Biomedical subjects

A Morales

Publications and source records attributed to A Morales.

At least 19 recordsLinked to original sources

PubMed1995

Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Animals↗

PubMed1995

Evidence that the rat hepatic mitochondrial carrier is distinct from the sinusoidal and canalicular transporters for reduced glutathione. Expression studies in Xenopus laevis oocytes.

Mitochondrial GSH derives from a mitochondrial transport system (RmGshT), which translocates cytosol GSH into the mitochondrial matrix. Mitochondria of oocytes, isolated 3-4 days after microinjection of total liver mRNA, expressed a RmGshT compared with water-injected oocytes. The expressed RmGshT exhibited similar functional features as reported in isolated mitochondria of rat liver such as ATP stimulation, inhibition by glutamate, and insensitivity to inhibition by sulfobromophthalein-glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed RmGshT is localized in the inner mitochondrial membrane since expression is still observed in mitoplasts prepared from total liver mRNA-injected oocytes. Fractionation of poly(A)+ RNA identified a single mRNA species of approximately 3-3.5 kilobases encoding for the RmGshT, which was stimulated by ATP and inhibited by glutamate but not by BSP-GSH or DNP-GSH. Microinjection of this fraction did not lead to expression of plasma membrane GSH transport in intact oocytes, and conversely, oocytes microinjected with cRNA for rat liver sinusoidal GSH transporter (RsGshT) or rat liver canalicular GSH transporter (RcGshT) did not express mitochondrial GSH transport activity. Thus, our results show the successful expression of the rat hepatic mitochondrial GSH carrier, which is different from RsGshT and RcGshT, and provide the strategic basis for the cloning of this important carrier.

Animals↗

PubMed1995

Feeding S-adenosyl-L-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes.

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-L-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and periportal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 +/- 0.7 to 7.3 +/- 0.6 and 2.1 +/- 0.3 to 6.1 +/- 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.

Administration, Oral↗

PubMed1995

Recovery of erectile function by the oral administration of apomorphine.

OBJECTIVES: Apomorphine has been reported to be effective in causing erections in animals and man when administered parenterally. The side effects, notably nausea, have seriously limited its clinical usefulness. We formulated apomorphine for controlled sublingual absorption and herein report on four preliminary studies evaluating efficacy and side effects in men with no documentable organic cause of erectile dysfunction. METHODS: Patients complaining of erectile dysfunction underwent a careful evaluation. Those with measurable organic dysfunction or known organic factors were excluded. Men with primarily psychogenic impotence were tested with one of four protocols of an apomorphine preparation (preliminary sublingual liquid, preliminary 5 mg tablet, aqueous nasal spray, and new 3 and 4 mg controlled absorption tablets). The erectile response of these men to the drug with visual erotic or sexually neutral stimulation was studied with the Rigiscan. RESULTS: Seven of 10 evaluable patients responded to the sublingual liquid preparation but the majority experienced significant nausea. The preliminary 5 mg tablet and aqueous forms did not produce useful responses free of side effects. The newly formulated controlled absorption 3 and 4 mg tablets were tested in 12 men. Eight of 12 (67%) developed erections in response to apomorphine. Erectile activity was seen during sexually neutral visual stimulation to a significantly greater extent than with placebo. Home trial use was found to be successful and sustained by 7 of 11 (64%) patients. CONCLUSIONS: We have shown that apomorphine will act as an erectogenic agent when absorbed through the oral mucosa. In a carefully selected group of impotent patients with no documentable organic causes of erectile dysfunction, but with proven erectile potential, 67% will experience significantly durable erections with a dose of 3 or 4 mg of apomorphine when formulated for controlled absorption. The results in these small groups appear to justify larger clinical studies of this proprietary formulation.

Administration, Sublingual↗

PubMed1995

A personal computer-based system for parallel line analysis of bioassay data.

A program is described for symmetric parallel line analysis of bioassay data with a logistic dose-response relationship. Dose-response relationship is transformed to semilog or log-log. A regression line can be calculated for the dose-response curve for standard and test samples, and produces potency estimate of test sample preparation relative to the standard preparation based on parallel-line bioassay statistical methods [3,4], together with detailed analysis of variance, estimates of slope, intercept and chi 2 test which permits a very sensitive test for linearity and parallelism of standard and test sample and facilitates detection of 'outliers', i.e. samples exhibiting non-parallelism. The general comparison of dose-response relationships produced by the program are a feature of particular interest.

Analysis of Variance↗

PubMed1995

Impotence.

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Erectile Dysfunction↗

PubMed1995

Heparin neutralization by recombinant platelet factor 4 and protamine.

Protamine is the only available drug to reverse heparin-induced anticoagulation. Platelet factor 4 (PF4) is a basic polypeptide stored in platelets that reverses heparin. To investigate its potential as a reversal drug, we studied recombinant PF4 on anticoagulated blood obtained during cardiac surgery. Blood was obtained from 33 different venous reservoirs, and activated clotting time (ACT), heparin concentrations, and heparinase-ACT were determined. Anticoagulation was reversed by adding incremental PF4:heparin and protamine:heparin ratios to the heparinized blood, and the ACTs were determined (n = 21). Viscoelastic analysis of anticoagulation reversal was performed by adding protamine or PF4 at reversal ratios of 1.3:1 protamine:heparin, and 3.2:1 PF4:heparin using thromboelastography (n = 12). PF4 reversal ratios of 3:1 and 3.5:1 and protamine reversal ratios of 1:1, 1.5:1, 2:1 were not statistically different from heparinase-ACT values. There were no significant differences in viscoelastic measurements of clot formation between protamine and PF4. Recombinant PF4 at a 3.0:1 ratio reverses heparin-induced anticoagulation after cardiopulmonary bypass, and represents a potential alternative, especially for the protamine allergic patient.

Blood Coagulation↗

PubMed1995

Isolation and perfusion of the pudendal vasculature in male rats.

PURPOSE: The present study outlines a novel in situ technique to assess the regulation of vascular resistance in the penile vascular bed of the Wistar rat. MATERIALS AND METHODS: The isolation and perfusion of the pudendal artery were achieved by ligating all branches of the external iliac artery not directly connected to the internal pudendal artery. RESULTS: A linear flow-perfusion pressure curve was generated to ensure a viable preparation. A cumulative concentration-vascular response curve to the alpha 1-adrenoceptor agonist methoxamine (0.5-64 micrograms./ml.) was obtained. CONCLUSIONS: This novel methodology will allow reproducible and consistent quantitative assessments of the numerous factors (both neural and hormonal) that are proposed to govern the flow of blood in the penile vascular bed.

Animals↗

PubMed1995

Serum prolactin levels and neonatal seizures.

To assess the effects of neonatal seizures on the hypothalamus and to test clinical use of prolactin as a neonatal seizure marker, we studied postictal and recovery baseline serum prolactin levels in 19 neonates whose seizures were classified according to their clinical and EEG features. Postictal prolactin levels were obtained 30 min after the seizure, and recovery levels were ascertained 2-4 days later. The ratio of postictal prolactin level to recovery baseline level (prolactin ratio) was used as an indicator of postictal prolactin increase. The specificity and sensitivity of a prolactin ratio of > 2 was compared with the current standard of diagnosis (seizure discharges recorded by ictal EEG). Infants with electroclinical seizures had significantly higher prolactin ratios than control infants or infants with seizures without EEG correlation. Marked prolactin increases were noted only in infants with focal tonic seizures and temporal electrode involvement. A prolactin ratio of > 2 had a specificity of 100% and a sensitivity of 40%. We conclude that neonatal seizures have variable effects on the hypothalamus and that the low sensitivity and the need to await recovery levels limit the clinical value of prolactin ratio as a neonatal seizure marker.

Biomarkers↗

PubMed1995

Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes.

Mitochondria are an important source of reactive oxygen intermediates because they are the major consumers of molecular oxygen in cells. Respiration is associated with toxicity, which is related to the activation of oxygen to reactive intermediates. The purpose of the present study was to examine the role of reduced glutathione (GSH) in the maintenance of mitochondrial functions during oxidative stress induced through selective inhibition of the complex III segment of the electron transport chain. Hydrogen peroxide monitored by the fluorescence of dichlorofluorescein increased in a time- and dose-dependent manner on incubation of mitochondria with antimycin A (AA), an inhibitor of complex III. However, blockade of complex I or II with rotenone or thenoyltrifluoroacetone, respectively, did not result in accumulation of hydrogen peroxide. Depletion of mitochondrial GSH to 10-20% of control by preincubation with diethylmaleate (0.8 mM) or ethacrynic acid (250 microM) also increased dichlorofluorescein and malondialdehyde levels and resulted in an additional (2-3-fold) increase after AA. Similar results were obtained when mitochondrial GSH depletion was produced by treatment with buthionine L-sulfoximine before mirochondria isolation. The endogenous oxidative stress induced by AA was accompanied by a moderate loss of activity of ATPase complex (77% of control) and complex IV of respiration (75% of control), which was accentuated after depletion of mitochondrial GSH (51% and 45% of control, respectively). Similar results were observed in isolated hepatocytes in which depletion of mitochondrial GSH and AA led to peroxidation and mitochondrial dysfunction. In addition, with electrophoretic mobility shift assay of the transcription factor nuclear factor-kappa B (NF-kappa B), we detected its activation in response to AA (2-3-fold). Depletion of mitochondrial GSH in hepatocytes (20% of control) led to further enhancement of NF-kappa B activation (2-4-fold), which correlated with generation of hydrogen peroxide. Thus, our results suggest that GSH protects mitochondria against the endogenous oxidative stress produced at the ubiquinone site of the electron transport chain. Mitochondrial GSH depletion potentiates oxidant-induced loss of mitochondrial functions. Oxidant stress in mitochondria can promote extramitochondrial activation of NF-kappa B and therefore may affect nuclear gene expression.

Animals↗

PubMed1995

Oral and topical treatment of erectile dysfunction. Present and future.

A great deal of progress has been made in the pharmacological treatment of erectile dysfunction. At present, however, the most effective therapies require intracavernosal injections with a number of associated drawbacks. An increasing number of oral and transdermal agents have been introduced clinically or are at various phases in their development. It is evident that severe end-organ disease probably will not result in successful systemic therapy. Nevertheless, in men with intact or mildly dysfunctional erectile mechanisms, noninvasive treatments can offer some measure of success. Further study of individual and synergistic activity of available compounds is underway.

Administration, Oral↗

PubMed1995

Immunotherapy of an experimental adenocarcinoma of the prostate.

We have developed and tested a fractionated, purified and deproteinized emulsion of a mycobacterial cell wall (MCW) and report on a controlled study of this compound in the treatment of the experimental R3327-H adenocarcinoma of the prostate. The intraperitoneal route of administration was found ineffective at the weekly dose of 500 micrograms. The intratumoral administration of 1000 micrograms of MCW exhibited significant antitumor activity. Tumors larger than 2.2 cm.3 in volume showed evidence of temporary regression, but no cures were recorded in these animals. Complete tumor regression was found in 50% of the rats with tumor volumes less than 2.2 cm.3 at the onset of treatment. The animals in this group not responding initially were treated with a second 3-week course of MCW which resulted in complete tumor regression in one-half of the animals, for a total cure rate, in the smaller tumor cohort, of 75%. Mycobacterial cell wall is an effective biological response modifier in the Dunning R3327-H adenocarcinoma of the prostate in Copenhagen rats. Additional studies to elucidate the effect of the compound in relation to dose and tumor volume are underway.

Adenocarcinoma↗

PubMed1994

Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.

The properties of glycine receptors (GlyRs) from newborn and adult rat spinal cord were studied in Xenopus oocytes injected with whole mRNA or the heavy (H) or light (L) mRNA fractions encoding their respective GlyRs. Mean open times and conductances of channels gated by H- or L-GlyRs were determined by noise analysis or voltage jumps. We found that adult H- and L-GlyRs opened channels that differed in their mean open time but had the same channel conductance. Both H- and L-GlyRs gated Cl- currents that displayed a similarly strong outward rectification. Nevertheless, single channels of adult H- and L-GlyRs did not rectify and their mean open times were only slightly altered by voltage. It follows that the outward rectification of adult GlyRs is due mainly to a reduction in the number of open channels. In contrast to H-GlyRs, whose characteristics seem to remain essentially unchanged with age, L-GlyRs from newborn and adult rats have different properties. Channels of newborn L-GlyRs have a higher conductance, longer open time, and greater voltage dependency than those from the adult. Interestingly, properties of newborn GlyRs expressed by whole mRNA were markedly different from those encoded by newborn or adult L or H mRNA. These results demonstrate that the functional heterogeneity of GlyRs is developmentally regulated.

Age Factors↗

PubMed1994

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes.

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the gamma subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the gamma subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the gamma subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the gamma subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.

Acetylcholine↗

PubMed1994

Diurnal penile tumescence recording in the etiological diagnosis of erectile dysfunction.

Rapid eye movement sleep occurs during napping. We investigated the appearance of penile tumescence during periods of day napping in a population of 18 impotent men who, in addition, underwent a comprehensive sleep investigation for impotence, including polysomnographic recording and nocturnal penile tumescence monitoring. Of the subjects 16 (88%) had rapid eye movement sleep during the night. Four patients who did not have erections on 2 separate sessions of nocturnal sleep recording also did not experience penile tumescence during the day. Of the 12 patients with documented erections at night 9 (75%) also exhibited erectile episodes during napping. Diurnal penile tumescence recording is less cumbersome, less expensive and more convenient to perform than its nocturnal counterpart. Diurnal penile tumescence appears to be a summary reflection of nocturnal penile tumescence episodes. The consistency between nocturnal and diurnal penile tumescence suggests that further study of this new technique is worthwhile. Validation of diurnal penile tumescence may offer a viable alternative to the comprehensive assessment of impotent men.

Circadian Rhythm↗

PubMed1994

Oral androgens in the treatment of hypogonadal impotent men.

A study was designed to assess the effect of supplemental oral methyltestosterone in the treatment of impotence associated with low total serum androgen levels. A total of 22 hypogonadal impotent men underwent a comprehensive investigation of erectile dysfunction, including an evaluation of the pituitary-gonadal axis. The patients then received a 1-month course of 2 different commercial preparations of oral methyltestosterone. Hormonal changes induced by the medication were assessed on days 15 and 30 of treatment. The patients kept daily records of sexual activity, and completed visual analogue scales to assess energy levels, mood and sensation of well being on a weekly basis. Supra-physiological levels of total serum testosterone were achieved in every patient but the free fraction of the hormone did not increase proportionally and in many cases a marked decrease was recorded. In all but 1 subject there was a decrease in circulating sex hormone binding globulin. Pituitary gonadotropin levels showed a marked decrease at the end of treatment. The clinical response was disappointing. Only 9% of the patients reported a complete recovery of sexual function. Visual analogue scales did not reveal noticeable changes for any individual in the levels of energy, mood or feeling of well being between pretreatment and posttreatment assessments. Oral methyltestosterone is of limited effectiveness in men with hypogonadal impotence. The positive responses in this study were recorded in men with the most profound deficiency. Exogenous administration of androgens to impotent men should be limited to those with profound hypogonadism as documented by at least 2 abnormal serum free testosterone determinations.

Administration, Oral↗

PubMed1994

Impotence.

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Erectile Dysfunction↗

PubMed1994

Cimetidine in the treatment of interstitial cystitis.

OBJECTIVES: To assess the effect of the H2-antagonist cimetidine in the treatment of patients with interstitial cystitis (IC) refractory to other conservative therapies. METHODS: A group of 9 patients previously treated conservatively for IC without success were entered in the study. They were thoroughly investigated and treated with cimetidine at the dose of 300 mg orally twice a day for 1 month. RESULTS: Six of the 9 patients (66%) experienced various degrees of symptomatic relief while on the drug. Of these, 4 (44%) have noted a complete and sustained response to the medication. CONCLUSIONS: The encouraging results observed in this pilot study together with the simplicity and tolerance of the treatment makes it an alternative when other options have been exhausted. Its use as a first-line monotherapy remains speculative.

Administration, Oral↗