Cadherin switch from E- to N-cadherin in melanoma progression is regulated by the PI3K/PTEN pathway through Twist and Snail.

BACKGROUND: Transition of normal melanocytic cells to malignant melanoma has characteristic features of epithelial to mesenchymal transition. This includes the disruption of the adherens junctions caused by the downregulation of E-cadherin and the upregulation of N-cadherin. The cadherins have functional importance in normal skin homeostasis and melanoma development; however, the exact mechanism(s) that regulate the 'cadherin switch' are unclear. OBJECTIVES: To determine the mechanistic role of the PI3K/PTEN pathway in regulating the change in cadherin phenotype during melanoma progression. METHODS: Using a panel of cell lines representative of the phases of melanoma progression, we determined cellular expressions of the components of the PI3K/PTEN pathway, E- and N-cadherin, and the transcriptional regulators Twist, Snail and Slug with Western blot and immunofluorescence analysis. Transcriptional regulation of E-cadherin, N-cadherin, Twist and Snail by the PI3K/PTEN pathway was confirmed using quantitative reverse transcription-polymerase chain reaction. RESULTS: Loss or inactivity of PTEN correlated with the switch in cadherin phenotype during melanoma progression. PTEN-null or inactive cells exhibited high levels of phosphorylated protein kinase B (PKB)/AKT (Serine 473) (PKB-Ser473-P), undetectable levels of E-cadherin and high levels of N-cadherin. Re-introduction of PTEN or treatment with the PI3K inhibitor Wortmannin resulted in the re-expression of E-cadherin and downregulation of N-cadherin. This cadherin switch was regulated at the transcriptional level by Twist and Snail which were, in turn, transcriptionally regulated by the PI3K pathway. Although E-cadherin was re-expressed, it failed to localize to the plasma membrane. CONCLUSIONS: The PI3K/PTEN pathway transcriptionally regulates the 'cadherin switch' via transcriptional regulation of Twist and Snail but does not regulate the localization of E-cadherin to the plasma membrane.

Drug utilization patterns in pregnant women: a case study at the Mount Hope Women's Hospital in Trinidad, West Indies / Patrones de utilización de medicamentos en las mujeres embarazadas: un caso de estudio en el Hospital de Mujeres Mount Hope en Trinidad, West Indies

OBJECTIVE: To explore drug (prescription, over-the-counter and herbal) utilization in pregnant women attending a public sector tertiary healthcare institution. METHODS: This was a cross-sectional case study in women attending antenatal clinics at the Mount Hope Women's Hospital. Women (506) who consecutively presented for routine care at the antenatal clinic were interviewed on the medication they took. Descriptive statistics and logistic regression for predictors of drug use were done using SPSS 16. RESULTS: There were 200 (39.5%) primigravidae, 306 (60.5%) multigravidae and 299 (59%) women were in the third trimester of pregnancy. Most women (69.8%) were between 20-35 years of age. Women took an average of 1.32, 1.22 and 0.94 prescribed drugs in each trimester, respectively. Multivitamins (59.8%) and iron/folic acid (54.2%) were the most frequently prescribed drugs. Regardless of trimester, only 20% of women took supplemental calcium. Very few women (2.4%) took herbal medications. Paracetamol was the most common over-the-counter (OTC) medication in all trimesters. Women with secondary level education were most likely to use OTC iron/folic acid (p = 0.02), paracetamol and histamine2 receptor antagonists [H2RAs] (p = 0.001). More primigravidae took non-steroidal anti-inflammatory drugs (p = 0.02) and more women in the first trimester used antiemetics (p = 0.001). Age group (p = 0.048), marital status (p = 0.001) and the trimester of pregnancy (p = 0.001) were predictors of drug utilization. CONCLUSION: Overall, women in tertiary healthcare institutions took medication as prescribed particularly multivitamins and iron/folic acid. More women with higher education took OTC paracetamol, iron/folic acid and vitamin supplements. Herbal supplements were rarely used. Research on drug utilization in primary care facilities is recommended.

OBJETIVO: Explorar el uso de los medicamentos (con prescripción, sin receta médica, herbarios) en mujeres embarazadas que asisten a una institución terciaria de atención a la salud pública dentro del sector público. MÉTODOS: Se trató de un estudio transversal de mujeres que asisten a las clínicas prenatales en el Hospital de Mujeres Mount Hope. Las mujeres (506) que consecutivamente se presentaron para cuidados de rutina en la clínica prenatal, fueron entrevistadas acerca de la medicación que tomaban. Se hicieron estadísticas descriptivas y se hizo una regresión logística para los predictores del uso del medicamento usando SPSS 16. RESULTADOS: Había 200 (39.5%) primerizas, 306 (60.5%) multíparas, y 299 (59%) embarazadas en su tercer trimestre. La mayoría de las mujeres (69.8%) tenían entre 20-35 años de edad. Las mujeres tomaban un promedio de 1.32, 1.22 y 0.94 medicamentos prescritos en cada trimestre, respectivamente. Las multivitaminas (59.8%) y el hierro/ácido fólico (54.2%) fueron los medicamentos más frecuentemente prescritos. Con independencia del trimestre, sólo 20% de las mujeres tomaron suplemento de calcio. Muy pocas mujeres (2.4%) tomaban medicaciones herbarias. El paracetamol fue el medicamento sin receta más común en todos los trimestres. Las mujeres con nivel de educación secundaria presentaban una mayor probabilidad de usar hierro/ácido fólico (p = 0.02), el paracetamol y los antagonistas de los receptores de la histamina-2- [H2RAs] (p = 0.001). Un mayor número de primerizas tomaron medicamentos anti-inflamatorios no esteroideos (p = 0.02) y más mujeres en el primer trimestre usaron anti-eméticos (p = 0.001). El grupo etario (p = 0.048), el estado matrimonial (p = 0.001) y el trimestre de embarazo (p = 0.001) fueron predictores de la utilización de medicamentos. CONCLUSIÓN: En general, las mujeres en las instituciones terciarias de atención a la salud tomaron la medicación como fue prescrita, en particular las multivitaminas y el hierro/ácido fólico. Más mujeres con mayor escolaridad tomaron medicamentos sin recetas: paracetamol, hierro/ácido fólico y suplementos de vitamina. Raramente se usaron suplementos herbarios. Se recomienda la investigación del uso de medicamentos en centros de atención primaria.

Drug utilization patterns in pregnant women: a case study at the Mount Hope Women's Hospital in Trinidad, West Indies.

OBJECTIVE: To explore drug (prescription, over-the-counter and herbal) utilization in pregnant women attending a public sector tertiary healthcare institution. METHODS: This was a cross-sectional case study in women attending antenatal clinics at the Mount Hope Women's Hospital. Women (506) who consecutively presented for routine care at the antenatal clinic were interviewed on the medication they took. Descriptive statistics and logistic regression for predictors of drug use were done using SPSS 16. RESULTS: There were 200 (39.5%) primigravidae, 306 (60.5%) multigravidae and 299 (59%) women were in the third trimester of pregnancy. Most women (69.8%) were between 20-35 years of age. Women took an average of 1.32, 1.22 and 0.94 prescribed drugs in each trimester respectively. Multivitamins (59.8%) and iron/folic acid (54.2%) were the most frequently prescribed drugs. Regardless of trimester only 20% of women took supplemental calcium. Very few women (2.4%) took herbal medications. Paracetamol was the most common over-the-counter (OTC) medication in all trimesters. Women with secondary level education were most likely to use OTC iron/folic acid (p = 0.02), paracetamol and histamine2 receptor antagonists [H2RAs] (p = 0.001). More primigravidae took non-steroidal anti-inflammatory drugs (p = 0.02) and more women in the first trimester used antiemetics (p = 0.001). Age group (p = 0.048), marital status (p = 0.001) and the trimester of pregnancy (p = 0.001) were predictors of drug utilization. CONCLUSION: Overall, women in tertiary healthcare institutions took medication as prescribed particularly multivitamins and iron/folic acid. More women with higher education took OTC paracetamol, iron/folic acid and vitamin supplements. Herbal supplements were rarely used. Research on drug utilization in primary care facilities is recommended.

Sources of stress and psychological disturbance among dental students in the West Indies

The aim of this study was to investigate sources of stress and psychological disturbance in dental students across the five years of undergraduate study at a dental school in Trinidad. Eighty-three percent of students completed a modified version of the Dental Environment Stress questionnaire (DES) and the Brief Symptom Inventory (BSI). On a scale ranging from 0 (not stressful) to 5 (highly stressful), overall mean DES scores for each of the five years of study were 1.58, 1.83, 2.65, 2.39, and 2.61 respectively, suggesting that levels of stress increase over the five years with a noticeable spike at the transition between the preclinical and clinical phases. Significant differences were found between specific stressors across the five years of study. Seven specific stressors and the stressor domains of Academic work and Clinical factors were more stressful for female students (t-test p < 0.05). The Global Severity Index of the BSI indicated that 54.8 percent of males and 44.2 percent of females were in the clinical range indicating significant psychological disturbance. Psychological disturbance was significantly associated with stress levels for male students (Spearmans rank correlation r = 0.56; p < 0.001), but not generally for female students. Further development is needed of dental educational programs that enhance students' psychosocial well-being.

Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343.

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB- serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.

Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells.

PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We recently have demonstrated that the integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. We now demonstrate that the activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy.

Alterations in cardiac membrane beta-adrenoceptors and adenylyl cyclase due to hypochlorous acid.

Although neutrophils and eosinophils are known to produce hypochlorous acid (HOCI) at the site of cardiac injury, the exact role of this toxic oxidant on the signal transduction mechanism in the heart is not clear. In this study, the effects of HOCI on beta-adrenoceptors, G-proteins and adenylyl cyclase activity were assessed by incubating rat heart membranes with HOCl. The basal as well as forskolin-, NaF-, 5-guanylylimidodiphosphate-, and isoproterenol-stimulated adenylyl cyclase activities were depressed by incubating cardiac membranes with HOCl. While both the density and affinity of the beta1-adrenoceptors were decreased by treatment of cardiac membranes with HOCl, the characteristics of the beta2-adrenoceptors were not modified significantly. Although cholera toxin-stimulated adenylyl cyclase activity, cholera toxin-catalyzed ADP-ribosylation and stimulatory guanine nucleotide binding protein immunoreactivity were depressed by HOCl, the pertussis toxin-stimulated adenylyl cyclase activity, pertussis toxin-catalyzed ADP ribosylation and inhibitory guanine nucleotide binding protein immunoreactivity were unaltered by HOCl. The presence of L-methionine in the incubation medium prevented the HOCl-induced alterations in adenylyl cyclase activities and characteristics of beta1-adrenoceptors. These results suggest that HOCl may be one of the factors attenuating the beta-adrenoceptor linked signal transduction mechanism in conditions such as ischemic heart disease.

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

Role of H2O2 in changing beta-adrenoceptor and adenylyl cyclase in ischemia-reperfused hearts.

In view of the accumulation of H2O2 in the myocardium due to ischemia-reperfusion and changes in beta-adrenoceptor mechanisms in the ischemic-reperfused heart, we investigated the effects of H2O2 on the beta-adrenoceptor, G-protein and adenylyl cyclase complex. Rat hearts were perfused with 1 mM H2O2 for 10 min before isolating membranes for measuring the biochemical activities. The stimulation of adenylyl cyclase by different concentrations of isoproterenol was depressed upon perfusing hearts with H2O2. Both the affinity and density of beta1-adrenoceptors as well as the density of the beta2-adrenoceptors were decreased whereas the affinity of beta2-adrenoceptors was increased by H2O2 perfusion. Competition curves did not reveal any effect of H2O2 on the proportion of coupled receptors in the high affinity state. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities were depressed by perfusing the heart with H2O2. Catalase alone or in combination with mannitol was able to significantly decrease the magnitude of alterations due to H2O2. The positive inotropic effect of 1 microM isoproterenol was markedly attenuated upon perfusing hearts with 200-500 microM H2O2 for 10 min. These results suggest that H2O2 may depress the beta1-adrenoceptor, Gs-proteins and catalytic subunit of the adenylyl cyclase enzyme and thus may play an important role in attenuating the beta-adrenoceptor linked signal transduction due to ischemia-reperfusion injury.

Modification of cardiac beta-adrenoceptor mechanisms by H2O2.

From the role of oxidative stress in cardiac dysfunction, we investigated the effect of H2O2, an activated species of oxygen, on beta-adrenoceptors, G proteins, and adenylyl cyclase activities. Rat heart membranes were incubated with different concentrations of H2O2 before the biochemical parameters were measured. Both the affinity and density of beta 1-adrenoceptors were decreased, whereas the density of the beta 2-adrenoceptors was decreased and the affinity was increased by 1 mM H2O2. Time- and concentration-dependent biphasic changes in adenylyl cyclase activities in the absence or presence of isoproterenol were observed when membranes were incubated with H2O2; however, activation of the enzyme by isoproterenol was increased or unaltered. The adenylyl cyclase activities in the absence or presence of forskolin, NaF, and Gpp(NH)p were depressed by H2O2. Catalase alone or in combination with mannitol was able to significantly decrease the magnitude of alterations due to H2O2. The cholera toxin-stimulated adenylyl cyclase activity and ADP ribose labeling of Gs proteins were decreased by treatment with 1 mM H2O2, whereas Gi protein activities, as reflected by pertussis toxin-stimulation of adenylyl cyclase and ADP ribosylation, were unaltered. The Gs and Gi protein immunoreactivities, estimated by labeling with respective antibodies, indicate a decrease in binding to the 45-kDa band of Gs protein, whereas no change in the binding of antibodies to the 52-kDa band of Gs protein or the 40-kDa subunit of Gi protein was evident when the membranes were treated with 1 mM H2O2. These results suggest that H2O2 in high concentrations may attenuate the beta-adrenoceptor-linked signal transduction in the heart by changing the functions of Gs proteins and the catalytic subunit of the adenylyl cyclase enzyme.

A telemedicine consultative service for the evaluation of patients with urolithiasis.

OBJECTIVES: A 6-month pilot teleconsultative project linking Georgetown University Medical Center (GUMC) in Washington, DC, and City Hospital in Martinsburg, West Virginia, 90 miles away, was designed to assess the effectiveness of telemedicine on the clinical decision-making process for patients with urolithiasis. METHODS: The telemedicine system designed and tested for this project was based on a PC-based platform. Videoconferencing and review of the patient's imaging studies were performed over an Integrated Service Digital Network (ISDN) with 3 Basic Rate (BRI) ISDN lines providing a 336-kilobytes/s bandwidth through an Inverse Multiplexor (IMUX). Treatment options were recorded for the clinical trial group and a simulated study group by the consulting urologist after the initial telephone consultation, after the telemedicine consultation, and after examination of those patients transferred to GUMC. RESULTS: A total of 32 telemedicine consultations were performed: 14 in the clinical trial group and 18 in the simulated study group. The recommendation of the consulting urologist at the tertiary center was altered in 12 patients (37.5%) after the telemedicine consultation compared with the recommended treatment after the initial telephone consultation. CONCLUSIONS: In the evaluation of patients with urolithiasis, this telemedicine application enhanced the clinical decision-making process by allowing for improved quality of care through immediate access and effective transfer of information between the referring urologist, the patient, and the stone center specialist.

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid.

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.

Biphasic alterations in cardiac beta-adrenoceptor signal transduction mechanism due to oxyradicals.

To assess the effects of oxyradicals on cardiac beta-adrenoceptors, G-proteins and adenylyl cyclase, rat heart membranes were incubated with xanthine (X) plus xanthine oxidase (XO) for different intervals. The basal as well as forskolin-, NaF-, 5'-guanylylimidodiphosphate and isoproterenol-stimulated adenylyl cyclase activities showed an increase at 10 min and a decrease at 30 min of incubation with X plus XO. Treatment of membranes with H2O2 also produced biphasic changes in adenylyl cyclase activities. The density of beta1-adrenoceptors was decreased when cardiac membranes were treated with X plus XO for 10 and 30 min whereas the affinity of beta1-adrenoceptors was increased after 10 min and reduced after 30 min of incubation. The beta2-adrenoceptors were not modified at 10 min whereas incubation of cardiac membranes with X plus XO for 30 min increased the affinity and decreased the density. Cholera toxin-stimulated adenylyl cyclase activity, cholera toxin-catalyzed ADP-ribosylation and stimulatory guanine nucleotide binding protein immunoreactivity in cardiac membranes were increased at 10 min and decreased at 30 min of incubation with X plus XO. However, the pertussis toxin-stimulated adenylyl cyclase activity, pertussis toxin-catalyzed ADP ribosylation and inhibitory guanine nucleotide binding protein immunoreactivity were not affected on treatment of membranes with X plus XO. Addition of superoxide dismutase plus catalase in the incubation medium prevented the X plus XO-induced alterations in adenylyl cyclase activities, stimulatory guanine nucleotide binding protein-related ADP-ribosylation and changes in the characteristics of beta-adrenoceptors except the increased affinity of beta1-adrenoceptors at 10 min of incubation. These data suggest that alterations in the beta1-adrenoceptor-linked stimulatory guanine nucleotide binding protein-adenylyl cyclase pathway due to X plus XO are biphasic in nature and these changes may likely be due to the formation of H2O2.

Alternations in beta-Adrenoceptor Mechanisms in Hearts Perfused With Xanthine Plus Xanthine Oxidase.

BACKGROUND: Although beta-adrenoceptors and adenylyl cyclase are known to be affected upon exposing cardiac membranes to some oxyradical generating systems, the results are conflicting. Furthermore, functional significance of alterations in the beta-adrenoceptor-adenylyl cyclase systems in terms of changes in the inotropic responses to catecholamines is not clear. METHODS AND RESULTS: The positive inotropic effect of isoproterenol was augmented on perfusing the isolated rat hearts with xanthine (X) plus xanthine oxidase (XO) for 5 minutes but was attenuated by perfusion for 15 minutes. The isoproterenol-stimulated adenylyl cyclase activity in cardiac membranes showed an increase at 10 minutes and a decrease at 30 minutes perfusion of hearts with X plus XO. The density of beta-adrenoceptors in cardiac membraners was reduced after 10 minutes and 30 minutes of perfusion with X plus XO, whereas the affinity of beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors were unaltered by 10 minutes of perfusion with X plus XO, their affinity was increased and density was decreased by 30 minutes of perfusion. The agonist competition curves using isoproterenol indicated an increase in the number of coupled receptors in the high affinity state on 10 minutes of perfusion and an increase in the low affinity state of coupled receptor due to 30 minutes of perfusion with X plus XO. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities in cardiac membranes exhibited an increase after 10 minutes and decrease after 30 minutes of perfusion with X plus XO. Although the presence of superoxide dismutase plus catalase in the perfusion medium prevented most of the alterations due to X plus XO, it did not alter the increased affinity of the beta-adrenoceptor upon perfusing hearts for 10 minutes with X plus XO. CONCLUSIONS: The results in this study suggest the biphasic nature of the oxyradical-induced alterations in both the inotropic responses to catecholamines and the beta-adrenoceptor-mediated signal transduction mechanism in the heart.

Beta-adrenoceptor-linked signal transduction in ischemic-reperfused heart and scavenging of oxyradicals.

In order to examine the mechanisms of ischemia-reperfusion induced changes in beta-adrenoceptor-linked signal transduction pathway, isolated rat hearts perfused in the absence or presence of superoxide dismutase (SOD) plus catalase (CAT) were made ischemic for 30 min and then reperfused for 60 min. The left ventricular developed pressure as well as the rare of contraction and rate of relaxation were markedly decreased, whereas the left ventricular end-diastolic pressure increased in the ischemic hearts. A significant increase in the density and affinity of beta 1-adrenoceptors without any changes in the characteristics of beta 2-adrenoceptors was evident in cardiac membranes obtained from the ischemic hearts. The recovery of contractile abnormalities in the ischemic heart was depressed upon reperfusion; the ischemic-reperfused hearts also showed attenuated inotropic responses to isoproterenol. The affinities and densities of beta- and beta-adrenoceptors were decreased in the ischemic-reperfused hearts; the magnitude of changes in beta 1-adrenoceptors was greater than that in beta 2-adrenoceptors. The isoproterenol-stimulated adenylyl cyclase activity was depressed in both ischemic hearts and ischemic-reperfused hearts. The basal and forskolin-stimulated adenylyl cyclase activities were unaltered due to ischemia but were increased upon reperfusion. The NaF- and 5'-Guanylyl-imidodiphosphate[Gpp(NH)p]-stimulated adenylyl cyclase activities were depressed in the ischemic hearts and increased in the ischemic reperfused hearts. Cholera toxin (CT)-stimulated adenylyl cyclase as well as the CT-catalysed ADP-ribosylation activity and stimulatory G protein (Gs protein) immunoreactivity were decreased in the ischemic hearts and increased in the reperfused hearts. Pertussis toxin (PT)-stimulated adenylyl cyclase activity was unaltered in both ischemic and ischemic-reperfused hearts, whereas the PT-catalysed ribosylation and inhibitory G protein (Gi protein) immunoactivity were slightly increased in the reperfused myocardium. Thus the inability of isoproterenol to stimulate adenylyl cyclase in the ischemic-reperfused hearts may be due to alterations mainly in the characteristics of beta 1-adrenoceptors including density, affinity and coupling with the adenylyl cyclase. Scavenging of oxyradicals by the addition of SOD plus CAT in the perfusion medium prevented the reperfusion-induced changes in contractile function, inotropic responses of the heart to isoproterenol, activation of adenylyl cyclase by isoproterenol, as well as densities and affinities of beta-adrenoceptors in cardiac membranes. These results suggest that the depressed contractile activity and the attenuated inotropic responses of ischemic-reperfused hearts to isoproterenol as well as the defects in beta-adrenoceptor-linked signal transduction may be due to the formation of oxyradicals in the myocardium.

Case study of major gynaecologic endoscopy in obese patients - abstract

A retrospective study of gynaecologic laparoscopy in obese patients is undertaken to determine results. A discussion of the unique challenges presented by the obsese patient is discussed and suggestions offered to minimize compications. All the cases were performed at a private 400 bed community hospital, affiliated with Emory University. Procedures were done on 36 obese patients over the last two years by the same surgeon. Twenty-two were LAVH, others were a variety of other major gynaecologic procedures. All patients weighed more than 200 lbs; 14 weighed more than 250 lbs. The results and complications are discussed in detail. There was one serious complication, colon laceration, which was diagnosed at laparoscopy and repaired by laparatomy. This was a case of post hysterectomy retroperitoneal ovary. There was no mortality. Details of technical approaches of laparoscopy in obese patients are presented. Clinical details of all the patients are presented (AU)

Status of Ca(2+)-channels in hearts perfused with Ca(2+)-free medium as well as upon reperfusion (Ca(2+)-paradox).

Although a wide variety of biochemical changes have been observed during the occurrence of Ca(2+)-paradox, very little is known about membrane alterations during Ca(2+)-free perfusion which may predispose the heart to the development of intracellular Ca(2+)-overload in the Ca(2+)-paradox phenomenon. In view of the marked influx of Ca2+ into the myocardial cell during Ca(2+)-paradox and the involvement of Ca(2+)-channels in the entry of Ca2+, we determined the status of Ca(2+)-channels by measuring the binding of a Ca(2+)-antagonist, [3H] PN200-110, with membranes obtained from rat hearts perfused with Ca(2+)-free medium. The density of Ca(2+)-channels in the membranes was increased upon perfusing the heart with Ca(2+)-free medium for > 2 min or when the perfusion medium contained less than 25 microM concentration of Ca2+. The increase in Ca(2+)-channel density was attenuated when the hearts were perfused with Ca(2+)-free medium in the presence of a low concentration (35 mM) of Na+ or at low temperature (21 degrees C); two conditions which are known to prevent the occurrence of Ca(2+)-paradox. These results indicate that increased density of Ca(2+)-channels due to Ca(2+)-free perfusion may contribute towards the massive Ca(2+)-influx into the myocardial cell for the induction of intracellular Ca(2+)-overload associated with Ca(2+)-paradox during reperfusion with Ca(2+)-containing medium.

Abnormal synthesis of N-methylated phospholipids during calcium paradox of the heart.

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activity that synthesizes phosphatidylcholine (PtdCho) via formation of methylated intermediates (phosphatidyl-N-monomethylethanolamine, PtdEtnMe and phosphatidyl-N,N-dimethylethanolamine, PtdEtnMe2) was comparatively studied in rat heart sarcolemmal (SL), sarcoplasmic reticular (SR) and mitochondrial fractions during Ca2+ paradox. Perfusion (5 min) with Ca(2+)-free medium followed by reperfusion (5 min) with Ca(2+)-containing medium produced a marked rise in resting tension without any recovery of contractile force. Methyltransferase catalytic sites I, II and III which synthesize PtdEtnMe, PtdEtnMe2 and PtdCho, respectively, were assayed by measuring the [3H] methyl group incorporation from 0.055, 10 and 150 microM S-adenosyl-L-[3H-methyl] methionine into membrane PtdEtn molecules. Five minutes of perfusion with Ca(2+)-free medium did not affect either SL or SR N-methyltransferase systems. Ca(2+)-readmission for 1 to 5 min induced a selective, time-dependent depression of SL site II and SR site I methyltransferase activities. Individual N-methylated phospholipids specifically formed at the two sites reflected these changes. The above abnormalities were differently influenced by the duration (1-5 min) of Ca(2+)-free perfusion and were characterized by different kinetic alterations. The mitochondrial methylation system was not affected under Ca2+ paradox. The results suggest that reduced synthesis of SL N-methylated phospholipids may contribute to the contractile dysfunction observed in Ca2+ paradox.

Ocular timolol levels after drug withdrawal: an experimental model.

Carbon 14-labelled timolol maleate was instilled into both eyes of 12 pigmented rabbits daily for 42 days. Drug levels in the aqueous humour and ocular tissues were measured up to 42 days after drug withdrawal. The results indicate that timolol concentrates mainly in melanotic tissues, with slow release. Even 42 days after withdrawal the drug was still present in pigmented ocular tissues. Timolol was detected in the aqueous up to 5 days after withdrawal. These findings explain the long-term depressant effect of topically administered timolol on aqueous production. We conclude that lower or less frequent doses of timolol should be considered in patients with glaucoma.