has become one of the most extensively characterized model systems for studying the regulation of gene expression in eukaryotic cells. However, specific details of gene regulation by steroid hormones are often complex and may be unique in specific cell types. Diverse regulatory mechanisms leading to either activation or repression of particular genes frequently involve interactions between steroid hormone receptors and other ubiquitous and/or cell-specific transcription factors that act on the complex promoter of the regulated gene. Interplay between steroid receptor-mediated and other signal transduction pathways may also be involved. In addition, recent novel results indicate that moderate variations in the intracellular concentration of pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, can have pronounced modulatory effects on steroid-induced gene expression. Specifically, elevation of intracellular PLP levels leads to decreased transcriptional responses to glucocorticoid, progesterone, androgen, or estrogen hormones. Conversely, cells in a vitamin B6-deficient state exhibit enhanced responsiveness to steroid hormones. One aspect of the mechanism by which these transcriptional modulatory effects of PLP occur has recently been shown to involve interruption of functional interactions between steroid hormone receptors and the nuclear transcription factor NF1. These findings--that the vitamin B6 nutritional status of cells modulates their capacity to respond to steroid hormones--impose an additional level of cell-specific control over steroid hormone regulation of gene expression and will serve as the focal point for this review.

Animals↗

PubMed1994

Regulation of the human glucocorticoid receptor by long-term and chronic treatment with glucocorticoid.

HeLa S3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.

Blotting, Northern↗

PubMed1994

Multiple signaling pathways activate the chicken progesterone receptor.

We have previously reported that treatment of CV1 cells, transiently transfected with DNA encoding the chicken progesterone receptor (cPR) and a reporter plasmid PREtkCAT, with either 8-Br-cAMP or okadaic acid resulted in ligand-independent transcriptional activation of the cPR. The surprising finding that cPR can be activated in the absence of hormone has been followed by numerous studies from other laboratories examining the effects of modulators of kinases and phosphatases on the activity of other steroid receptors. These studies have yielded mixed results: ligand-independent activation has been observed in some cases, but not in others. In order to determine whether the ligand-independent activation of cPR was restricted to a specific reporter and cell type and to better characterize this phenomenon, the studies in this report were undertaken. Using both the original reporter, PREtkCAT, and a simpler reporter, GRE2E1bCAT, we found that ligand-independent activation of the cPR can be induced in both CV1 and HeLa cells. The magnitude of the response and the response of the reporter alone differed in the two cell types. Further analysis of the activation of cPR by inhibitors of protein phosphatases showed that inhibition of phosphatase 1 rather than phosphatase 2A was necessary for activation of cPR. Finally, treatment with vanadate, an inhibitor of phosphotyrosine phosphatases, or epidermal growth factor resulted in activation of cPR. These studies suggest that signals transduced through multiple signaling pathways can activate cPR.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Modulation by vitamin B6 of glucocorticoid receptor-mediated gene expression requires transcription factors in addition to the glucocorticoid receptor.

We have investigated the mechanism by which vitamin B6 acts to modulate steroid hormone-mediated gene expression. We show that the level of glucocorticoid-induced gene expression from simple promoters, containing only hormone response elements and a TATA sequence, was not affected by alterations in intracellular vitamin B6 concentration. However, modulation of hormone-induced gene expression was restored with the inclusion of a binding site for the transcription factor nuclear factor 1 (NF1) within the hormone-responsive promoter; glucocorticoid-induced gene expression was reduced by 44% under conditions of elevated intracellular vitamin B6 concentration and enhanced by 98% in mild vitamin deficiency. Under these conditions, neither glucocorticoid receptor sedimentation characteristics, receptor activation, nor DNA binding capacity was affected. Quantitatively analogous effects were detected with estrogen-induced gene expression when an NF1 binding site was removed from or introduced into an estrogen-responsive promoter. NF1-mediated constitutive transcription was not affected by alterations in vitamin concentration. The modulatory effect of vitamin did not require strict positioning of or spacing between the glucocorticoid response element and NF1 binding site. Moreover, a heterologous transcriptional activator, composed of the viral E1a transactivation domain and the GAL4 DNA binding domain, does not substitute for NF1 in restoring vitamin B6 modulation of hormone-induced gene expression. These results suggest that vitamin B6 modulates steroid hormone-mediated gene expression through its influence on a functional or cooperative interaction between steroid hormone receptors and the transcription factor NF1.

Base Sequence↗

PubMed1992

Vitamin B6 modulates transcriptional activation by multiple members of the steroid hormone receptor superfamily.

Recent studies have shown that vitamin B6 modulates transcriptional activation by the human glucocorticoid receptor in HeLa S3 cells. We have now examined the possibility that vitamin B6 might similarly influence transcriptional activation by the glucocorticoid receptor in other cell types, as well as gene expression mediated by other members of the steroid hormone receptor superfamily. We show that elevated vitamin B6 concentrations suppress by 40-65% the level of transcription mediated through the endogenous murine L cell glucocorticoid receptor, as well as the human receptor transfected into E8.2 and T47D cells. In contrast, glucocorticoid receptor-mediated transcription was enhanced 60-110% in mild vitamin deficiency. The level of hormone-independent constitutive gene expression was not affected by these same alterations in vitamin B6 concentration. These studies indicated that the transcriptional modulatory effects of the vitamin were neither restricted to specific cell types nor limited to the human form of the glucocorticoid receptor. We next determined if hormone-induced transcription by several other steroid receptors (androgen, progesterone, and estrogen receptors) was analogously affected by alterations in vitamin B6 concentration. Analysis of gene expression mediated through the mouse mammary tumor virus promoter revealed that transcriptional activation of both the androgen and progesterone receptors was reduced by 35-40% under conditions of elevated vitamin B6 and enhanced by 60-90% in deficiency, again under conditions where constitutive expression was unaffected. Using a different promoter, the estrogen-regulated vitellogenin promoter, we found that transcriptional activation of the estrogen receptor was similarly affected. Estrogen-induced gene expression was reduced by 30% under conditions of elevated intracellular vitamin B6 and enhanced by 85% in vitamin deficiency. Thus, vitamin B6 modulates transcriptional activation by multiple classes of steroid hormone receptors. The similarities in vitamin B6 effects on transcription mediated through different promoters, the mouse mammary tumor virus and vitellogenin promoters, suggest that this vitamin may modulate the expression of a diverse array of hormonally responsive genes. These observations together support the hypothesis that vitamin B6 represents a physiological modulator of steroid hormone action.

Animals↗

PubMed1990

Vitamin B6 influences glucocorticoid receptor-dependent gene expression.

We have examined the influence of intracellular vitamin B6 concentration on glucocorticoid receptor function in HeLa S3 cells transfected with a glucocorticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The intracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was elevated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibitor 4-deoxypyridoxine. Analysis of glucocorticoid responsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The modulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concentration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin B6 selectively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor concentration or ligand binding capacity.

Gene Expression↗

PubMed1990

The influence of vitamin B6 on the structure and function of the glucocorticoid receptor.

Pyridoxal phosphate influences several properties of steroid hormone receptors in vitro, but its role in vivo has not been clearly established. In an effort to address this issue, we have investigated the in vivo effects of vitamin B6 on the physical properties and biological function of the human glucocorticoid receptor. We demonstrate that vitamin B6 treatment of whole cells in culture produces an alteration in the isoelectric point of the receptor, as well as changes in the steroid and DNA binding capacities. Furthermore, glucocorticoid dependent transcriptional activation properties of the receptor are also altered by modulation of the vitamin B6 status. High concentrations of vitamin B6 suppress activation of transcription, while vitamin deficiency enhances responsiveness to steroid hormone. Together, these studies imply a physiological role for vitamin B6 in glucocorticoid hormone action.

Animals↗