Rhabdomyolysis: a rare adverse effect of diphenhydramine overdose.

Drug- and toxin-induced rhabdomyolysis is a nonspecific clinical syndrome resulting from the release of skeletal muscle cell contents into the plasma and urine. Antihistamine drugs are the active ingredients in a number of over-the-counter preparations and are frequently ingested in suicide attempts. We report rhabdomyolysis as a rare adverse effect of diphenhydramine toxicity in a 29-year-old man who ingested an unknown quantity of an over-the-counter sleep preparation in a suicide attempt. The patient had documented toxic concentration of diphenhydramine in his cerebrospinal fluid and no history of seizures, coma, or hypothermia. A high index of suspicion and an evaluation for rhabdomyolysis is warranted in antihistamine toxicity.

Correlation of chromosome 17p loss with clinical outcome in medulloblastoma.

Medulloblastomas are primitive neuroectodermal tumors that arise in the cerebella of children. Cytogenetic and loss of heterozygosity (LOH) studies have shown that deletions on the short arm of chromosome 17 occur in 25-50% of cases, suggesting that loss of a tumor suppressor gene located on 17p plays a role in the genesis or progression of medulloblastoma. We report here on an LOH analysis of 21 patients with medulloblastoma using markers for 15 polymorphic loci previously ordered on chromosome 17 by meiotic break point mapping. Although the frequency of LOH in our patients (48%) was consistent with previous reports, our maps showed much larger deletions, the smallest spanning a 38-cM genetic distance distal to marker UT49 (D17S731). Survival analysis did not show a significant association between LOH on 17p and survival, although the sample size was too small to rule out a difference of less than 10-fold. Clinical risk factors were better prognostic indicators than LOH, with significantly prolonged survival in patients free of craniospinal metastasis following gross total tumor resection.

Treatment of mouse brain cytosol with dextran-coated charcoal and high salt does not reveal a new glucocorticoid binder.

Incubation of steroid-free whole mouse brain cytosol from adrenalectomized-ovariectomized mice with saturating concentrations of tritiated dexamethasone was found to label all Type I as well as all Type II adrenocorticosteroid receptors. The quantitative and brain regional distribution of residual dexamethasone binding in cytosols pre-treated with dextran-coated charcoal (DCC) and 300 mM KCl was indistinguishable from that for tritiated aldosterone-Type I receptor complexes under the same conditions. We therefore conclude that the dexamethasone binding sites remaining after DCC and KCl treatment of steroid-free brain cytosol are due to the presence of Type I receptors. The differential sensitivity of Type I and Type II receptors to the DCC/KCl treatment paradigm may be useful in the purification of Type I receptors.

Initial characterizations of nontransformed and transformed [3H ]aldosterone-type I receptor complexes in brain cytosol.

Incubation of [3H]aldosterone-type I receptor complexes in mouse brain cytosol with the chaotropic anion thiocyanate increased the fraction of receptors retained by DNA-cellulose from less than 10% to over 40%, whereas it decreased the fraction retained by protamine sulfate from more than 90% to less than 10%. Thiocyanate-induced transformation to the DNA-binding species was also accompanied by a 2.1-fold decrease in the rate of [3H]aldosterone dissociation from type I receptors as well as by an increase in the apparent positive charge and hydrophobicity of the surface of these receptors, as revealed by DEAE Bio-Gel ion exchange and pentyl agarose hydrophobic interaction chromatography. Sucrose density gradient sedimentation and Sephacryl S-300 gel exclusion chromatography revealed a reduction in the sedimentation coefficient and Stokes radius of the steroid-receptor complex from 9.6S and 8.0 nm before to 4.7S and 6.1 nm after transformation, respectively. These changes in hydrodynamic parameters were found to correspond to a 2.8-fold reduction in the apparent molecular mass from 331,000 before to 120,000 after transformation. In view of these various findings as well as the known differential affinity of protamine sulfate for the 90K heat shock protein, we suggest that thiocyanate-induced transformation is initiated by the dissociation of two molecules of heat shock protein from each steroid/DNA-binding type I receptor subunit.

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol.

Recently we reported that adding molybdate to crude steroid-free cytosol at 0 degree C results in a dose-dependent reduction in the binding of [3H]aldosterone ([3H]ALDO) to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30-50% increase in the subsequently measured maximal specific binding capacity (BMAX) of [3H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (Kd) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the Kd and over a 50% reduction in the subsequently measured BMAX of [3H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [3H]ALDO binding capacity compared with non-gel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22 degrees C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [3H]ALDO-Type I receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Further chemical differentiation of type I and type II adrenocorticosteroid receptors in mouse brain cytosol: evidence for a new class of glucocorticoid receptors.

There are at least two classes of intracellular receptors for adrenocorticosteroid hormones in brain. Type I receptors have a high affinity for the naturally occurring gluco- and mineralocorticoids, corticosterone (CORT) and aldosterone (ALDO), respectively, and a very low affinity for synthetic glucocorticoids such as dexamethasone (DEX). type II receptors have a high affinity for the synthetic glucocorticoids, a lower affinity for CORT and a very low affinity for ALDO. In recent studies with mouse brain cytosol we have found a number of other biochemical differences between these two receptor types. In the present study, brain cytosol from adrenalectomized mice was prepared in HEPES buffer and subjected to various potentially inactivating treatments prior to assessment of Type I and Type II receptor specific binding capacity by incubation for 24 h at 0 degrees C with [3H]ALDO +/- [1H]RU 26988 (to prevent or permit the cross-binding of [3H]ALDO to Type II receptors) or [3H]DEX +/- [1H]Prorenone (to prevent or permit the cross-binding of [3H]DEX to Type I receptors), respectively. These studies revealed that 10-20% of the high-affinity (Kd = 3 nM) [3H]DEX specific binding capacity remained even after extensive, high concentration and repeated pretreatments with dextran-coated charcoal (DDC. to remove endogenous sulfhydryl-reducing reagents and other biochemicals). These procedures had little effect on Type I receptors. Further analyses revealed that DCC-resistant [3H]DEX binders were not Type I receptors since they were not saturated by [1H]Prorenone. These binders were also not inactivated by aging steroid-free cytosol at 0 degree C or by treating it with buffers containing 0.3 M KCl. Since these

Effects of polyhydric and monohydric compounds on the stability of type I receptors for adrenal steroids in brain cytosol.

We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aldosterone ([3H]ALDO) in a time- and temperature-dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0 degree C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22 degrees C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 degrees C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0 degree C on the subsequent binding of [3H]ALDO yielded an inverse U-shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of the [3H]aldosterone-type I receptor complex to a DNA-binding species.

For most steroid receptor complexes, the transformation to a DNA-binding species can be achieved readily in vitro by incubation at elevated temperatures and/or salt concentrations. Although the aldosterone-Type I receptor complex forms a clear exception to this generalization, a marked increase in its transformation can be achieved by incubation with the chaotropic anion, thiocyanate. Time and concentration-response analyses with brain cytosol revealed that over 40% of the complexes were retained in DNA-cellulose assays after a 15 min pre-incubation at 0 degree C with 100 mM thiocyanate. As expected, molybdate prevented this transformation; however, in contrast to results with heat- and/or salt-induced transformation of other steroid receptors, the molybdate effect was only partially removed by gel filtering the cytosol prior to thiocyanate addition. Thiocyanate-induced transformation should prove useful in the biochemical characterization and purification of non-transformed and transformed aldosterone-Type I receptor complexes.

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol.

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.

Effects of metal ions and chelating agents on in vitro stability of glucocorticoid receptors in brain cytosol.

In vitro studies in a variety of tissues and cell types suggest that glucocorticoid receptor binding capacity is not static and that binding sites are subject to up- and down-regulatory mechanisms. The interpretation of such studies, however, is often complicated by factors affecting the stability of the receptor. This situation is particularly acute in the absence of ligand because of the increased lability of the unoccupied receptor. Studies reported here investigate effects of various metal ions and chelating agents on the stability of unoccupied [3H]dexamethasone binding sites in whole mouse brain cytosol. Variation in the ionic strength of cytosol, as created by the additions of various monovalent cations (Li+, Na+, K+, Rb+ and Cs+), was found to be an important factor affecting the increased stability of the receptor in vitro. Additions of divalent (Mg++, Ca++, Ba++, and Mn++) and trivalent (La , Cr and Al ) cations to cytosol, however, were generally found to produce a dose-dependent decrease in the stability of the unoccupied receptor. Additions of the chelating agents EDTA, EGTA and 1,10-phenanthroline to cytosol, resulted in differential, and sometimes complex, dose-dependent effects on receptor stability. The complex effects of various combinations of cations and the chelator EDTA were also investigated.

Efficacy of five methods for the bound-free separation of gluco- and mineralocorticoids from type I, II and III receptors found in hepes- and tris-buffered mouse brain cytosol.

We compared the efficacy of G-25 and LH-20 column chromatography, dextran-coated charcoal adsorption, and DEAE-cellulose and glass fiber filter disc assays to separate unbound steroids from three classes of brain cytosolic receptors prepared in HEPES and TRIS buffers and labeled selectively as follows: Type I = [3H]aldosterone + unlabeled RU26988, Type II = [3H]triamcinolone acetonide and Type III = [3H]corticosterone + unlabeled Prorenone and RU26988. Prorenone and RU26988 were added to reduce unwanted [3H]steroid binding to Type I and Type II receptors, respectively. In each case total, non-specific and specific binding and free steroid were compared individually. No single assay was found to be best for all three receptor classes, but both buffers and most assays could be used with appropriate correction factors. Variations between the results with different assays suggest fundamental differences between the three classes of adrenosteroid receptors and their ligands.

Increased binding to DNA-cellulose of the unactivated and activated glucocorticoid-receptor complex from mouse brain following sucrose density gradient ultracentrifugation.

The binding to DNA-cellulose of both the unactivated and activated forms of the molybdate-stabilized glucocorticoid-receptor complex increases markedly after subjecting these preparations to sucrose density gradient ultracentrifugation. We speculate that this increase results from the removal of endogenous macromolecular factors which competitively inhibit glucocorticoid receptor binding to DNA and which may normally be involved in regulating the genomic responses of these steroids in brain.