Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model.

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.

Evaluación del tráfico de mujeres, adolescentes y niños/as en bolivia

Este trabajo presenta el estudio realizado por la consultora Científica, para la Organización Internacional para las Migraciones (OIM/BOLIVIA) y la Organización de Estados Americanos (OEA) , en el que se describe y analiza distintas formas de tráfico de mujeres, niños/as y adolescentes con fines de explotación laboral sexual, también las estrategias de reclutamiento, los traficantes y la situación de migración e inmigración por la que pasan éstas personas en Bolivia.

Evaluación del tráfico de mujeres, adolecentes y niños/as en Bolivia / Evaluation of trafficking in women, adolescents and children / as in Bolivia

El problema planteado por el tráfico de personas constituye ya tema de permanente análisis y debate, tanto en las agendas de organizaciones internacionales e intergubernamentales interesadas en los derechos humanos, como en los programas institucionales de un mayor número de Estados y gobiernos a lo largo y ancho del planeta.

Myomectomy as a pregnancy-preserving option in the carefully selected patient.

OBJECTIVES: To present the indications for myomectomy during pregnancy and to discuss complications possibly related and unrelated to the procedure. METHOD AND RESULTS: A 33-year-old patient at 18 weeks of gestation underwent removal of a 1,570-gram symptomatic fundic myoma. Histologically the patient had a leiomyomatous neoplasm of uncertain malignant potential. The pregnancy was continued under sequential observation with magnetic resonance imaging and ultrasound. At 36 weeks of gestation a healthy girl with an upper extremity limb defect was born via cesarean section. Follow-up of the mother and the child was uneventful. CONCLUSIONS: Certain known risk factors in pregnant women with myomas can predispose to complications during pregnancy. Women with such risk factors or women who have failed medical therapy should be offered the option of undergoing myomectomy as a pregnancy-preserving procedure.

Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase.

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.

The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site.

Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O(2)) via the PAI-1 promoter region -175/-159 containing a hypoxia response element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an adjacent response element (HRE-1) binding a so far unknown factor. The aim of the present study was to identify this factor and to investigate its role in the regulation of PAI-1 expression. It was found by supershift assays that the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 of the PAI-1 promoter and to a lesser extent to HRE-2. Overexpression of USF-2a inhibited PAI-1 messenger RNA and protein expression and activated L-type pyruvate kinase expression in primary rat hepatocytes under normoxia and hypoxia. Luciferase (Luc) gene constructs driven by 766 and 276 base pairs of the 5'-flanking region of the PAI-1 gene were transfected into primary hepatocytes together with expression vectors encoding wild-type USF-2a and a USF-2a mutant lacking DNA binding and dimerization activity (DeltaHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of DeltaHU2a did not influence Luc activity. Mutation of the HRE-1 (-175/-168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (-165/-158) was less effective. Cotransfection of a HIF-1alpha vector could compete for the binding of USF at HRE-2. These results indicated that the balance between 2 transcriptional factors, HIF-1 and USF-2a, which can bind adjacent HRE sites, appears to be involved in the regulation of PAI-1 expression in many clinical conditions.

Safety and preliminary efficacy of one month glycoprotein IIb/IIIa inhibition with lefradafiban in patients with acute coronary syndromes without ST-elevation; a phase II study.

AIMS: Oral glycoprotein IIb/IIIa inhibitors might enhance the early benefit of an intravenous agent and prevent subsequent cardiac events in patients with acute coronary syndromes. We assessed the safety and preliminary efficacy of 1 month treatment with three dose levels of the oral GP IIb/IIIa blocker lefradafiban in patients with unstable angina or myocardial infarction without persistent ST elevation. METHODS: The Fibrinogen Receptor Occupancy STudy (FROST) was designed as a dose-escalation trial with 20, 30 and 45 mg lefradafiban t.i.d. or placebo. Five hundred and thirty-one patients were randomized in a 3:1 ratio to lefradafiban or placebo in a double-blind manner. Efficacy was assessed by the incidence of death, myocardial infarction, coronary revascularization and recurrent angina. Safety was evaluated by the occurrence of bleeding classified according to the TIMI criteria and by measuring clinical laboratory parameters. RESULTS: There was a trend towards a reduction in cardiac events with lefradafiban 30 mg when compared with placebo and lefradafiban 20 mg. The benefit was particularly apparent in patients with a positive (> or = O.1 ng. ml(-1)) troponin I test at baseline and less so in those with a negative test result. In patients receiving lefradafiban, the cardiac event rate decreased with increasing minimal levels of fibrinogen receptor occupancy. There was a dose-dependent increase in the incidence of bleeding: the composite of major or minor bleeding occurred in 1% of placebo patients, 5% of patients receiving lefradafiban 20 mg and in 7% of patients receiving 30 mg, with an excessive risk (15%) in the 45 mg group which resulted in early discontinuation of this dose level. Gingival and arterial or venous puncture site bleedings were most common and accounted for more than 60% of all haemorrhagic events. There was an increased incidence of neutropenia (neutrophils <1. 5 x 10(9)/l) in the lefradafiban groups (5.2% vs 1.5% in the placebo group), which did not result from bone marrow depression but rather from a reversible redistribution of neutrophils by margination or clustering. CONCLUSION: One month's treatment with the oral glycoprotein IIb/IIIa inhibitor lefradafiban in patients with unstable angina and myocardial infarction without persistent ST elevation resulted in a decrease in cardiac events with lefradafiban 30 mg and a dose-dependent increase in haemorrhagic events. The observed favourable trend towards a reduction in cardiac events in patients with elevated troponin levels requires confirmation in a large clinical trial.

New calixarene-bonded stationary phases in high-performance liquid chromatography: comparative studies on the retention behavior and on influences of the eluent.

The chromatographic behavior of six calixarene-bonded stationary phases is reported. Varying analyte selectivities (i.e., for phenols, substituted aromatics, polycyclic aromatic hydrocarbons, barbituric acid derivatives, xanthines) exist as a function of the ring-size of the calix[n]arenes (n=4, 6, 8) and the substitution at the "upper rim" with para-tert.-butyl groups. Although eluents with unusually high proportions of water were used, a comparison with conventional reversed-phase (RP) columns shows a predominantly reversed-phase character with remarkable selectivities of these phases. The influences of several organic solvents on retention variations of solutes are compared for RP-C18, phenyl and calixarene phases.

Measurement of respiratory impedance by impulse oscillometry--effects of endotracheal tubes.

Impulse Oscillometry is a new, noninvasive method to measure respiratory impedance, i.e. airway resistance and reactance at different oscillation frequencies. These parameters are potentially useful for the monitoring of respiratory mechanics in the critically ill patent with respiratory dysfunction. The endotracheal tube, used to mechanically ventilate these patients, however, represents an additional nonlinear impedance that introduces artifacts into the measurements. The objective of this work was therefore to investigate the effects of clinically available endotracheal tubes on resistance and reactance of an in vitro analogue of the respiratory system. Additionally, the effects of decreasing the compressible gas volume in this experimental model, as a simulation of decreased lung capacity and compliance, was investigated. Impulse oscillometric measurements of the test analogue gave highly reproducible results with and without an endotracheal tube. The tubes had significant influence on the measurement of the test object at all frequencies investigated. Changes of low frequent reactance were negligible - at least if repetitive measurements of the same system are performed - for realistic measurement of airway resistance, a correction of the tube impedance or measurement of the pressure distal of the tube is required. Resistance increased and low frequent reactance decreased significantly with decreasing gas volume. These changes were of magnitudes higher than the variations due to the introduction of the endotracheal tubes. Our results suggest that changes of respiratory reactance measured with impulse oscillometry may be used as a monitoring parameter in intubated patients.

Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O(2) tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O(2)). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5'-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1alpha vector and the PAI-1-LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O(2) deficiency.