[Homozygous C6 deficiency disclosed by Streptococcus A infection].
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Adolescent↗
Biomedical subjects
A Munck
Publications and source records attributed to A Munck.
Stabilization of thymic glucocorticoid-receptor complexes by the calcium-activated protease inhibitor, calpastatin.
We previously described a heat-stable factor from WEHI-7 mouse thymoma, rat liver, spleen, and human chronic lymphocytic leukemia cells that prevents degradation of glucocorticoid-receptor complexes (GRC) in cytosols from rat thymus and acute non-lymphocytic leukemia cells. We now show that the factor has many properties in common with calpastatin, a naturally occurring inhibitor of a family of neutral calcium-activated proteases called calpains. Liver GRC-stabilizing activity and calpastatin activity, in addition to surviving boiling, co-chromatography on columns of DEAE-cellulose ion exchange or agarose A-0.5M gel filtration matrices, and have identical isoelectric points of 5.1. This factor should be especially useful for studying GRC function in the presence of calcium.
Adrenalectomy↗
[Esophageal manometry in infants and children].
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Child↗
[Treatment of virilizing adrenal hyperplasia in adolescents. Use and side-effects of dexamethasone].
Dexamethasone (DXM) has been used to reduce clinical and/or biological hyperandrogenism in 18 adolescent patients treated by hydrocortisone and fludrocortisone for congenital virilizing adrenal hyperplasia (CAH). The doses and duration of treatment did vary among these patients. Androgen excess has been suppressed in all cases, even treated with low doses. Side-effects have been noticed in 9 patients, 5 having large purple striae, and 4 having mild and transient hypercortisolism. The analysis of data shows that DXM may be used for the treatment of CAH in pubertal patients provided that its use remains limited to cases in which the usual treatment does not allow to avoid the androgen excess. In such patients, the dose of DXM at the onset of treatment should be 0.25 mg given once a day, at night, and should not exceed 0.5 mg daily; it has to be adjusted according to clinical results, avoiding to obtain subnormal androgen levels. Hydrocortisone and/or fludrocortisone have to be prescribed for short periods in situations of stress, but not as permanent association. Within these limits, DXM may be considered as a valuable means to obtain hormonal adjustment in CAH adolescent patients.
17-alpha-Hydroxyprogesterone↗
Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors.
We have found that nonactivated and activated forms of the rat thymus glucocorticoid-receptor complex (GRC) will react with reactive sulfhydryl matrices to form covalently immobilized complexes that can subsequently be eluted with reducing agents. The interaction of GRCs with these matrices depends on the nature of both the immobilized sulfhydryl group and the type of leaving group attached. One matrix, agarose CL-4B-diaminoethyl-succinyl-thioethylamine-2-thiopyridyl+ ++ (DSTT), binds total receptor-bound steroid. A second matrix, agarose CL-4B-diaminoethyl-succinyl-cysteinyl-2-thiobenzoic acid (DSCT), binds activated but not nonactivated complexes. The reaction of activated complexes with the DSCT matrix is apparently through a sulfhydryl group located near the DNA binding domain, as soluble DNA interferes with the reaction. This sulfhydryl group(s) appears to be located in a portion of the GRC that is resistant to degradation, since proteolytic digestion of activated GRC to a point where DNA binding is lost results in only a moderate decrease in binding with the DSCT matrix. Purified receptor, covalently labeled with [3H]dexamethasone to the sulfhydryl associated with the steroid binding domain, was able to bind to DSCT matrix, providing evidence for distinct sulfhydryl groups associated with the steroid and DNA binding domains.
Animals↗
Forskolin stimulates adenylate cyclase in human colonic crypts: interaction with VIP.
Forskolin in the 10(-8)-10(-4) M concentration range (ED50 2 microM) strongly stimulated the cyclic AMP production of epithelial crypts isolated from the human colon. At a maximal dose, production increased up to 500 and 700 times the basal cyclic AMP levels at 15 and 37 degrees C, respectively. Forskolin was thus much more efficient than VIP, which is the physiological regulator of this system. Forskolin (ED50 7 microM) also stimulated colonic membrane adenylate cyclase. The stimulation was immediate, did not require guanyl nucleotides and was inhibited by calcium (10(-5)-10(-3) M). In the concentration range between 10(-9) and 10(-5) M (ED50 0.04 microM), forskolin strongly potentiated the stimulation of adenylate cyclase by VIP. We conclude that: (1) forskolin exerts a double dose-dependent action on cyclic AMP production in human colonic crypts, i.e. direct activation of basal adenylate cyclase activity and potentiation of VIP effect; (2) forskolin may be a unique pharmacological tool to investigate the cyclic AMP-dependent processes in human intestine.
Adenylyl Cyclases↗
Sulfhydryl-modifying reagents reversibly inhibit binding of glucocorticoid-receptor complexes to DNA-cellulose.
Glucocorticoid -receptor complexes from intact rat thymus cells incubated with [3H]dexamethasone at 0 degree C are in the nonactivated form and do not bind to DNA-cellulose. Upon being warmed, they are transformed to activated complexes that bind to DNA-cellulose at 0 degree C. We have found that treatment of dexamethasone-receptor complexes with the sulfhydryl-modifying reagents methyl methanethiosulfonate ( MMTS ) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), either before or after the warming, inhibits subsequent binding to DNA-cellulose. The effects of these reagents can be reversed at 0 degree C by dithioerythritol and other sulfhydryl-containing compounds. These results provide the first clear evidence that sulfhydryl-modifying reagents inhibit the binding of activated dexamethasone-receptor complexes to DNA-cellulose and suggest that sulfhydryl groups may be located in or near the DNA binding domain of the rat thymus glucocorticoid-receptor complex. Furthermore, addition of dithioerythritol at 0 degree C to nonactivated receptor complexes that have been treated with MMTS or DTNB produces a substantial increase in the capacity of these complexes to bind to DNA-cellulose, raising the possibility that sulfhydryl groups may be associated with a region on the receptor that plays a critical role in the activation process.
Animals↗
Glucocorticoid-receptor complexes in rat thymus cells. Rapid kinetic behavior and a cyclic model.
We have studied the kinetics, on time scales of minutes and seconds, of formation and interconversion of glucocorticoid-receptor complexes in rat thymus cells under physiological conditions. Nonactivated and activated complexes were measured by a minicolumn technique that permits rapid, multiple simultaneous assays. The rate-limiting step in formation of nuclear complexes was activation, which at 37 degrees C had a half-time of 30-60 s. Activation in cells at 25 degrees C followed first order kinetics. Nuclear binding at 37 degrees C was too fast to measure, and probably has a half-time below 10 s. Earlier findings suggesting that triamcinolone acetonide and dexamethasone give higher steady state ratios of activated to nonactivated complexes than cortisol and corticosterone have been supported by showing that these ratios are concentration-independent, and are unlikely to be due to degradation or dissociation of complexes after cell disruption. A simple cyclic model of receptor kinetics, in which each glucocorticoid is characterized by its dissociation rate constant, accounts quantitatively for these results and many others. The model is based on the assumptions that activation is irreversible, and that energy is required for regenerating functional receptors after each cycle. It yields steady state ratios of activated to nonactivated complexes in agreement with experiment without introducing steroid-specific allosteric influences on activation, and suggests a new mechanism for explaining agonist-antagonist relationships.
Adrenalectomy↗
Nonactivated and activated glucocorticoid-receptor complexes in WEHI-7 and rat thymus cells.
Our own results and those of others have indicated that nonactivated glucocorticoid-receptor complexes are oligomeric proteins with Stokes radius Rs = 8-9 nm, and that activation is accompanied by a reduction in size to Rs = 5-6 nm. The most convincing evidence for the large size of the nonactivated compared to the activated complex has been obtained with cytosols stabilized with molybdate. It has been suggested, however, that molybdate causes aggregation of complexes. Here we show that nonactivated rat thymus complexes in cytosols with molybdate and 400 M KCl have Rs = 8 nm. Furthermore, cytosols from WEHI-cells, which are exceptionally stable, show clear indications of 8 nM nonactivated complexes even without molybdate. The principal complexes in thymus cells under physiological conditions are the nonactivated, activated and nuclear-bound forms. We have studied the rapid kinetics of formation and interconversion of these complexes in intact cells at 37 degrees C, using our newly-developed mini-column procedure to assay nonactivated and activated complexes. These kinetic results, along with many earlier results, can be accounted for quantitatively with a simple cyclic (irreversible) model in which the dissociation rate constant of the steroid plays a key role. The model predicts correctly the different degrees of activation in the cell with glucocorticoids such as triamcinolone acetonide and dexamethasone on the one hand, and cortisol and corticosterone on the other, without assuming steroid-specific allosteric influences of each of these steroids on the receptor.
Animals↗
Stabilization of glucocorticoid-receptor complexes in rat thymus cytosol by a factor from WEHI-7 cells.
Using a variety of physico-chemical techniques we have recently characterized three distinct forms of glucocorticoid-receptor complexes present in the cytosol from rat thymus cells incubated with glucocorticoid; the relative proportions of these complexes are dependent on the conditions to which the cells or cytosols are exposed. Two of these complexes correspond to the well established nonactivated and activated receptor forms, while the third has properties consistent with mero-receptor. Based on their differential affinities for DNA- and DEAE-cellulose we have developed a rapid mini-column chromatographic procedure for separating these three forms and have used it to examine the stability of complexes in cytosol preparations. We have found that activated glucocorticoid-receptor complexes from rat thymus cells are relatively unstable under cell-free conditions in that they undergo time-dependent losses in DNA binding and are converted to mero-receptor. In contrast, cytosolic glucocorticoid-receptor complexes prepared from WEHI-7 mouse thymoma cells are remarkably stable under similar conditions. Mixing experiments with equal portions of rat thymus and WEHI-7 cytosol revealed that the difference between the two tissues cannot be accounted for merely by differences in amounts of proteolytic enzymes, since addition of rat thymus cytosol to WEHI-7 cytosol containing activated glucocorticoid-receptor complexes does not result in their conversion to mero-receptor. However, the WEHI-7 cytosol affords considerable protection to activated glucocorticoid-receptor complexes in thymus cytosol. The stabilizing factor from WEHI-7 cytosol is heat stable (survives 100 degrees C for 30 min), insensitive to pH over a wide range (4.0-10.0), and appears to be macromolecular. It does not inhibit activation, and thus appears distinct from the previously described endogenous glucocorticoid receptor stabilizing factor responsible for stabilization of thymocyte receptor binding capacity (Leach et al., J. Biol. Chem. 257: 381-388, 1982). We propose that the factor is an endogenous inhibitor of the protease(s) responsible for mero-receptor formation.
Animals↗
Physiological functions of glucocorticoids in stress and their relation to pharmacological actions.
Almost any kind of threat to homeostasis or stress will cause plasma glucocorticoid levels to rise. The increased levels have traditionally been ascribed the physiological function of enhancing the organism's resistance to stress, a role well recognized in glucocorticoid therapy. How the known physiological and pharmacological effects of glucocorticoids might accomplish this function, however, remains a mystery. A generalization that is beginning to emerge is that many of these effects may be secondary to modulation by glucocorticoids of the actions of numerous intercellular mediators, including established hormones, prostaglandins and other arachidonic acid metabolites, certain secreted neutral proteinases, lymphokines, and a variety of bioactive peptides. These mediators participate in physiological mechanisms--endocrine, renal, immune, neural, etc.--that mount a first line of defense against such challenges to homeostasis as hemorrhage, metabolic disturbances, infection, anxiety, and others. Contrary to the traditional view that glucocorticoids enhance these defense mechanisms, however, it has become increasingly clear that glucocorticoids at moderate to high levels generally suppress them. This paradox, which first emerged when glucocorticoids were discovered to be antiinflammatory agents, remains a major obstacle to a unified picture of glucocorticoid function. We propose that stress-induced increases in glucocorticoid levels protect not against the source of stress itself but rather against the body's normal reactions to stress, preventing those reactions from overshooting and themselves threatening homeostasis. This hypothesis, the seeds of which are to be found in many discussions of particular glucocorticoid effects, immediately accounts for the paradox noted above. Furthermore, it provides glucocorticoid physiology with a unified conceptual framework that can accommodate such apparently unrelated physiological and pharmacological effects as those on carbohydrate metabolism, inflammatory processes, shock, and water balance. It also leads us to suggest that some of the enzymes rapidly induced by glucocorticoids, such as glutamine synthetase, detoxify mediators released during stress-induced activation of primary defense mechanisms. These mediators would themselves lead to tissue damage if left unchecked.
Animals↗
Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones.
The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.
Animals↗
Characteristics, stability and regulation of glucocorticoid-receptor complexes in normal and neoplastic cells as studied with a new mini-column technique.
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Acute Disease↗
Glucocorticoid actions on lymphoid tissue and the immune system: physiologic and therapeutic implications.
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Glucocorticoids↗
Stabilization of labile glucocorticoid-receptor complexes from acute nonlymphocytic leukemia cells by a factor from chronic lymphocytic leukemia cells.
Glucocorticoid-receptor complexes in cytoplasm from normal lymphoid and leukemia cells incubated with glucocorticoid can be resolved into three different components, activated, nonactivated, and mero-receptor complexes, in relative amounts, dependent on the conditions to which the cells or cytosols are exposed. Recently, we reported that cytosols of acute nonlymphocytic leukemia (ANLL) cells contained high levels of mero-receptor complexes relative to those of chronic lymphocytic leukemia (CLL) or normal lymphoid cells. In the present study, we examined the cause for the lability of cytosolic complexes of ANLL cells. Mero-receptor accumulated rapidly in ANLL cytosols in a time-dependent fashion. The accumulation was most rapid in cytosols which contained activated receptor complexes, but it also occurred in cytosols containing only nonactivated receptor forms. Molybdate (20 mM) slowed but did not prevent the conversion to mero-receptor. Cytosols of ANLL specimens of the M4 French-American-British class (with monocytoid differentiation properties), in general, contained more stable complexes than did specimens of the M1 to M3 French-American-British classes (primarily myelocytic differentiation) suggesting that lability may in part be related to the state or direction of differentiation of the leukemic cells. In keeping with this hypothesis, cytosols of polymorphonuclear cells isolated from normal blood were much more labile than were those of monocytes. Mixing experiments with ANLL and CLL cells showed that the lability of ANLL complexes is not due simply to a higher content of proteolytic enzymes in these cells, because addition of ANLL cells or cytosols to CLL specimens did not result in increased mero-receptor. To the contrary, addition of CLL cells to ANLL specimens greatly stabilized the cytosolic complexes. These findings indicate the presence of an endogenous factor, present in CLL but lacking in ANLL cells, which is capable of stabilizing cytosolic complexes.
Acute Disease↗
Effects of ATP and pyrophosphate on properties of glucocorticoid-receptor complexes from rat thymus cells.
Cytosols from rat thymus cells incubated with glucocorticoid contain nonactivated and activated receptors and mero-receptor complexes, in relative amounts that depend on the incubation conditions. These forms can be separated by a rapid minicolumn chromatographic technique based on their differential affinities for DNA, DEAE, and hydroxylapatite. We have used this method to examine the effects of ATP, pyrophosphate (PPi), and related compounds on cytosolic complexes. In addition to ATP, already known to promote activation at 0 degrees C, PPi, ADP, and other triphosphates at millimolar concentrations promoted activation of nonactivated complexes. AMP and Pi had little effect. ATP and PPi at millimolar concentrations also reduced binding of activated complexes to DNA. Characterization of the ATP- and PPi-activated complexes by gel filtration and ion exchange chromatography revealed two DNA-binding forms. One was essentially identical (Stokes radius of approximately 5.4 nm, elution from DEAE at approximately 50 mM KCl) to the normal activated complex obtained directly from cells incubated at 37 degrees C. The other had a Stokes radius of approximately 3.1 nm and had no affinity for DEAE. Analysis by minicolumns and gel filtration showed that ATP and PPi prevented formation of mero-receptor complexes, a process which occurs relatively rapidly in untreated thymus cytosols. These compounds did not alter properties of preformed mero-receptor. The accumulation of 3.1-nm complexes in thymus cytosols in which formation of mero-receptor is prevented suggests that this form is an intermediate, normally short-lived, in the conversion of 5.4 nm complexes to mero-receptor.
Adenosine Triphosphate↗
Characterization of nonactivated and activated glucocorticoid-receptor complexes from intact rat thymus cells.
In cells exposed to glucocorticoids at 37 degrees C activated glucocorticoid-receptor complexes (complexes with affinity for nuclei and DNA) are formed after nonactivated complexes. Activation thus appears to be an obligatory physiological process. To investigate this process we have characterized cytoplasmic complexes formed in rat thymocytes at 0 and 37 degrees C. Complexes in cytosols stabilized with molybdate were analyzed by sucrose gradient centrifugation and by chromatography on DNA-cellulose, DEAE-cellulose, and agarose gels. Two major complexes were observed: the nonactivated complex, eluted from DEAE at approximately 200 mM KCl, was formed at 0 and 37 degrees C, gave S20,w = 9.2 S, Stokes radius = 8.3 nm, and calculated Mr = 330,000; the activated complex, eluted from DEAE at approximately 50 mM KCl, appeared only at 37 degrees C, gave S20,w = 4.8 S, Stokes radius = 5.0 nm, and Mr = 100,000. A third, minor complex, probably mero-receptor, which appeared mainly at 37 degrees C, bound to neither DNA nor DEAE, and gave S20,w = 2.9 S, Stokes radius = 2.3 nm, and Mr = 27,000. With three small columns in series (DNA-cellulose, DEAE-cellulose and hydroxylapatite), the three complexes can be separated in 5-10 min. By this method we have examined the stability of complexes under our conditions. We conclude that in intact thymus cells glucocorticoid-receptor complexes occur principally in two forms, nonactivated and activated, and that activation is accompanied by a large reduction in size. The origin of the mero-receptor complex remains uncertain.
Animals↗
Analysis of activated and nonactivated cytoplasmic glucocorticoid-receptor complexes from human leukemia cells by rapid DNA-diethylaminoethyl minicolumn chromatography.
Glucocorticoid-receptor complexes in cytoplasm from normal lymphoid cells incubated with [3H]dexamethasone can be resolved into three different components. Two of these correspond to the well-established activated and nonactivated forms, while the third appears similar to the mero-receptor complex first described by Sherman et al. (Fed. Proc., 37: 167-173, 1978). Based on their differential affinities for DNA- and DEAE-cellulose in buffers of low ionic strength (the activated complex binds to DNA- and DEAE-cellulose; the nonactivated complex binds to only DEAE-cellulose; the mero-receptor-like complex binds to neither), we have developed a rapid minicolumn chromatographic procedure for separating these forms, and have applied it to examine the relative proportion of different complexes in cytosols from cells of leukemia patients. All samples from nine patients with chronic lymphocytic leukemia contained these three complexes in proportions similar to those seen with normal lymphoid tissue. Cytosols from six of eight specimens from patients with acute nonlymphocytic leukemia contained a lower proportion of activated complexes and a higher proportion of mero-receptor-like complexes than cytosols from normal or chronic lymphocytic leukemia cells. Whether such differences in the properties of cytosolic complexes are due to degradation taking place after the cells are broken, and whether they can be correlated to in vivo therapeutic response, is not known, but studies are in progress to answer these questions. The minicolumn procedure described here offers a simple and reliable method for these purposes.
Adolescent↗