Buspirone metabolite structure profile using a standard liquid chromatographic-mass spectrometric protocol

A rapid and systematic LC-MS protocol is utilized to profile buspirone metabolites. Analysis of rat bile, urine and liver S9 samples using a standard LC-MS method provides structural information for 25 metabolites. The resulting buspirone metabolite structure database contains characteristic retention time, molecular mass and MS-MS product ion information for each compound. Metabolites are categorized according to profile groups, which illustrate that substitution reactions are primarily associated with the azaspirone decane dione and pyrimidine substructures. Structures of new buspirone metabolites are reported and include the despyrimidinyl, despyrimidinylpiperazine, glucuronide, hydroxyglucuronide (four isomers), methoxyglucuronide and hydroxymethoxyglucuronide (two isomers) buspirone metabolites.     

Galpha12 and Galpha13 mediate differentiation of P19 mouse embryonal carcinoma cells in response to retinoic acid

P19 mouse embryonal carcinoma cells can be stimulated to differentiate into endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens. At low concentrations, retinoic acid stimulates P19 embryonal cells to differentiate to cells displaying an endodermal phenotype, whereas at higher concentrations it stimulates differentiation to neuroectoderm. The Galpha12 and Galpha13 subunits of heterotrimeric G-proteins are expressed in the embryonal P19 cells and stimulated in response to retinoic acid as the cells differentiate to endodermal or neuroectodermal phenotypes. Suppression of the expression of either Galpha12 or Galpha13 by antisense RNA is shown to promote cell detachment from substratum and apoptosis. Expression of the constitutively active, mutant form of Galpha12 (Q229L), in contrast, stimulates loss of the embryonal phenotype. Expression of the constitutively active form of Galpha13 (Q226L) stimulates differentiation of the cells from embryonal to endodermal, in the absence of retinoic acid. Thus, both Galpha12 and Galpha13 are essential to stimulation of cell differentiation by retinoic acid. Deficiency of either Galpha12 or Galpha13 increases programmed cell death.     

Secretion of cardiac plasminogen activator during hypoxia-induced right ventricular hypertrophy

In the circulation, fibrinolytic activity is determined to a large degree by the relative levels of tissue plasminogen activator (tPA) and its major inhibitor (PAI-1). Vascular beds in different organs secrete tPA and PAI-1 into the circulation, and the total secretory rate of each protein is balanced by its half-life in the bloodstream. We are testing the hypothesis that in the heart, ventricular hypertrophy will alter the rates of formation of tPA and/or PAI-1 and the rates of their release into the cardiac vasculature. In this study, we have examined the effects of continuous hypoxia on PA activity in extracts of rat heart ventricles, on the activity secreted into the cardiac vasculature of perfused hearts, and on the levels of mRNAs for tPA and PAI-1. Rats were subjected to hypobaric hypoxia at 0.5 atm for 1-21 days. The treatment caused polycythemia within 1-3 days, and right ventricular hypertrophy by 3 days. PA activity in extracts of both right and left ventricles was significantly elevated after 3 days of hypoxia, continued to increase for 4 additional days, and remained elevated for 3 weeks. The actions of inhibitors of urokinase and tPA indicated that the PA activity in heart extracts was exclusively tPA. Fibrin zymography confirmed that result. The mRNAs for tPA and for PAI-1 were elevated after 1 day of hypoxia and then returned to near control levels on days 2 and 3. After 7 days, hearts from hypoxic rats secreted more tPA activity into perfusates than did hearts from controls. The difference in secretory rates was proportional to the differences in the levels of tPA in the corresponding heart extracts.     

Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. Relationship To rat carboxylesterase ES-2

A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, Phenyl-Sepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver (Alexson, S. E. H., Finlay, T. H., Hellman, U., Svensson, L. T., Diczfalusy, U., and Eggertsen, G. (1994) J. Biol. Chem. 269, 17118-17124). Antibodies against this enzyme also react with the purified NREH. Purified NREH shows a substrate preference for retinyl palmitate over triolein and did not catalyze the hydrolysis of cholesteryl oleate. With retinyl palmitate as substrate, the enzyme had a pH optimum of 7 and showed apparent saturation kinetics, with half-maximal activity achieved at substrate concentrations (Km) of approximately 70 microM.     

Long-term phonatory instability in individuals with multiple sclerosis

This paper uses a new approach to describe and quantify the long-term phonatory instability of speakers with MS. Sustained vowel phonations of 20 individuals with a definite diagnosis of multiple sclerosis (MS) and 20 age- and gender-matched individuals with normal speech were recorded. The phonations were f0 and intensity analyzed and subjected to spectral analysis using the Fast Fourier Transform. Three methods for analyzing the instabilities are presented, compared, and related to perceptual judgments: (a) coefficients of variation, (b) magnitude-based analysis of spectral energy, and (c) frequency-based analysis of spectral components. All measures reliably distinguished between individuals with MS and persons with normal speech. A single factor based on a linear discriminant analysis of the frequency-based measures was especially useful in distinguishing these groups. Critical frequency bands of instability, corresponding to wow (1-2 Hz), tremor (around 8 Hz), and flutter (17-18 Hz), distinguished the MS group from those of the control group.     

Activating Smoothened mutations in sporadic basal-cell carcinoma

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.     

Teacher discipline and child misbehavior in day care: untangling causality with correlational data

The report shows that Alzheimer's disease (AD) brain creatine kinase (CK) is modified such that the nucleotide binding site of CK is blocked and that abnormal partitioning of CK between the soluble and pellet fractions occurs. First, CK activity was 86% decreased in AD brain homogenates in comparison to age-matched controls. Secondly, over a 23.5 fold greater 32P photoincorporation of [alpha 32P]8N3ATP was observed into CK of control vs. AD samples. Also, a 7.4-fold increase of enzyme induced 32P incorporation was observed in controls vs. AD samples by incubation with [gamma 32P]ATP. Thirdly, Western blot analysis showed that CK copy numbers in the AD homogenate were decreased by less than 14% in comparison to controls. However, analysis showed that control supernatant and pellet fractions contained 10.3 and 0.4 times the CK copy number found in the corresponding AD fractions. 32P incorporation by both photolabeling and enzyme catalyzed incorporation of radiolabel followed CK activity and not CK copy number. Further, [alpha 32P]ADP and [gamma 32P]ATP incorporated 32P into control brain and purified brain CK equally well, indicating that a mechanism different from gamma-phosphoryl transfer is involved in the enzymatic incorporation of radiolabel. Also, the level of abnormal partitioning of CK into AD brain pellet correlated with the decreased [32P]8N3GTP photolabeling and abnormal partitioning of beta-tubulin, a protein known to be aberrantly modified in the AD brain. This indicates that a common chemistry is affecting both CK and tubulin in AD.     

Activity of medullary respiratory neurons during ventilator-induced apnea in sleep and wakefulness

Mechanical ventilation of cats in sleep and wakefulness causes apnea, often within two to three cycles of the ventilator. We recorded 137 medullary respiratory neurons in four adult cats during eupnea and during apnea caused by mechanical ventilation. We hypothesized that the residual activity of respiratory neurons during apnea might reveal its cause(s). The results showed that residual activity depended on 1) the amount of nonrespiratory inputs to the cell (cells with more nonrespiratory inputs had greater amounts of residual activity); 2) the cell type (expiratory cells had more residual activity than inspiratory cells); and 3) the state of consciousness (more residual activity in wakefulness and rapid-eye-movement sleep than in non-rapid-eye-movement sleep). None of the cells showed an activation during ventilation that could explain the apnea. Residual activity of approximately one-half of the cells was modulated in phase with the ventilator. The strength of this modulation was quantified by using an effect-size statistic and was found to be weak. The patterns of modulation did not support the idea that mechanoreceptors excite some respiratory cells that, in turn, inhibit others. Indeed, most cells, inspiratory and expiratory, discharged during the deflation-inflation transition of ventilation. Residual activity failed to reveal the cause of apnea but showed that during apnea respiratory neurons act as if they were disinhibited and disfacilitated.     

Tissue reactions to bacteria-challenged implantable leads with enhanced infection resistance

Tissue reactions to implantable pacemaker leads were investigated in an early infection model in rabbits. Both standard leads and surface-modified leads were used. The surface modification technique was applied to achieve controlled release of the antibiotic gentamicin. The insulating polyurethane tubing material of the leads was provided with an acrylic acid/acrylamide copolymer surface graft and then loaded with gentamicin. Implantation periods varied from day 4, to week 3 1/2, to week 10. We investigated tissue reactions in the absence of an infectious challenge and also the efficacy of surface-modified leads in preventing infection after challenge with Staphylococcus aureus was evaluated. It was demonstrated that the applied surface modification did not induce adverse effects although during early postimplantation an increase in infiltration of granulocytes and macrophages and wound fluid and fibrin deposition were observed. After bacterial challenge, standard leads were heavily infected at each explantation period, denoted by abscesses, cellular debris, and bacterial colonies. In contrast, little or no infection was observed, either macroscopically or by bacterial cultures, with the surface-modified leads. Microscopy showed little evidence of the bacterial challenge, and that primarily at day 4. It was concluded that the applied surface modification demonstrated enhanced infection resistance and thus represents a sound approach to the battle against infectious complications with biomaterials.