Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Cortisone-induced osteoporosis: effects on bone adenylate cyclase

The majority of patients with soft tissue or bone sarcomas of the upper extremity can be treated today with limb-saving procedures using combined modality therapies. For patients with a tumor in the shoulder area, sometimes an interscapulothoracic amputation is the only radical surgical treatment. However, in selected cases, in which the tumor does not involve the neurovascular bundle, a limb-sparing alternative might be the Tikhoff-Linberg resection. Normal function of the hand and forearm, with reasonable function of the elbow, can be maintained by this procedure. Four case histories are reported.     

Potentiation of a three drug chemotherapy regimen by radiation

The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.     

Reactivity of antibodies against conserved regions of pilins of Haemophilus influenzae type b

To identify epitopes on pilins of Haemophilus influenzae type b (Hib) that may also be immunologically available on assembled pili, antisera were developed against eight synthetic peptides that represent conserved and hydrophilic regions of Hib pilin. Seven of the eight peptides were immunogenic. Binding of the anti-peptide antibodies to purified pili of Hib strain Eagan was weak. However, when the purified pili were denatured by heating, binding of the anti-peptide antibodies improved considerably, suggesting that the epitopes defined by the peptides were more available for anti-peptide antibody binding on the denatured pilins than on purified pili. On Western blot analysis, strain variation was seen in the binding of some of the anti-peptide antibodies, notably those directed against peptides in the N-terminal half of the pilin. Thus, when pilins are assembled into pili, the epitopes defined by the seven immunogenic peptides appear to be altered so that binding of the anti-peptide antibodies is greatly reduced.     

A major isoform of the maize plasma membrane H(+)-ATPase: characterization and induction by auxin in coleoptiles

The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.     

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of angiotensin-converting enzyme gene I/D polymorphism with change in left ventricular mass in response to physical training

PURPOSE: To determine the radiologic characteristics of cystic dystrophy of the duodenal wall. MATERIALS AND METHODS: Ten patients with cystic dystrophy of the duodenal wall and chronic pancreatitis underwent ultrasonography (US) (n = 10), computed tomography (CT) (n = 10), endoscopic US (n = 5), and endoscopic retrograde cholangiopancreatography (ERCP) (n = 9). Cystic dystrophy of the duodenal wall was classified as either cystic or solid. The imaging findings were retrospectively analyzed and compared with findings at pancreatoduodenectomy (n = 10). RESULTS: The more frequent cystic type (n = 7) of cystic dystrophy of the duodenal wall was characterized by the presence of easily recognizable cystic lesions (diameter, more than 1 cm), located within the thickened wall of the second portion of the duodenum. The solid type (n = 3) of cystic dystrophy of the duodenal wall demonstrated fibrous thickening of the duodenal wall within which small cysts (diameter, less than 1 cm) were present. The intraduodenal cysts were usually elongated or bilobate with a thick wall. The thickening of the duodenal wall appeared as a solid layer between the duodenal lumen and the pancreas, hypoechoic at US, isoattenuating at unenhanced CT, and hypoattenuating in the early phase (after initiation of infusion of contrast material) and isoattenuating in the late phase (after completion of infusion) at contrast material-enhanced CT. Findings at retrospective analysis of CT and endoscopic US images were characteristic. CONCLUSION: Imaging modalities, notably CT and endoscopic US, helped establish the diagnosis of cystic dystrophy of the duodenal wall.     

Reassessment of cytochrome P450 2B2: catalytic specificity and identification of four active site residues

Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity. Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 --> 2B2 substitutions. To address these discrepancies, we have characterized two 2B2 variants. A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114. A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin. The expressed 2B2 variants metabolized most substrates at rates that were 1-9% of those of 2B1. When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16beta- and 16alpha-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme. Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF. On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity. In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.     

Resection of the right middle lobe and lingula for mycobacterial infection

BACKGROUND: In a series of 229 patients infected with mycobacterial organisms, we noted a specific female phenotype that involves isolated infections of the middle lobe and lingula. METHODS: Thirteen patients were found to have infections of the middle lobe, lingula, or both. All of them were infected with Mycobacterium other then Mycobacterium tuberculosis, all were women, 12 of the 13 were slender, and most had variable combinations of skeletal abnormalities. All underwent resection of the middle lobe, lingula, or both. RESULTS: There were no operative deaths. Only 2 patients have had reactivation requiring additional antibiotic therapy. All patients have had a decreased number of pulmonary infections in the postoperative period. Anatomic findings at operation included a complete major fissure and at least a partially complete minor fissure with middle lobe resections or an elongated lingula. CONCLUSIONS: Mycobacterial infection of the middle lobe and lingula is primarily a disease of asthenic women and is often associated with skeletal abnormalities and complete fissures or an elongated lingula. We recommend that surgical intervention be performed early once the condition is identified.