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J D Baxter

Publications and source records attributed to J D Baxter.

At least 181 records · Page 10Linked to original sources
PubMed1982

Differential regulation by glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary.

Glucocorticoids negatively regulate de novo synthesis of proopiomelanocortin (POMC) and the steady state POMC mRNA levels in the rat pituitary anterior lobe. The POMC system of the intermediate lobe is far less sensitive to alterations in circulating glucocorticoid levels. Possible mechanisms for this differential regulation of POMC gene expression in the two tissues are discussed.

Adrenalectomy↗
PubMed1982

Photoaffinity labelling of the rat liver nuclear thyroid hormone receptor with [125I]triiodothyronine.

[125I]Triiodothyronine (T3) was used as a photoreactive probe for the thyroid hormone nuclear receptor in photoaffinity labelling experiments. Autoradiograms of photolysis products electrophoresed on either one or two-dimensional gels showed that [125I]T3 covalently, but nonspecifically, labelled many proteins in the partially purified receptor preparations used. However, one of these proteins with an estimated molecular weight of 47,000 and an isoelectric point of approximately 6.2 +/- 0.5 pH units appears to be the thyroid hormone receptor, since, in contrast to the other proteins, its photoinduced labelling was blocked by concentrations of T3 and thyroxine (T4) similar to those that inhibit binding of [125I]T3 by the receptor in equilibrium binding assays. In addition, the isoelectric point of the photolabelled protein agrees with that determined in separate equilibrium isoelectric focusing studies. These results indicate that [125I]T3 can serve as a photoreactive probe for the thyroid hormone nuclear receptor, and they suggest that this receptor is a single polypeptide chain of molecular weight 47,000 with an isoelectric point of 6.2 +/- 0.5 pH units.

Affinity Labels↗
PubMed1982

A radioreceptor assay for direct measurement of plasma free glucocorticoid activity.

An assay for plasma free glucocorticoid receptor-binding activity has been developed with the use of specific glucocorticoid receptors in cultured rat pituitary tumor cells (GC line). Unlike other assays for corticosteroids, this assay measures the summed free (rather than the total) receptor-binding activity of all natural and synthetic steroids present in the sample. Plasma samples (0.5 ml) were incubated with [3H]dexamethasone and GC cells at 37 C for 45 min, and the nuclear bound dexamethasone was then measured. The free glucocorticoid receptor-binding activity was determined by the extent to which steroids present in the sample inhibit binding of the 3H-labeled steroids. Free cortisol concentrations of 0.3-3.6 micrograms/dl were accurately detected. That the assay measures free but not total steroids and that the free steroid provides the correct index of receptor-occupying capability were verified by two findings: 1) the assay yields similar results when the free cortisol is separated from plasma and then run in the assay, and 2) the receptor-binding activity of dexamethasone in plasma is reduced to the extent of plasma binding of this steroid measured directly. Free receptor-binding activity of adult plasma taken at various times determined by the assay correlated well with that expected from the total cortisol levels (measured by the corticosteroid-binding globulin-isotope assay) and the extent of plasma binding. The utility of the assay for measuring plasma dexamethasone levels after oral administration of the steroid is shown. The assay should be useful to determine the free glucocorticoid receptor-binding activity in plasma due to cortisol, synthetic glucocorticoids, or unidentified medication. In most circumstances, i.e. when antagonists are not present in sufficient quantities, such activity should equal the free glucocorticoid activity as well.

Animals↗
PubMed1981

Development of support matrices for affinity chromatography of thyroid hormone receptors.

Certain cellular responses to thyroid hormones appear to be mediated by non-histone chromatin protein receptors. Purification of these proteins is important for an investigation of the detailed mechanisms of their regulatory role. In the present studies, we report the development of an affinity chromatographic system that can be used to purify thyroid hormone receptors solubilized from nuclei. Amine-substituted hormone analogs were prepared with D and L isomers of T3; these bind to the receptor. This finding supports the hypothesis that thyroid hormones fit into the receptor with the amino groups accessible from outside the binding site. Although L-triiodothyronine (LT3) (the naturally occurring isomer) binds more tightly (relative Kd = 1.0 nM) to the nuclear receptor than D-triiodothyronine (DT3) (relative Kd = 2.0 nM), the amine-substituted analog of DT3 binds more tightly than the LT3 analog (DT3 analog, relative Kd = 40 nM; LT3 analog, relative Kd = 1500 nM). Agarose-based gels containing DT3 and LT3 covalently coupled by their amino groups were also prepared. Binding of receptor to these gels was biospecific in that it could be inhibited by prior incubation of the receptors with LT3. In addition, as predicted by the analog studies, the DT3 affinity gels were more effective than LT3 gels in adsorbing receptor. Elution of receptor from the LT3-derived gels was achieved in a predicted volume and concentration of counter-ligand in elution buffer. These results suggest that affinity chromatography can be applied to the purification of thyroid hormone receptors.

Animals↗
PubMed1981

Photoaffinity labeling of glucocorticoid receptors.

The cross-reactivity of progestins for glucocorticoid receptors was exploited to photoaffinity label glucocorticoid receptors from cultured rat hepatoma (HTC) and mouse lymphoma (S49) cell cytosol. The synthetic progestin, 17 alpha, 21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020), rapidly forms covalent bonds with protein upon irradiation of either cytosol with 350 nm light. Polyacrylamide gel electrophoresis under denaturing conditions reveals a single band photolabeled by R5020 that is not observed when excess nonradioactive dexamethasone is included in the incubation. This protein band corresponds to a molecular weight of about 87,000 in both HTC and S49 cell cytosol; it is entirely absent in cytosol from glucocorticoid-resistant S49(r-) cells which lack receptor-binding activity. Another steroid-resistant mutant, S49 (nti), which exhibits normal levels of steroid-binding activity but increased binding of receptor-steroid complexes by the nucleus, yields a receptor which, when photolabeled, has an apparent molecular weight of only 39,000. These results demonstrate that glucocorticoid receptors can be photoaffinity-labeled; the data are consistent with the notion that the binding form of the receptor consists of a single polypeptide chain, Mr = 87,000, in two different species, rat and mouse, and in cells of either hepatic or lymphoid origin. The data also suggest that the lesion in the steroid-resistant S49 (nti) lymphoma cell line is a mutation of the structural gene for the glucocorticoid receptor which results in the synthesis of a truncated protein.

Affinity Labels↗
PubMed1981

Human prolactin. cDNA structural analysis and evolutionary comparisons.

Prolactin (Prl), growth hormone, and chorionic sommatomammotropin form a set (the "Prl set") of hormones which is thought to have evolved from a common ancestral gene. This assumption is based on several lines of evidence: overlap in their biological and immunological properties, similarities in their amino acid sequences, and homologies in the nucleic acid sequences of their structural genes. In the current study we report the cloning, amplification in bacteria, and sequence analysis of DNA complementary to Prl mRNA isolated from human pituitary Prl-secreting adenomas. The cloned DNA contains 914 bases, which includes the entire coding sequence of human prePrl as well as portions of the 5- and 3'-untranslated regions of the mRNA. The amino acid sequence predicted by our data differs from a previously reported amino acid sequence in 8 positions. With the results of this study we can now compare in one species the nucleotide sequences of the structural gene coding for each of the hormones of the Prl set. The sequence divergence at replacement sites is used to establish an evolutionary clock for the Prl set of genes. Using this clock, we postulate that the chromosomal segregation of human Prl and human growth hormone occurred about 392 million years ago and that growth hormone and chorionic sommatomammotropin underwent an intrachromosomal recombination within the last 10 million years.

Adenoma↗
PubMed1981

Regulation of calcium-binding protein messenger RNA by 1,25-dihydroxycholecalciferol.

1,25-Dihydroxycholecalciferol modulates the synthesis of the intestinal calcium-binding protein. To determine if this effect is due to an increase in calcium-binding protein mRNA activity, we measured total cytoplasmic protein-specific mRNA activity from chick intestine in an in vitro wheat germ translation system. 1,25-Dihydroxycholecalciferol enhances calcium-binding protein specific mRNA activity. The sterol does not induce a general increase in mRNA synthesis since the concentration of only a few proteins increased. Thus 1,25-dihydroxycholecalciferol regulates only a subset of genes and specifically affects a transcriptional process involving calcium-binding protein synthesis.

Animals↗
PubMed1981

Primary structure and evolution of rat growth hormone gene.

The rat growth hormone gene was isolated on a cloned 11.4-kilobase EcoRI-generated DNA fragment from a bacteriophage "library" of chromosomal DNA. The structural gene sequence, approximately 2.1 kilobases long, was identified by hybridization to the corresponding cloned rat growth hormone cDNA and shown to contain four intervening sequences. The complete primary structure of the gene and the 5' and 3'-flanking regions was determined. The mosaic structure of exons and introns can be related to the different biological activities of growth hormone and to the evolution from ancestral sequences of a gene that was the precursor to the growth hormone and the related prolactin and placental lactogen (chorionic somatomammotropin) genes. The largest intron was found to contain a dispersed repetitive DNA sequence flanked by perfect 18-base pair direct repeats. The mobility of sequences of this kind could play a role in the observed variation of intron sizes and in rearrangements of mammalian genes.

Amino Acid Sequence↗
PubMed1981

Cloning of bovine prolactin cDNA and evolutionary implications of its sequence.

Prolactin, growth hormone, and chorionic somatomammotropin (placental lactogen) constitute a set of related polypeptides believed to derive from a common evolutionary ancestor protein. We have cloned and sequenced DNA complementary to the mRNA coding for bovine prolactin. This cDNA contains 702 bases corresponding to 10 amino acids in the leader peptide, all 199 amino acids of the hormone, and 75 nucleotides in the 3' untranslated region of the mRNA. Nucleotide sequence analysis of this cDNA permitted the identification of 10 amino acids in the signal peptide, plus the correction or elucidation of amino acid assignments at 16 sites where aspartic and glutamic acids had not been distinguished from their amides by amino acid sequencing. Codon usage in bovine prolactin mRNA is nonrandom, but, similarly to rat and human prolactins, it does not exhibit the strong preference for G or C in codon third positions seen in bovine, rat, and human growth hormone mRNAs. The translational termination signal in bovine prolactin in UAA, also the same as in rat and human prolactins and differing from the UAG "stop" codon used in bovine, rat, and human growth hormones and human chorionic somatomammotropin. The amino acid and mRNA nucleotide sequences of bovine, rat, and human prolactins and growth hormones were compared by several techniques based on various theories of molecular evolution. The comparison of prolactin to growth hormone is consistent in all three species, suggesting that the genes for these two hormones diverged about 350 million years ago. However, comparisons among the three prolactins or among the three growth hormones to determine the times of evolutionary divergence of the three species generated values that were inconsistent with each other and with the fossil record. Analysis of these discrepancies suggests that the genes for prolactin and growth hormone may now be evolving by different mechanisms.

Amino Acid Sequence↗