Conductive and Kinetic Properties of Connexin45 Hemichannels Expressed in Transfected HeLa Cells

Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca²⁺]_o revealed an extra current. Its sensitivity to extracellular Ca²⁺ and gap junction channel blockers (18α-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, I _hc, were determined adopting a biphasic pulse protocol. I _hc activated at positive V _m and deactivated partially at negative V _m. The analysis of the instantaneous I _hc yielded a linear function g _hc,inst = f(V _m) with a hint of a negative slope (g _hc,inst: instantaneous conductance). The analysis of the steady-state I _hc revealed a sigmoidal function g _hc,ss = f(V _m) best described with the Boltzmann equation: V _m,0 = −1.08 mV, g _hc,min = 0.08 (g _hc,ss: steady-state conductance; V _{m, 0}:V _m at which g _hc,ss is half-maximally activated; g _hc,min: minimal conductance; major charge carriers: K⁺ and Cl⁻). The g _hc was minimal at negative V _m and maximal at positive V _m. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. I _hc deactivated exponentially with time, giving rise to single time constants, τ_d. The function τ_d = f(V _m) was exponential and increased with positive V _m (τ_d = 7.6 s at V _m = 0 mV). The activation of I _hc followed the sum of two exponentials giving rise to the time constants, τ_a1 and τ_a2. The function τ_a1 = f(V _m) and τ_a2 = f(V _m) were bell-shaped and yielded a maximum of ≅ 0.6 s at V _m ≅ −20 mV and ≅ 4.9 s at V _m ≅ 15 mV, respectively. Neither τ_a1 = f(V _m) nor τ_a2 = f(V _m) coincided with τ_d = f(V _m). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.     

Horseflies of Jordan

The horsefly (Diptera: Tabanidae) fauna of Jordan consists of 21 species belonging to seven genera. The present study adds 17 new records to this little-known group of haematophagous insects. Most of the new records were collected from several localities within the Jordan Valley. These new records are Chrysops flavipes Meigen, Atylotus farinosus (Szilády), Dasyrhamphis umbrinus (Meigen), Haematopota coronata Austen, Hybomitra decora (Loew), Hybomitra mendica (Villeneuve), Tabanus accensus Austen, T. albifacies Loew, T. bifarius Loew, T. darimonti Leclercq, T. laetetinctus Becker, T. leleani Austen, T. pallidipes Austen, T. regularis Jaennicke, T. rupinae Austen, T. sufis Jaennicke and Therioplectes tunicatus (Szilády). Tabanus albifacies and T. sufis were the most common species collected by hand nets (19.85% and 17.73%, respectively) and D. umbrinus, Haematopota minuscula Austen, and Haematopota coronata were the least common species (0.12% for each). Zoogeographical analysis of the recorded species showed that 12 species are of Mediterranean origin, four are of Afrotropical origin, two of European origin and at least three species can be considered as endemic to the Middle East.     

Cloning and characterization of an inversion breakpoint at 6q23.3 suggests a role for Map7 in sacral dysgenesis

Here we report on a male patient with sacral dysgenesis (SD) and constitutional pericentric inversion of chromosome 6 (p11.2;q23.3). SD is a heterogeneous group of congenital anomalies with complex genetic etiology. Previously, a patient with sacral abnormalities and an interstitial deletion of 6q23-->q25 region has been described. We speculated that a susceptibility gene for SD lies in 6q23.3 region (disrupted in both patients), and therefore, cloning of the breakpoint in our patient would lead to the identification of the disrupted gene. We performed FISH analysis followed by Southern blot analysis and inverse PCR to clone the breakpoint. The 6p11.2 breakpoint mapped very close to the centromere, and the 6q23.3 breakpoint localized in the ninth intron of the MAP7 gene. We then evaluated the involvement of MAP7 in SD by further screening of the gene in several patients with a similar phenotype. Two nucleotide changes causing Ile257Asn and Glu571Ala substitutions in the protein, both affecting amino acid residues conserved in the mouse homolog, were identified in two patients. Both changes are either very rare polymorphisms or true mutations, since they were not detected in 167 normal individuals nor found in the SNP database. Therefore, our study suggests MAP7 as a candidate gene for SD. However, we were unable to detect any sacral defects in the MAP7 knockout mice.     

Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Background  

The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision.     

Chronic endothelin receptor blockade reduces end-organ damage independently of blood pressure effects in salt-loaded heterozygous Ren-2 transgenic rats

The present study was performed to evaluate the role of an interaction between the endothelin (ET) and the renin-angiotensin systems (RAS) in the development and maintenance of hypertension and in hypertension-associated end-organ damage in heterozygous male and female transgenic rats harboring the mouse Ren-2 renin gene (TGR). Twenty-eight days old heterozygous TGR and age-matched transgene-negative normotensive Hannover Sprague-Dawley rats (HanSD) were randomly assigned to groups with normal-salt (NS) or high-salt (HS) intake. Nonselective ET(A)/ET(B) receptor blockade was achieved with bosentan (100 mg.kg(-1).day(-1)). All male and female HanSD as well as heterozygous TGR on NS exhibited 100 % survival rate until 180 days of age (end of experiment). HS diet in heterozygous TGR induced a transition from benign to malignant phase hypertension. The survival rates in male and in female heterozygous TGR on the HS diet were 46 % and 80 %, respectively, and were significantly improved by administration of bosentan to 76 % and 97 %, respectively. Treatment with bosentan did not influence either the course of hypertension (measured by plethysmography in conscious animals) or the final levels of blood pressure (measured by a direct method in anesthetized rats) in any of the experimental groups of HanSD or TGR. Administration of bosentan in heterozygous TGR fed the HS diet markedly reduced proteinuria, glomerulosclerosis and attenuated the development of cardiac hypertrophy compared with untreated TGR. Our data show that the ET receptor blockade markedly improves the survival rate and ameliorates end-organ damage in heterozygous TGR exposed to HS diet. These findings indicate that the interaction between the RAS and ET systems plays an important role in the development of hypertension-associated end-organ damage in TGR exposed to salt-loading.     

Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species

Hypoxia sensing and related signaling events, including activation of hypoxia-inducible factor 1 (HIF-1), represent key features in cell physiology and lung function. Using cultured A549 cells, we investigated the role of NAD(P)H oxidase 1 (Nox1), suggested to be a subunit of a low-output NAD(P)H oxidase complex, in hypoxia signaling. Nox1 expression was detected on both the mRNA and protein levels. Upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by enhanced reactive oxygen species (ROS) generation. A549 cells, which were transfected with a Nox1 expression vector, revealed an increase in ROS generation accompanied by activation of HIF-1-dependent target gene expression (heme oxygenase 1 mRNA, hypoxia-responsive-element reporter gene activity). In A549 cells stably overexpressing Nox1, accumulation of HIF-1alpha in normoxia and an additional increase in hypoxia were noted. Interference with ROS metabolism by the flavoprotein inhibitor diphenylene iodonium (DPI) and catalase inhibited HIF-1 induction. This suggests that H2O2 links Nox1 and HIF-1 activation. We conclude that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways.     

Altered neutrophil homeostasis in kinin B1 receptor-deficient mice

The kallikrein-kinin system is activated during inflammation and plays a major role in the inflammatory process. One of the main mechanisms of kinin action includes the modulation of neutrophil function employing both receptors for kinins, B1 and B2. In this report we show by the use of B1 receptor-deficient mice that neutrophil migration in inflamed tissues is dependent on kinin B1 receptors. However, there is no change in circulating leukocyte number and composition after genetic ablation of this receptor. Furthermore, apoptosis of neutrophils necessary for the resolution of persistent inflammatory processes is impaired in mice lacking the B1 receptor. We also show that this receptor is expressed on neutrophils, thus it may be directly involved in the induction of apoptosis in these cells after prolonged activation at inflamed sites. In conclusion, our data show that the kinin B1 receptor modulates migration and the life span of neutrophils at sites of inflammation and may be therefore an important drug target in the therapy of inflammatory diseases.     

Determination of molecular changes in soft tissues under strain using laser Raman microscopy

The paper presents a non-contact technique to examine the molecular changes in a collagen fibre subjected to in vitro axial tension. Laser Raman microscopy was employed to monitor the vibrational changes in specific assignments of the Raman spectrum of collagen. Results were presented in the form of Raman wavenumber shift as a function of applied tensile strain. Two distinct responses were observed depending on whether the vibrations were axial to, or normal to, the collagen backbone. The former response produced a decrease in wavenumber values, indicating tension, whereas the latter produced an increase, indicating compression. The rate of wavenumber shift with applied strain was non-linear in form, with a marked increase at higher levels of applied strain, for example, a strain 4% in the case of axial vibrations. This technique can prove to be a powerful tool for examining deformation at the molecular level in collagenous tissues.