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Biomedical subjects

M T Gilbert

Publications and source records attributed to M T Gilbert.

14 recordsLinked to original sources
PubMed1999

Glycosylation is important for binding to human calcitonin receptors.

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.

Alanine↗
PubMed1995

Cross-validation of bioanalytical methods between laboratories.

Increased reliance on pharmacokinetic studies in regulatory submissions emphasizes the need for cross-validating bioanalytical methods between different laboratories to allow comparison of data. Globalization of pharmaceutical development results in a greater need to define cross-validation standards. A strategy for performing cross-validation experiments using prepared biological samples of known concentration and "real" samples from clinical trials is presented. The statistical techniques used to compare data sets and establish acceptability of the assays are illustrated by practical examples.

Chemistry Techniques, Analytical↗
PubMed1994

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

Animals↗
PubMed1994

A Pit-1 binding site in the human renin gene promoter stimulates activity in pituitary, placental and juxtaglomerular cells.

One of the principal aims of our research is to determine the mechanisms which direct renin gene expression to different sites. We recently demonstrated that human renin (hRen) 5'-flanking DNA sequences -148 +/- 11 can drive the transient expression of a linked luciferase reporter gene transfected into pituitary GC cells. This activity was found to be dependent on the binding of Pit-1 to a site approximately 70 bp upstream from the transcription start site. Pit-1 is a pituitary-specific transcription factor which is involved in directing the cell-specific expression of growth hormone (GH) and prolactin (PRL) gene expression to somatotrope and lactotrope cells of the anterior pituitary. Thus, Pit-1 may be play a role in directing the expression of renin to primate lactotrope cells. Renin promoter-driven luciferase or CAT hybrid genes were found to be expressed following transfection into primary, or early passage cell cultures of placental chorionic membranes, and the renin-secreting renal tumor cell line As4.1. As with GC cells, deletion or mutagenesis of the Pit-1 site reduced activity several-fold in both placental and renal cells. These results suggest that members of the POU family of transcription factors, or some other closely related group such as the Hox proteins, participate in directing renin gene expression to placental and juxtaglomerular cells.

Binding Sites↗
PubMed1994

Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5'-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines.

Renin gene expression is limited to a number of specific tissues, including the kidney, adrenal glands, reproductive organs (of particular relevance to this study, the placenta), and the pituitary gland. In the present study, we investigated the human renin (hRen) 5'-flanking DNA sequences required to drive the expression of a luciferase reporter gene in placental and pituitary cells and in two cell lines, 293 and JEG-3, which have been proposed as model systems with which to study transcriptional regulation of renin genes. The activities of specific sequences in the hRen 5'-flanking DNA sequences in human placental cell primary cultures were very similar to those that we previously reported in pituitary cells, suggesting the involvement of common promoter elements and related transcription factors. Accordingly, the binding site for the pituitary-specific transcription factor (Pit-1) was the major determinant of renin promoter activity in both pituitary and placental cells. Gel mobility shift analysis showed a placental nuclear factor with a gel mobility different from that of Pit-1. However, Northern blot analysis failed to demonstrate abundant Pit-1-related mRNAs in renin-expressing cultures of chorionic and decidual cells, suggesting that the placental factor is not closely related to Pit-1. Although a factor from 293 cells also bound to the Pit-1 site, it had gel mobility shift characteristics different from Pit-1 and the placental factor. Moreover, the low promoter activity in 293 cells was independent of this site or, indeed, of sequences upstream from the TATA box. In JEG-3 cells, renin 5'-flanking DNA sequences showed virtually no transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1993

Promoter activity of human renin 5'-flanking DNA sequences is activated by the pituitary-specific transcription factor Pit-1.

Although the renal juxtaglomerular cell is the source of circulating renin, the renin gene is also expressed at a number of extrarenal sites including lactotrope cells of human and ovine pituitaries. In the present study, we demonstrate that GC cells, a pituitary lactotrope precursor cell line, efficiently express transiently transfected hybrid genes containing human renin 5'-flanking DNA sequences -148/+11. Gel mobility shift competition analyses show that a highly conserved sequence in human and rodent renin 5'-flanking DNAs (human coordinates: -80/-58) binds a nuclear factor from GC cells, most likely the pituitary-specific factor Pit-1. Deletional and mutational analyses demonstrate that this site is a major determinant of renin promoter activity in GC cells. Transfection of a Pit-1 expression construct into HeLa cells, where activity of the human renin promoter is low, stimulates expression of cotransfected renin-luciferase constructs. Moreover, activation of the human renin promoter by co-expression of Pit-1 is dependent on an intact Pit-1 site. Taken together, these data strongly suggest that Pit-1 activates pituitary renin gene expression. This finding raises the possibility that member(s) of the POU family of transcription factors, of which Pit-1 is an archetypal member, may direct renin expression in other tissues, including the kidney.

Animals↗
PubMed1991

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards.

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.

Journal Article↗
PubMed1977

Characterization of diastereomeric and enantiomeric ephedrines by gas chromatography combined with electron impact mass spectrometry and isobutane chemical ionization mass spectrometry.

The diastereomeric hydroxyamines, ephedrine and psi-ephedrine, were well differentiated by gas chromatography of their N-acetyl O-trimethylsilyl derivatives. For the analytical separation of enantiomers, conversion into N-(R)-alpha-phenylbutyryl O-trimethylsilyl derivatives was effective in the cases of ephedrine, psi-ephedrine and nor-psi-ephedrine (but not that of nor-ephedrine, on the two stationary phases used). Electron impact mass spectra showed structurally diagnostic fragmentations, but the two diastereomeric amides from each pair of enantiomers yielded generally similar spectra. Molecular ions were of low abundance, but under chemical ionization conditions (isobutane) abundant protonated molecular ions were produced.

Chemical Phenomena↗