The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1alpha.

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1alpha in respiratory-deficient RCC4-rho0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O(2)(-) in combination with NO to destabilize HIF-1alpha was corroborated in RCC4-rho0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1alpha required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O(2)(-) blocked HIF-1alpha degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O(2)(-) coformation triggers a calcium increase that activates calpain to degrade HIF-1alpha, independently of the proteasome.

An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone beta subunit gene.

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the alpha and LHbeta subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LHbeta promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LHbeta promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line alphaT3-1. The functional role of the promoter-distal site in regulating the full-length bovine LHbeta promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LHbeta promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.

The antiparkinsonian drug budipine binds to NMDA and sigma receptors in postmortem human brain tissue.

We have estimated the affinity of the antiparkinsonian drug budipine to the PCP binding site of the NMDA receptor and to sigma 1 binding sites in membranes from postmortem human brain frontal cortex. The affinity of budipine to both binding sites is in a concentration range that may be reached under therapeutic conditions (Ki-values of about 12 and 2 microM at the PCP and sigma 1 binding site, respectively). Interactions with NMDA receptors as well as sigma 1 binding sites may contribute to the antiparkinsonian effects of budipine.

Sequence of changes in body composition induced by testosterone and reversal of changes after drug is stopped.

OBJECTIVE: --To study the changes in body composition produced by large doses of testosterone and reversal of changes when the drug is discontinued. DESIGN: --Weekly injections of testosterone enanthate were given to young adult male volunteers for 12 weeks. Repeated assays of lean body mass (LBM) by potassium 40 counting were made during this period and at intervals during the ensuing 5 to 6 months. PARTICIPANTS AND SETTING: --Subjects who were living on their own, who were known to be free of significant disease, and who volunteered as controls for a study of patients with neuromuscular disease. Assays were done in the Clinical Research Center. MAIN OUTCOME MEASURES: --Changes in body weight, LBM, and (by subtraction) body fat. RESULTS: --Testosterone treatment produced a progressive increase in LBM and a progressive decrease in body fat. Body composition reverted slowly toward normal when the injections were stopped; thus, the effects of testosterone lingered for some time. The magnitude of the observed changes in LBM was in keeping with the change in urinary creatinine excretion reported for these same subjects. CONCLUSION: --Testosterone is a powerful anabolic agent that also serves to reduce body fat content.

Length of stay in a general hospital psychiatric unit.

Fifty psychiatric inpatients with a prolonged length of stay were compared to 50 control admissions for factors associated with prolonged hospitalizations in a general hospital. Seven variables were found to be significantly overrepresented among the long stayers, including treatment with electroconvulsive therapy, medical consultations, underemployment, dementia, disposition to a place other than home, absence of alcohol or drug abuse, and presence of psychosis without affective symptoms. The clinical and policy implications of these finding are discussed.

Enhancement of mammary carcinogenesis by high levels of dietary fat: a phenomenon dependent on ad libitum feeding.

Female 55-day-old Sprague-Dawley rats were treated with a single intravenous dose of 7,12-dimethylbenzanthracene (DMBA), 2 mg/100 g of body weight each. At 60 days of age, the rats were divided into four dietary groups (41-42 rats/group):I, 5% corn oil diet fed ad libitum; II, 20% corn oil diet fed ad libitum; III, 5% corn oil diet fed 12% less than group I; and IV, 20% corn oil diet fed 12% less than group II. The 5% and 20% corn oil diets were purified semisynthetic diets that were isonutrient on a caloric basis. All animals were housed individually in single cages; food consumption of each animal was computed daily throughout the study. Sixteen weeks after carcinogen treatment, mean numbers of mammary carcinomas per rat (+/- SE) in groups I, II, III, and IV were 4.1 +/- 0.6, 6.8 +/- 0.7, 3.0 +/- 0.3, and 4.1 +/- 0.5, respectively. Mean weight of mammary carcinomas per rat (g +/- SE) in groups I, II, III, and IV were 3.5 +/- 0.7, 8.0 +/- 1.3, 3.0 +/- 1.1, and 4.6 +/- 1.3, respectively. Mammary carcinoma number and weight were significantly (P less than .01) increased in the animals fed the 20% corn oil diet ad libitum when compared with those fed the 5% corn oil diet ad libitum; however, no significant differences in mammary tumor number or weight were observed between the animals fed a restricted, 20% corn oil diet and those fed a restricted, 5% corn oil diet. The study involving the animals fed the 12%-restricted diets was repeated (38-42 rats/group), with virtually identical results, i.e., the mean number of mammary carcinomas per rat in the groups fed the restricted 5% fat and 20% fat diets at termination of the study was 3.1 +/- 0.4 and 3.7 +/- 0.3, respectively, and the mean weight (g) of mammary carcinomas per rat was 4.3 +/- 1.2 and 4.0 +/- 1.1, respectively (no significant differences). Thus, high levels of dietary fat can significantly enhance mammary carcinogenesis in female rats, but only in animals on an ad libitum feeding protocol. A slight restriction in amount consumed (12% less than ad libitum) abolished the mammary carcinogenic differential between a high-fat and a low-fat diet.

Mechanism of muscle wasting in myotonic dystrophy.

Myotonic dystrophy is associated with progressive muscular atrophy. In order to determine the mechanism of muscle wasting in this condition, we measured fractional mixed skeletal muscle protein synthesis in the postabsorptive state in 8 patients with myotonic dystrophy, and compared the results with those of 10 normal subjects. Fractional muscle protein synthesis was determined by measuring the increment of 13C leucine in mixed skeletal muscle protein obtained by needle biopsy from the quadriceps muscle during a primed-continuous infusion of L-(1-13C) leucine. We used plasma 13C alpha-ketoisocaproate (representing intracellular leucine labeling) as the precursor pool for the calculation of fractional muscle protein synthesis and leucine kinetics. Fractional muscle protein synthesis was depressed in the patients with myotonic dystrophy (28% decrease, p less than 0.02). Leucine flux, leucine oxidation, and the nonoxidative portion of leucine flux were not different between the patients with myotonic dystrophy and the normal control subjects. Muscle atrophy in myotonic dystrophy reflects a selective decrease in muscle protein synthesis without any similar decrease in nonmuscle protein synthesis. This decrease may result from an impaired end-organ response to anabolic hormones or substrates.

Randomized controlled trial of testosterone in myotonic dystrophy.

Because testosterone has an anabolic effect in myotonic dystrophy, we conducted a 12-month, randomized, double-blind therapeutic trial of testosterone enanthate (3 mg/kg/wk) in 40 men with myotonic dystrophy. We evaluated strength by manual muscle tests, quantitative myometry, pulmonary function, and quantitative functional assessment. A sustained, significant elevation of testosterone levels was produced but there was no effect on any measurement of muscle strength. Muscle mass as estimated by creatinine excretion and lean body mass (40K method) increased significantly. We conclude that testosterone does not improve strength in myotonic dystrophy despite increasing muscle mass.

Effect of testosterone on muscle mass and muscle protein synthesis.

We have studied the effect of a pharmacological dose of testosterone enanthate (3 mg.kg-1.wk-1 for 12 wk) on muscle mass and total-body potassium and on whole-body and muscle protein synthesis in normal male subjects. Muscle mass estimated by creatinine excretion increased in all nine subjects (20% mean increase, P less than 0.02); total body potassium mass estimated by 40K counting increased in all subjects (12% mean increase, P less than 0.0001). In four subjects, a primed continuous infusion protocol with L-[1-13C]leucine was used to determine whole-body leucine flux and oxidation. Whole-body protein synthesis was estimated from nonoxidative flux. Muscle protein synthesis rate was determined by measuring [13C]leucine incorporation into muscle samples obtained by needle biopsy. Testosterone increased muscle protein synthesis in all subjects (27% mean increase, P less than 0.05). Leucine oxidation decreased slightly (17% mean decrease, P less than 0.01), but whole-body protein synthesis did not change significantly. Muscle morphometry showed no significant increase in muscle fiber diameter. These studies suggest that testosterone increases muscle mass by increasing muscle protein synthesis.

Lack of relationship of hypogonadism to muscle wasting in myotonic dystrophy.

Myotonic dystrophy is frequently associated with testicular atrophy. Since androgens may play a role in the maintenance of muscle mass, we have studied the levels of plasma testosterone and gonadotropins and of urinary 17-ketosteroids in 22 men with myotonic dystrophy, 36 normal men, and 16 men (control group) with muscle wasting. Results were correlated with muscle mass as estimated by creatinine excretion and total body potassium. Patients with myotonic dystrophy had significantly lower testosterone and higher gonadotropin levels than normal, and these changes were progressive in longitudinal studies. Testosterone levels were also lower than normal in disease control subjects. There was no correlation between low testosterone levels and diminished muscle mass in either myotonic dystrophy or disease control patients. The low plasma concentration of testosterone in men with myotonic dystrophy and other neuromuscular diseases does not appear to be directly related to their muscle wasting. This study does not exclude the possibility that an alteration in testosterone receptor or tissue effects may contribute to a loss of muscle tissue.

Myotonic dystrophy: effect of testosterone on total body potassium and on creatinine excretion.

Muscle wasting in myotonic dystrophy appears to reflect impaired anabolism rather than accelerated catabolism. We therefore investigated the effects of testosterone, an anabolic hormone, on muscle mass as estimated by creatinine excretion and total body potassium in nine patients with myotonic dystrophy. Weekly injections of testosterone for 10 to 13 weeks increased both creatinine excretion (19%) and total body potassium (16%) in all patients. Metabolic balance data showed a confirmatory accretion of nitrogen, potassium, and phosphorus. Because testosterone increases indirect measures of muscle mass, it may deserve a therapeutic trial in myotonic dystrophy.

Long term outcome in working and homemaking alcoholic women.

Retrospective and outcome data were examined for 48 homemaking and 24 working women who came for treatment for alcoholism to an Addiction Recovery Unit at an inpatient psychiatric hospital. Outcome was assessed at the end of each of 4 years following treatment via personal interview by an experienced social worker and was corroborated by at least one source. At admission, the homemakers had been drinking for significantly more years (p less than 0.05) than working women. Occupation at admission did not directly relate to outcome. However, for a small subgroup comprised of both homemaking and working women, a change in occupation following treatment was associated with improvement.

The cloak of competence: after two decades.

Fifteen mildly retarded persons originally studied in 1960-1961, and restudied in 1972-1973, were studied again in 1982. The quality of their lives over the past decade was reexamined with an emphasis on personal and social resources for coping with chronic or acute stress. We found that these persons were less dependent on others than they had been previously. Moreover, compared to other aging mentally retarded persons described in the literature, these persons were more hopeful, confident, and independent, despite ill health, stressful life events, and the lack of assistance from mental retardation service agencies.

The assessment of muscle mass in progressive neuromuscular disease.

We performed sequential studies of two methods used to estimate muscle mass in 34 patients with progressive neuromuscular disease for periods of up to 52 months. Creatinine excretion and total body potassium were low at the outset in virtually all patients. Creatinine excretion continued to decline in most patients, but total body potassium did not decline significantly. Creatinine excretion may measure declining muscle mass more accurately than total body potassium, but both measurements can estimate muscle mass for metabolic studies or therapeutic trials.

Decreased insulin sensitivity of forearm muscle in myotonic dystrophy.

Previous studies of patients with myotonic dystrophy have demonstrated hyperinsulinism after glucose loading. This hyperinsulinism has been attributed by some investigators to tissue insulin resistance. We have directly studied insulin sensitivity of forearm muscle in patients having such hyperinsulinism. The effect of an intrabrachial arterial insulin infusion (100 mu U/kg per min) on glucose uptake was determined in six cases of myotonic dystrophy, six normal subjects, and in seven disease control subjects with myotonia or wasting from other disorders. There was no significant difference in insulin tolerance comparing myotonic dystrophy patients to the normal and disease control groups. Glucose tolerance and basal insulin levels were normal in the myotonic dystrophy patients, but hyperinsulinism occurred after glucose ingestion. After 25 min of intra-arterial insulin, the mean peak muscle glucose uptake in myotonic dystrophy was 2.54 +/- 0.54 mu mol/min per 100 ml forearm compared to 5.24 +/- 0.86 mu mol/min per 100 ml for disease controls (P is less than 0.05). Myotonic dystrophy patients showed a peak glucose uptake increment of only 2.6 +/- 0.2-fold over basal contrasted with the disease control value of 6.5 +/- 1.0-fold (P is less than 0.02) and the normal control value of 8.8 +/- 1.1-fold (P is less than 0.01). Thus, there was an absolute as well as a relative decrease in muscle insulin sensitivity in myotonic dystrophy patients compared to both control groups. The peak increments in arterio-superficial venous glucose concentration differences after insulin infusion were not significantly different comparing myotonic dystrophy and control groups. These data suggest that in myotonic dystrophy, there is insulin insensitivity of skeletal muscle.

Distal myopathy: electron microscopic and histochemical studies.

This report describes the clinical, laboratory, and muscle biopsy histochemical and electron microscopic studies of one inherited and two sporadic cases of distal myopathy. Histopathologic and histochemical studies showed numerous myopathic alterations and no significant evidence of denervation. Electron microscopic studies showed a broad spectrum of nonspecific alterations similar to those in other forms of muscular dystrophy. Autophagic vacuoles were prominent in all cases. The inherited case was characterized by an unusual focal granular degeneration that, ultrastructurally, was composed of homogeneous fine granules devoid of other organelles or myofilamens.

A Bodian method for mounted frozen sections.

For application of the Bodian method to frozen sections, cut frozen peripheral nerve or muscle at 10 mum and mount. Fix for 4 days in 18 parts 80% ethanol, 1 part 10% formalin, and 1 part glacial acetic acid. Fix central nervous tissue in the same mixture prior to freezing and sectioning, and after mounting postfix for 4 days. Impregnate by the Bodian procedure. The results equal Bodian stains of paraffin sections. The technique is simple and reliable. The use of 10 mum frozen sections produces little artifact and allows alternate serial sections to be stained with other techniques.