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J D Baxter

Publications and source records attributed to J D Baxter.

At least 253 records · Page 14Linked to original sources
PubMed1976

Solubilized nuclear "receptors" for thyroid hormones. Physical characteristics and binding properties, evidence for multiple forms.

Tissues regulated by thyroid hormones contain chromatin-localized "receptors" that may be involved in the actions of these hormones. In this report, we describe some properties of these receptors after their solubilization from rat liver nuclei and their separation from nucleic acids and basic proteins. The nuclear extract and partially purified preparations contain a dominant class of binding sites which have a high affinity for triiodothyronine (3,5,3'-triiodo-L-thyronine, Kd approximately 1 nM) and for the biologically potent isopropyl diiodothyronine (3,5-diiodo-3'-isopropyl-L-thyronine, Kd congruent to 1 nM) and also bind thyroxine (3,5,3',5'-tetraiodo-L-thyronine, Kd approximately 5 nM) and reverse triiodothyronine (3,3',5'-triiodo-L-thyronine, Kd approximately nM). This binding activity elutes on Sephadex G-100 in an included peak which has a Stokes radius of 35 A and sediments on glycerol gradients at 3.5 S. From these data a molecular weight ratio of 50,500 and a frictional ratio of 1.4 were calculated, suggesting that the receptor is somewhat asymmetrical. There was a sharp decline in triiodothyronine binding by this component above pH 8.7 (optimum around pH 7.6) where there is marked dissociation of the 4' phenolic hydroxyl of triiodothyronine (pKalpha approximately 8.5). A similar decrease in thyroxine (pKalpha approximately 6.7) binding with pH increases in this range was not observed. Thus, ionization of the phenolic hydroxyl may influence binding. The solubilized preparations can also contain a minor specific-binding component that can be identified by binding analyses, and by G-100 or quaternary aminoethyl Sephadex chromatography. this component has a much lower affinity for triiodothyronine and isopropyl diiodothyronine than for thyroxine as compared to the major component. It probably has a pH optima around 6.0 and demonstrates and apparent tendency to aggregate. The minor component was not always identified by direct Scatchard analysis and may be generated in part from the major component as it was more commonly observed after storage or purification of the nuclear extract. Thus, at least two thyroid hormone-binding components can be present in extracts of purified rat liver nuclei; the minor component may be an altered form or subunit of the major component. The relative binding activities of triiodothyronine, isopropyl diiodothyronine, and thyroxine by the major component, similar to those in intact nuclei, parallel the biological potencies of these compounds, and suggest that the dominant binding is by biologically relevant receptors. Since ionization of the phenolic hydroxyl may influence binding, the lower activity of thyroxine relative to triiodothyronine may in part be due to the fact that at physiological pH, the phenolic hydroxyl of thyroxine is more dissociated than is that of triiodothyronine. The finding that this receptor is somewhat asymmetrical provides an indication of the shape of an intrinsic chromatin protein implicated in specific gene regulation...

Animals↗
PubMed1976

Chromatin receptors for thyroid hormones. Interactions of the solubilized proteins with DNA.

Thyroid hormone-responsive tissues contain chromatin "receptor" proteins that are concentrated in chromatin subfractions enriched in DNA. These receptors appear to be DNA-binding proteins. In the present study, we utilized a DNA-cellulose binding assay to further examine the interactions of solubilized receptors with DNA. [125I]Triiodothyronine associates with receptors bound to DNA-cellulose, whereas free [I]triiodothyronine and [125I]triiodothyronine associated with other proteins does not. The DNA-receptor interactions appear to be strong enough to exist at physiological ionic strength since binding is 50% maximal ag 0.175 M NaCl and is only partly inhibited by Ca2+ and Mg2+ in the 1 to 5 mM range. Most, if not all, of the receptors are capable of DNA binding, and there are at least 80,000 receptor binding sites/diploid DNA (assuming one triiodothyronine binding site/receptor). Binding of the receptor-[125I]triiodothyronine complexes to other DNAs and analogs was examined using a competition assay. There is similar binding by native and denatured DNA, gy eukaryotic DNA from different species and by prokaryotic DNA (Bacillus subtilis). Binding by natural DNAs is more avid than by cytoplasmic RNA, nuclear RNA, poly(dA-dT)-poly(dA-dT), or poly(dG-dC)-poly(dG-dC). Under these conditions, binding by tRNA and poly(dA) is insignificant, and the nucleotide monomers ATP and GTP have no detectable binding. These studies support the idea that the thyroid hormone receptor is a DNA-binding protein and that the interaction is a major determinant for receptor localization in chromatin. The competition studies suggest that the polynucleotide composition and/or conformation can have marked influences on the binding, and that multiple orders of binding affinity can exist. The presence of specific sequences cannot be excluded. However, the finding that receptors bind extensively and tightly to DNA suggests that receptors in chromatin may randomly bind to any available DNA, resulting in some of the receptors being at physiologically unimportant sites. If so, the several thousand hormone receptors present in each target cell may be required to enhance the possibility that some of the receptors are present at the actual sites of action.

Animals↗
PubMed1976

Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.

Binding Sites↗
PubMed1976

Role of steroids in hypertension: evaluation of plasma mineralocorticoid activity with aldosterone receptors.

1. A competitive "mineralocorticoid" receptor-binding technique has been used to study plasma "mineralocoroticoid" activity in normal and hypertensive states. The binding reaction mixture contains the competitor steroids in undiluted plasma, [3H]aldosterone and rat kidney slices. Thus plasma binding and receptor occupancy can be simultaneously considered. 2. Competitor activities relative to that of aldosterone (100%) were: deoxycorticosterone (DOC), 16%, cortisol, 0-4%; 18-hydroxydeoxycorticosterone (18-OH-DOC), 0-1%; 16beta-OH-dehydroepiandrosterone and 16-oxo-androstenediol, inactive. (Binding characteristics for aldosterone and the other steroids tested with the rat receptor were found to be similar to those with receptors in slices from human kidney, obtained at operation.) These steroids and the spironolactone SC14266 were less active in plasma than in buffer, suggesting that they bind significantly to plasma and that reduces their capacity to occupy the receptors. 3. These competition data also suggest that at normal and even mildly elevated concentrations, cortisol does, but DOC and 18-OH-DOC do not, contribute significantly to the plasma mineralocorticoid activity. Competitor activity in plasma samples taken at 12.00 hours from normal subjects (upright) was greater than that in those taken at 08.00 hours (supine). This physiological change, corresponding to an increase in aldosterone, was detected even though there was an associated decrease in plasma cortisol. Competitor activity in plasma from patients with primary aldosteronism was markedly elevated as compared with that of normal subjects. However, an increase in plasma steroids which bind to the mineralocorticoid receptor was not detected in plasma from patients with "low-renin essential hypertension".

Aldosterone↗
PubMed1976

Aldosterone receptors and the evaluation of plasma mineralocorticoid activity in normal and hypertensive states.

Aldosterone receptors from rat kidney slices were utilized in a competitive binding technique to analyze the contribution of various steroids to plasma "mineralocorticoid" activity and to assess their possible role in hypertension. To consider simultaneously the plasma binding, steroids were incubated with slices in undiluted plasma; competitor activities for [3H]aldosterone binding were aldosterone, 100%; deoxycorticosterone, 16.2%; cortisol, 0.4%; and 18-hydroxy-deoxy-corticosterone and d18-hydroxy-corticosterone, 0.1%. These steroids were more active in buffer than plasma, suggesting that they bind to plasma and that this reduces their receptor binding. Analysis of the competition data suggests that at normal plasma concentrations, aldosterone occupies the receptors to a major extent, cortisol occupies some of the receptors, and deoxycorticosterone and 8-hydroxydeoxycorticosterone contribute little to receptor occupancy. Two steroids implicated in low-renin essential hypertension, 16beta-hydroxy-dehydro-epiandrosterone and 16-oxoandrostenediol, did not have significant competitor activity. Competitor activity in plasmas from normal subjects taken at 12 noon (upright) was greater than that in those taken at 8 a.m. (supine). Since the 12 noon samples had higher aldosterone and lower cortisol levels than the 8 a.m. samples, the competitor activity under these physiological circumstances reflects aldosterone more than cortisol. The competitor activities of plasmas from patients relative to normal subjects (100+/-12.1%; mean+/-SEM) were: normal renin "essential" hypertension, 117+/-14%; low-renin essential hypertension, 101+/-6.6%; and primary aldosteronism, 176+/-14.3%. Thus a significant increase in activity of steroids that interact with mineralocorticoid receptors was detected in primary aldosteronism (P LESS THAN 0.01) BUT WAS NOT DETECTED IN LOW-RENIN OR NORMAL-RENIN ESSENTIAL HYPERTENSION.

Aldosterone↗
PubMed1976

Mineralocorticoid receptors in human kidney.

Cytosol binding of [3H]aldosterone was measured after incubation of the hormone at 37 C with slices of human kidney obtained after surgical removal. High affinity [3H]aldosterone binding with an apparent equilibrium dissociation constant (Kd) of approximately 0.5 nM was observed to probably "mineralo-corticoid receptors." [3H]aldosterone binding of lower affinity and higher capacity (probably to "glucocorticoid receptors") was also observed. Binding of other steroids by the mineralocorticoid receptors was determined by competitive analysis using a low concentration of [3H]aldosterone (so that [3H] binding is predominantly by the mineralocorticoid receptors). The binding activities relative to aldosterone (100%) were: deoxycorticosterone, 48%; cortisol, 1.5%; 18-hydroxy-deoxycorticosterone, 1.2%; and 18-hydroxy-corticosterone, 0.2%. The relative sodium-retaining potencies of these steroids in vivo correlate well with their binding activities. These data provide further support to the view that the major high affinity [3H]aldosterone binding is by mineralocorticoid receptors. Two steroids, 16beta-hydroxy-dehydroepiandrosterone and 16-oxo-androstenediol, recently shown to have sodium-retaining activity in the rat, and also implicated in low-renin "essential" hypertension in man, showed no competitive binding activity. The affinity of [3H]aldosterone for binding to these mineralocorticoid receptors and the relative steroid binding activities are similar to the values previously reported in the rat. Thus, human and rat mineralocorticoid receptors appear to be similar in their affinity for aldosterone and their specificity for binding a number of other steroids.

Aldosterone↗
PubMed1976

Cochlear and vestibular dysfunction resulting from physical exertion or environmental pressure changes.

Inner ear damage due to physical exertion or environmental pressure changes has been described only recently. According to Goodhill there are two possible mechanisms of injury: the explosive and the implosive. The former is triggered by increased C.S.F. pressure transmitted to the perilymph space; the latter by increased pressure in the middle ear transmitted to the perilymph space through the two windows. Sudden hearing loss or dizziness, or both, are the main symptoms. The diagnosis will be established by the history, the audiogram, and the electronystagmogram (ENG). The treatment is still controversial. As the formation of a fistula is a real possibility, an exploratory tympanotomy is suggestive on suspected cases. Due to the variety of symptoms every case has to be assessed individually. A detailed history is of utmost importance.

Adolescent↗
PubMed1976

Cellular receptors for lymphotoxin: correlation of binding and cytotoxicity in sensitive and resistant target cells.

The binding of radioactively labeled lymphotoxin (LT) to both lymphotoxin-sensitive and -resistant cell clones was examined. The sensitive clone had a low- capacity, high-affinity ("specific") binding component, the curve of which closely followed the cytotoxicity curve of the lymphocyte mediator. The capacity of this binding component was calculated to be about 600 molecules of LT/cell. In addition, there was a low-affinity, high-capacity ("nonspecific") binding component. In striking contrast, the high-affinity, low-capacity ("specific") component was absent or greatly diminished from the resistant clone, whereas the low-affinity, high-capacity ("nonspecific") component was present at a similar level as in the sensitive cells. These binding characteristics closely resemble those observed by us and other investigators working with a variety of steroid hormones in steroid-sensitive and- resistant cell lines.

Animals↗
PubMed1975

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.

Animals↗
PubMed1975

Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization.

Thyroid hormones have diverse effects on growth and metabolism. Specific "receptor" proteins which bind triiodothyronine and other biologically active analogs and which may be involved in thyroid hormone action have been recently found in nuclei of responsive tissues. This report presents studies of these receptors in rat liver nuclei. Confirming previous reports, a Scatchard analysis of the binding data suggests the reaction, triiodothyronine + specific receptor in equilibrium with triiodothyronine-receptor complex, with an apparent equilibrium dissociation constant (Kd) at 22 degrees of about 190 pM and a capacity of about 1 pmol of triiodothyronine-binding sites per mg of DNA. The kinetics of the binding were also examined. Triiodothyronine-receptor complex formation is second order and dissociation is first order. The apparent association (k+1) and dissociation (k minus 1) rate constants at 22 degrees are, respectively, 4.7 times 10-7 m-minus 1 min-minus 1 and 7.6 times 10-minus 3 min-minus 1. The apparent Kd, estimated from the ratio of the rate constants (k minus 1:k+1), was about 150 pM, similar to that determined from the equilibrium data. These data support the expression written above for the interaction of thyroid hormone with its receptor. Additional kinetic experiments indicate that some of the triiodothyronine binding by cell-free nuclei is to sites previously occupied by hormone in the intact animal, providing further evidence that the intact cell and cell-free reactions are the same. It was previously found that nuclear-bound triiodothyronine is localized in chromatin. We found that isolated chromatin retains specific binding activity similar to that of isolated nuclei. Thus, binding may not require cytoplasmic, nucleoplasmic, or nuclear membrane factors. These findings may imply that chromatin localization of the receptor does not depend on the hormone. This idea is supported by an earlier finding that binding activity is present in nuclei from thyroidectomized animals. However, many stimuli such as steroid hormones, bacterial inducers, and cyclic adenosine 3':5'-monophosphate in bacteria influence regulatory proteins at the gene level by promoting the protein's addition to or removal from chromatin. Thus, we studied the effect of thyroid hormone on the nuclear content of receptors under assay conditions of receptor stability and reversible binding. Receptor levels in hypothyroid animals are identical with those in euthyroid animals. These data suggest that the hormone does not influence the nuclear localization of receptors. Thus, the basis for thyroid hormone action may be to regulate the activity of receptors resident in chromatin rather than to promote receptor addition to or removal from chromatin.

Animals↗
PubMed1975

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.

Animals↗
PubMed1975

A radioreceptor assay for evaluation of the plasma glucocorticoid activity of natural and synthetic steroids in man.

An assay for plasma glucocorticoid activity has been developed using specific glucocorticoid receptors. Unlike other assays for cortisol and certain synthetic corticosteroids, this radioreceptor assay measures the glucocorticoid activity of all natural an synthetic steroids. Steroids extracted from as little as 0.05 ml of plasma are incubated with 3H-dexamethasone and cytosol receptors from cultured rat hepatome cells. From 0.5 to 50 ng of cortisol are accurately detected. Glucocorticoid activities of adult determined by the assay correlate closely with corticoid levels obtained in the CBG-isotope and fluorometric assays. Other steroids are measured in proportion to both concentration and potency as glucocorticoids. Relative activities include: cortisol 100, dexamethasone 940, prednisolone 230, prednisone 3, estradiol 1 and androstenedione 1. A similar ranking of steroids was found using receptors from a human source (fetal lung). The assay has been useful in detecting glucocorticoid activity in unidentified medications and in measuring plasma glucocorticoid levels after administration of synthetic corticosteroids. A modification of the assay is described for also estimating the level of free (unbound) glucocorticoid activity in plasma. Assays are performed before and after removal by charcoal of steroids not bound to plasma transcortin. This consideration is shown to be particularly important with prednisolone where the unbound steroid level is much less than the total. Thus, the radioreceptor assay provides a relatively simple technique for measuring the total of free plasma of glucocorticoid activity in man due to any natural or synthetic steroid or combination of steroids.

Aldosterone↗