Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors.

The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.

Adjuvant chemotherapy for high-risk squamous-cell carcinoma of the head and neck.

A prospective clinical trial was developed to evaluate efficacy, toxicity, and patient compliance to adjuvant chemotherapy following surgery and postoperative radiation therapy in patients with squamous-cell carcinoma of the head and neck with extracapsular spread of tumor in cervical metastases. Following postoperative radiation therapy, 18 courses of methotrexate (MTX) and 5-fluorouracil (5-FU) were administered over 6 months. Fifty patients were registered. A total of 771 doses were administered. Dose reduction was required 72 times. Therapy was stopped in one patient (2%) because of toxicity. Three patients (6%) refused to complete the adjuvant therapy. Adjusted 2-year no evidence of disease (NED) survival is 66%. This study demonstrates that patients with advanced squamous-cell carcinoma of the head and neck can undertake an aggressive program of adjuvant MTX/5-FU with acceptable compliance and toxicities. Preliminary data generated in this nonrandomized study support the call for a prospective randomized multiinstitutional trial of this program.

Preliminary crystallographic studies of the ligand-binding domain of the thyroid hormone receptor complexed with triiodothyronine.

A truncated, recombinant form of the thyroid hormone receptor, including the hormone binding domain, has been co-crystallized with the hormone T3. The crystals are monoclinic, most likely space group P2, with two molecules per asymmetric unit and cell dimensions a = 63.6 A, b = 80.8 A, c = 100.9 A and beta = 92.1 degrees. The crystals diffract to only medium resolution and decay rapidly in the X-ray beam using laboratory sources. By contrast, high resolution, high-quality data are obtained using synchrotron radiation in conjunction with cryocrystallography.

A novel TR beta mutation (R383H) in resistance to thyroid hormone syndrome predominantly impairs corepressor release and negative transcriptional regulation.

Resistance to thyroid hormone (RTH) is characterized by elevated serum thyroid hormones, failure to suppress pituitary TSH secretion, and variable T3 responsiveness in peripheral tissues. The disorder is associated with diverse mutations that cluster within three areas of the thyroid hormone beta(TR beta) receptor. Here, we report a novel RTH mutation (R383H), which is located in a region not known to harbor naturally occurring mutations. Although the R383H mutant receptor activated positively regulated genes to an extent comparable to wild-type (WT), negative transcriptional regulation of human TSH alpha and TRH promoters was impaired in either TR beta 1 or TR beta 2 contexts, and WT receptor function was dominantly inhibited. T3-dependent changes in basal transcription with R383H were also impaired: on the TRH promoter, basal activation by unliganded R383H was not reversed by T3 to the same extent as WT; similarly transcriptional silencing by an unliganded Gal4-R383H fusion was not relieved at a T3 concentration that derepressed WT. In keeping with this, ligand-dependent corepressor release by R383H, either in a protein-protein interaction assay or as a DNA-bound heterodimer with retinoid X receptor on either positive or negative thyroid hormone response elements, was disproportionately impaired relative to its ligand-binding affinity, whereas its T3-dependent recruitment of coactivator was unimpaired. These properties were shared by another previously described RTH mutant (R429Q), and in the crystal structure of TR alpha the homologous residues interact in a polar invagination. Our data indicate a role for these residues in mediating negative transcriptional regulation and facilitating corepressor release and suggest that predominant impairment of these functions may be the minimal requirements for causation of RTH.

Hormone selectivity in thyroid hormone receptors.

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRbeta) for Ser (TRalpha). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRbeta receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.

Mastocytosis: diverse presentations and outcomes.

Within the general category of mastocytosis lies an array of clinical presentations with differing prognostic implications. We report 3 cases of systemic mastocytosis distinguished by novel aspects of the disease. Case 1 documents the first successful orthotopic liver transplantation in a patient with mastocytosis; case 2 depicts a potential hereditary component of mastocytosis; and case 3 documents the progression of mastocytosis with hematologic abnormality to mast cell leukemia. Future investigations, such as the early definition of c-kit receptor mutations, may provide additional insight as to the molecular basis for this heterogeneous disease and guidance for prognostic implications and targeted therapies.

Right and left ventricular compliance in the hereditary cardiomyopathy of the Syrian hamster.

Ventricular pressure-volume curves were examined in 10 pre-oedematous cardiomyopathic Syrian hamsters, aged 120 d, and 10 oedematous cardiomyopathic hamsters, aged 210 d, and compared with 10 and 8 age-matched controls, respectively. Previous studies had shown filling pressures and cardiac output to be normal in the pre-oedematous stage. In contrast, the oedematous stage was characterised by elevated filling pressures and increased cardiac output, raising the question whether this stage represents true myocardial failure or circulatory congestion associated with decreased ventricular compliance. Compliance, defined as dV/dP, was derived simulataneously for both ventricles from post-mortem pressure-volume curves from 0 to 2.66 kPa (0 to 20 mmHg). Left ventricular tissue elastic modulus, E, defined as dsigma/depsilon or the incremental stress (sigma) for an increment of strain (epsilon), was derived from the compliance curves and certain linear dimensions of the heart. At isobaric intervals, compliance did not differ between control and myopathic ventricles. At pressures corresponding to previously measured, naturally prevailing end-diastolic pressures, the myopathic ventricles were significantly dilated in both stages; end-diastolic compliance was normal in pre-oedematous hamsters, but was significantly decreased in the oedematous animals. Left ventricular elastic modulus was elevated at all values of sigma in both groups of myopathic animals.

Murumuru (Astrocaryum murumuru) meal as an additive to elephant grass silage / Torta de murumuru (Astrocaryum murumuru) como aditivo en el ensilado de pasto elefante / Torta de murumuru (Astrocaryum murumuru) como aditivo em silagens de capim elefante

Abstract Background: Tropical grasses, such as elephant grass, have high moisture content during its ideal phenological state for silage. High moisture content hinders proper preservation and reduces the nutritive value of silage due to secondary fermentation and production of effluents. Adding feed materials with high dry matter content, such as murumuru (Astrocaryum murumuru) meal, is a potential alternative to improve silage yield. Objective: To determine the effects of including murumuru meal (0, 7, 14, 21, and 28%) on the fermentative characteristics, microbiological activity, aerobic stability, and chemical composition of elephant grass silages. Methods: A completely randomized design with five treatments and five replicates was used. Elephant grass was collected at 60 d of age, minced, and homogenized with murumuru meal. The mass was placed in experimental 15-L silos. The silos were collected and analyzed 45 d later. Results: Effluent production decreased (p<0.05) as the proportions of murumuru meal in silage increased. A quadratic effect (p<0.05) was observed on dry matter recovery. An increase (p<0.05) was observed in dry matter content, a decrease (p<0.05) in the neutral detergent fiber content, and an increase (p<0.05) in the non-fibrous carbohydrate content with the inclusion of murumuru meal. Conclusions: Addition of murumuru meal improves chemical composition and does not affect the fermentative characteristics of elephant grass silage, while it reduces effluent losses. Nevertheless, the inclusion of murumuru meal in the elephant grass silage decreased the time of aerobic stability.

Resumen Antecedentes: los pastos tropicales, tales como el pasto elefante, tienen alto contenido de humedad cuando están en su estado fenológico ideal para ensilar. Esto dificulta su adecuada preservación en el silo, reduciendo el valor nutritivo del ensilaje debido a fermentaciones secundarias y generación de efluentes. Una posible solución sería incluir materiales con alto contenido de materia seca, tales como la torta de murumuru (Astrocaryum murumuru). Objetivo: determinar el efecto de la inclusión de torta de murumuru (0; 7; 14; 21 y 28%) sobre las características fermentativas, microbiológicas, estabilidad aeróbica y composición química de los ensilajes de pasto elefante. Métodos: se utilizó un diseño completamente al azar, con cinco tratamientos y cinco repeticiones. El pasto elefante fue colectado a los 60 días de edad, luego picado y homogeneizado con la torta de murumuru. La masa fue colocada en silos experimentales con capacidad de 15 L. Luego de 45 días de ensilado, los silos fueron abiertos y las muestras fueron colectadas para su posterior análisis. Resultados: hubo reducción (p<0,05) en la producción de efluentes a medida que se incrementó la proporción de torta de murumuru en el ensilado. Se observó un efecto cuadrático (p<0,05) en la recuperación de materia seca. Hubo aumento (p<0,05) en los contenidos de materia seca con la adición de torta de murumuru. Se observó disminución (p<0,05) en el contenido de fibra detergente neutra y aumento (p<0,05) de carbohidratos no fibrosos. Conclusión: La adición de torta de murumuru mejora la composición química, reduce las perdidas por efluentes y no afecta las características fermentativas de ensilado de pasto elefante. Sin embargo, la inclusión de la torta de murumuru en el ensilado de pasto elefante disminuye el tiempo en estabilidad aeróbica.

Resumo Antecedentes: capins tropicais, como por exemplo o capim-elefante apresentam alto teor de umidade quando possuem produtividade compatível e estão no estádio fenológico adequado para ensilagem. Isso dificulta a adequada preservação no silo, ocasionando a redução do valor nutritivo da silagem devido a fermentações secundárias e a produção de efluentes. Uma possível solução seria o uso de aditivos com alto teor de matéria seca, como bolo de murumuru (Astrocaryum murumuru). Objetivo: determinar o efeito da inclusão da torta de murumuru (0; 7; 14; 21 e 28%) na ensilagem de capim-elefante sobre as características fermentativas, microbiológicas, estabilidade aeróbia e a composição química das silagens. Métodos: utilizou- se um delineamento inteiramente casualizado, com cinco tratamentos e cinco repetições. O capim-elefante foi colhido aos 60 dias de idade, o mesmo foi picado e homogeneizado à torta de murumuru. A massa foi ensilada em silos experimentais com capacidade de 15 L. Após 45 dias de ensilagem, os silos foram abertos e amostras foram coletadas para posteriores análises laboratoriais. Resultados: houve redução (p<0,05) na produção de efluentes à medida que se elevaram as proporções da torta de murumuru na ensilagem. Observou-se efeito quadrático (p<0,05) na recuperação de matéria seca. Houve aumento (p<0,05) nos teores de matéria seca com a adição da torta murumuru. Observou-se diminuição (p<0,05) nos teores de fibra em detergente neutro e aumento (p<0,05) nos teores de carboidratos não fibrosos. Conclusão: torta de murumuru como aditivo melhora a composição química, reduz perdas principalmente por efluente e não afeta as características fermentativas de silagens de capim- elefante. Entretanto, a inclusão da torta de murumuru na ensilagem de capim-elefante diminui o tempo em estabilidade aeróbia.

Antipsychotic-withdrawal akathisia versus antipsychotic-induced akathisia: further evidence for the existence of tardive akathisia.

The authors present a study in which 33 chronic schizophrenic patients who, when withdrawn from antipsychotic drug treatment for more than 2 weeks, presented with concurrent signs of akathisia and tardive dyskinesia; however, signs of akinesia, facial masking, rigidity, or dystonia were not concurrent with the patients' akathetic presentation. In a subsequent study phase, these patients were treated with antipsychotics for up to 6 weeks. The dyskinetic signs that had been dramatically more severe in those patients exhibiting akathisia following withdrawal from antipsychotic medication continued for up to 6 weeks following the renewal of antipsychotic drug therapy. These findings help to confirm a relationship between tardive dyskinesia and a persistent akathisia of later onset known as tardive akathisia.

Lymphocyte monoamine oxidase and plasma prolactin and growth hormone in tardive dyskinesia.

Twelve elderly women with tardive dyskinesia were matched with 12 patients without dyskinesia. Lymphocyte monoamine oxidase (MAO) activity and plasma prolactin and growth hormone concentrations were determined "blind" in these 12 pairs of patients. Chronic schizophrenic patients with tardive dyskinesia had significantly lower lymphocyte MAO activity as compared to controls. Organic brain syndrome patients with dyskinesia did not differ from controls in the lymphocyte MAO activity. These results with lymphocyte MAO parallel our earlier findings on platelet MAO. No significant differences were found between dyskinesia group and controls in the plasma prolactin and growth hormone concentrations. Possible implications of our findings are discussed.

Serum neuroleptic concentrations and tardive dyskinesia.

Using a liquid chromatographic assay, we measured serum neuroleptic concentrations in eight middle-aged or elderly female inpatients with tardive dyskinesia (TD) and eight controls. All 16 patients were receiving either thioridazine or mesoridazine at stable doses. TD patients were found to have a significantly higher ratio of serum concentration to daily dose of neuroleptics compared with controls. A 1-year follow-up revealed that this ratio did not change appreciably in those patients who continued to receive neuroleptics. Differences in serum neuroleptic levels were not related to peripheral inflammatory activity as indicated by serum alpha-1-acid glycoprotein concentrations. Of the various thioridazine metabolites, sulforidazine (which is reportedly the most potent in terms of affinity for dopaminergic and alpha-noradrenergic receptors) seemed to be significantly elevated in the serum of TD patients as compared with non-TD patients. Our data suggest a need for further pharmacokinetic investigations to study neuroleptic metabolism in patients with TD.

X-ray crystallographic and functional studies of thyroid hormone receptor.

We have solved several X-ray crystallographic structures of TR ligand-binding domains (LBDs), including the rat (r) TR alpha and the human (h) TR beta bound to diverse ligands. The TR-LBD folding, comprised mostly of alpha-helices, is likely to be general for the superfamily. The ligand, buried in the receptor, forms part of its hydrophobic core. Tight fitting of ligand into the receptor explains its high affinity for the TR, although the structure suggests that ligands with even higher affinities might be generated. The kinetics of 3,5,3'-triiodo-L-thyronine (T3) and 3,5,3',5'-tetraiodo-L-thyronine (T4) binding suggest that folding around the ligand, rather than receptor opening, is rate-limiting for high affinity binding. TR beta mutations in patients with resistance to T3 cluster around the ligand; these different locations could differentially affect on other receptor functions and explain the syndrome's clinical diversity. Guided by the structure, mutations have been placed on the TR surface to define interactions with other proteins. They suggest that a similar surface in the LBD is utilized for homo- or heterodimerization on direct repeats and inverted palindromes but not on palindromes. Coactivator proteins that mediate TR transcriptional activation bind to a small surface comprised of residues on four helices with a well-defined hydrophobic cleft, which may be a target for pharmaceuticals. The coactivator-binding surface appears to form upon ligand-binding by the folding of helix 12 into the scaffold formed by helices 3, 4 and 5. The analysis of most currently used antagonists suggest that although they probably fit into the ligand-binding pocket, they possess a group that may alter proper folding of the receptor, with disruption of the coactivator-binding surface (the 'extension model').

Neuropathological studies of schizophrenia: a selective review.

Over the past 20 years, there has been a relatively quiet but persistent effort to investigate anatomical neuropathology in schizophrenia. This effort has involved post-mortem histopathology using novel preparation procedures, pneumoencephalography, and computed tomography. The bulk of this research is critically reviewed here. The evidence presented suggests that the brains of schizophrenic patients frequently contain abnormalities. The limbic region is especially likely to show pathological changes. However, the changes are variable and nonspecific. The implications of these findings are discussed in terms of our limited knowledge of the clinical manifestations of subtle limbic pathology. It is concluded that pathology of limbic regions is associated with schizophrenia and that the more gross the pathology, the more neurologically impaired are the patients.

Serum haloperidol concentration and clinical response in schizophrenia.

Previous studies have reported a therapeutic window (i.e., a curvilinear relationship between clinical response and drug level) for haloperidol concentrations in serum or plasma. The authors treated 30 acutely decompensated schizophrenic inpatients with a fixed dose of haloperidol (.4 mg/kg/day). After 6 weeks there was no statistically significant correlation between clinical improvement and serum haloperidol concentration. Fifteen subjects with serum concentrations of 5-15 ng/ml did not differ in clinical improvement compared with 15 subjects who had concentrations above 15 ng/ml. These data are consistent with a therapeutic plateau, rather than a window, and suggest that in most cases there is no clinical advantage to the use of haloperidol doses greater than approximately 30 mg/day in schizophrenic patients.

Cisplatin--5-fluorouracil chemotherapy for advanced inoperable squamous carcinoma of the head and neck.

Patients with locally advanced, inoperable squamous cell carcinoma of the head and neck were offered three courses of cisplatin and 96-h 5-fluorouracil (5-FU) infusion. Subsequent therapy included surgery when feasible, irradiation therapy, and a maintenance program of methotrexate (MTX)-5-FU. Thirty-three patients were evaluated prospectively. Seven patients underwent a single course of chemotherapy. Five patients underwent two courses of chemotherapy. Twenty-one patients underwent three courses of adjuvant chemotherapy. The overall response rate was 48% (16 of 33). Fifteen of 21 patients (76%) receiving three courses of chemotherapy evidenced a response; this included three complete responses (CRs) (9%). No responses were seen in patients receiving only one or two courses of chemotherapy. Among responding patients, the initial favorable response to chemotherapy was apparent after the first course of chemotherapy. Patients who failed to demonstrate any response after two courses of chemotherapy did not respond after a third course. A significant group of patients fail to respond and should be offered participation in other investigational protocols as they become available.

3-carboethoxy-beta-carboline (beta-CCE) elicits electroencephalographic seizures in rats: reversal by the benzodiazepine antagonist CGS 8216.

Intravenous administration of 3-carboethoxy-beta-carboline (beta-CCE, 10 mg/kg) to rats resulted in multiple bursts of rhythmic waves (2-4 second duration, 5-7 Hz) with amplitudes of 100-250 microV. Pretreatment of animals with the benzodiazepine receptor antagonists CGS 8216 prevented the electroencephalographic seizures elicited by beta-CCE. This dose of CGS 8216 did not produce any electroencephalographic abnormalities when administered alone. These observations suggest that the electroencephalographic seizures elicited by beta-CCE are mediated via an interaction with benzodiazepine receptors. An in vitro study of the rate of degradation of beta-CCE and 3-carbomethoxy-beta-carboline (beta-CCM) in rat plasma demonstrated that the rate of degradation of the former compound was three times more rapid than the latter. These observations, taken together with previous studies demonstrating that parenteral administration of beta-CCM elicits tonic and clonic seizures, suggests that pharmacokinetic factors may be involved in defining the pharmacologic profile of beta-carboline-3-carboxylic acid esters.

Flow cytometric evaluation of adenoid cystic carcinoma: correlation with histologic subtype and survival.

Adenoid cystic carcinoma is an unusual but capricious tumor of salivary gland origin. Characteristically, these tumors follow a relentless clinical course, although some patients experience prolonged disease-free survival. Tumor size, site, and grade have been shown to correlate with tumor behavior. Recent investigation by others has suggested that DNA ploidy as determined by flow cytometry may provide an additional biologic marker of tumor behavior. This study was undertaken to investigate the relationship of DNA ploidy to tumor grade, biologic behavior, and patient outcome. A retrospective comparison of flow cytometric evaluations of paraffin-embedded formalin-fixed tumor specimens with patient outcome and histopathologic grade was undertaken. Follow-up of 4 to 7 years in 26 patients confirmed that the presence of a solid histologic component in the tumor correlated with the presence of recurrent or persistent disease (p = 0.04). Twenty-two of 28 tumors (78%) were found to be aneuploid on at least one section. Comparison of DNA ploidy with either patient outcome or the presence of a solid component did not achieve statistical significance, although a trend was suggested. This study confirms previous studies demonstrating the validity of histopathologic evaluation of tumor grade in the prediction of the biologic behavior of adenoid cystic carcinoma. However, our findings suggest that DNA ploidy has only limited value as an additional marker of tumor behavior in this patient population.

A probable neuroleptic effect on platelet monoamine oxidase in chronic schizophrenic patients.

Platelet monoamine oxidase activity (MAO) was studied serially over time in 16 chronic schizophrenic patients when medication free and then when medicated. Thirteen of the 16 patients had significant decreases in platelet MAO activity following neuroleptic drug treatment. The change in MAO activity was found to be correlated with response to treatment and to dose of medication.

Etiologic factors in head and neck wound infections.

The use of antibiotic prophylaxis does not prevent all postoperative wound infections. The records of 245 patients undergoing major head and neck surgery were reviewed to determine the cause of wound infection which developed in 17 patients. Patients with Stage IV tumors had an increased infection rate as compared to lesser stages (p less than .01). Reconstruction with a myocutaneous flap was associated with an increased infection rate when compared to primary closure or split-thickness skin graft reconstruction (p less than .001). Probable errors in surgical technique were identified in 10 of 17 infected patients. Neither weight loss, diabetes mellitus, prior radiation therapy, nor prior tracheotomy were found to increase the risk of wound infection (p greater than .05). This study lends insight into factors that may potentiate the development of wound infection despite antibiotic prophylaxis. A strategy to minimize postoperative infection is offered.

SCC-RIA in the diagnosis of squamous cell carcinoma of the head and neck.

A reliable circulating tumor marker, appropriate for head and neck malignancy, is not yet available. This manuscript reports the efficacy of using circulating squamous cell carcinoma antigen in evaluating patients with squamous cell carcinoma of the head and neck. Serum samples from 89 patients with squamous cell carcinoma of the head and neck were obtained before treatment and at intervals following treatment. Squamous cell carcinoma antigen levels were determined by radioimmunoassay. Elevated pretreatment serum levels of squamous cell carcinoma antigen were identified in 39 of 89 patients (44%) who had head and neck squamous cell carcinoma. Of the 16 patients evaluated from this group with recurrent or persistent disease, 15 (93.5%) demonstrated elevated levels of squamous cell carcinoma antigen. However, of the 21 evaluable patients who remained disease free, only 10 (48%) had post-therapy levels within the normal range. The possible use of this marker in aiding the clinical follow-up of head and neck cancer patients is discussed.