Contribution of the gap junction proteins Connexin40 and Connexin43 to the control of blood pressure

Summary Cells in tissues and organs coordinate their activities by communicating with each other through intercellular channels named gap junctions. These channels are conduits between the cytoplasmic compartments of adjacent cells, allowing the exchange of small molecules which may be crucial for hormone secretion. Renin is normally secreted in a regulated manner by specific cells of the juxtaglomerular apparatus located within the renal cortex. Gap junctional communication may be requisite to maintain an accurate functioning in coordination of renin-producing cells, more especially as renin is of paramount importance for the control of blood pressure. Connexin43 (Cx43) and Cx40 form gap junctions that link in vivo the cells of the juxtaglomerular apparatus. Cx43 links the endothelial cells, whereas gap junctions made of Cx40 connect the endothelial cells, the renin secreting cells, as well as the endothelial cells of to the renin-secreting cells of the afferent arteriole. The observation that loss of Cx40 results in chronic hypertension associated with altered vasomotion and signal conduction along arterioles, has lead us to suggest that connexins may contribute to control blood pressure by participating to the integration of various mechanical, osmotic and electrochemical stimuli involved in the control of renin secretion and by mediating the adaptive changes of the vascular wall induced by elevated blood pressure and mechanical stress. We therefore postulated that the absence of Cx40 could have deleterious effects on the coordinated functioning of the renin-containing cells, hence accounting for hypertension. In the first part of my thesis, we reported that Cx40-deficient mice (Cx40) are hypertensive due to increased plasma renin levels and numbers of renin-producing cells. Besides, we demonstrated that prostaglandins and nitric oxide, which are possible mediators in the regulation of renin secretion by the macula densa, exert a critical role in the mechanisms controlling blood pressure ín Cx40 knockout hypertensive mice. In view of previous studies that stated avessel-specifc increase in the expression of Cx43 during renin-dependent hypertension, we hypothesized that Cx43 channels are particularly well-matched to integrate the response of cells constituting the vascular wall to hypertensive conditions. Using transgenic mice in which Cx43 was replaced by Cx32, we revealed that the replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin and prevent the renin-dependent hypertension which is normally induced in the 2K1C model. To gain insights into the regulation of connexins in two separate tissues exposed to the same fluid pressure, the second part of my thesis work was dedicated to the study of the impact of chronic hypertension and related hypertrophy on the expression of the cardiovascular connexins (Cx40, Cx37, Cx43 and Cx45) in mouse aorta and heart. Our results documented that the expression of connexins is differentially regulated in mouse aorta. according to the models of hypertension. Thus, blood pressure induces mechanical forces that differentially alter the expression of vascular connexins in order to respond to an adaptation of the aortic wall observed under pathological conditions. Altogether these data provide the first evidences that intercellular communication mediated by gap junctions is required for a proper renin secretion from the juxtaglomerular apparatus in order to control blood pressure.

Local Renal Circadian Clocks Control Fluid-Electrolyte Homeostasis and BP.

The circadian timing system is critically involved in the maintenance of fluid and electrolyte balance and BP control. However, the role of peripheral circadian clocks in these homeostatic mechanisms remains unknown. We addressed this question in a mouse model carrying a conditional allele of the circadian clock gene Bmal1 and expressing Cre recombinase under the endogenous Renin promoter (Bmal1(lox/lox)/Ren1(d)Cre mice). Analysis of Bmal1(lox/lox)/Ren1(d)Cre mice showed that the floxed Bmal1 allele was excised in the kidney. In the kidney, BMAL1 protein expression was absent in the renin-secreting granular cells of the juxtaglomerular apparatus and the collecting duct. A partial reduction of BMAL1 expression was observed in the medullary thick ascending limb. Functional analyses showed that Bmal1(lox/lox)/Ren1(d)Cre mice exhibited multiple abnormalities, including increased urine volume, changes in the circadian rhythm of urinary sodium excretion, increased GFR, and significantly reduced plasma aldosterone levels. These changes were accompanied by a reduction in BP. These results show that local renal circadian clocks control body fluid and BP homeostasis.

Pore size distribution in soils irrigated with sodic water and wastewater

Soil porosity, especially pore size distribution, is an important controlling factor for soil infiltration, hydraulic conductivity, and water retention. This study aimed to verify the effect of secondary-treated domestic wastewater (STW) on the porosity of a sandy loam Oxisol in the city of Lins, state of São Paulo, Brazil. The two-year experiment was divided into three plots: soil cultivated with corn and sunflower and irrigated with STW, soil cultivated and irrigated with sodic groundwater, and non-irrigated and non-cultivated soil (control). At the end of the experiment, undisturbed core samples were sampled from 0 to 2.0 m (8 depths). The water retention curves were obtained by tension plates and Richard's pressure plate apparatus, and the pore size distribution inferred from the retention curves. It was found that irrigation with treated wastewater and treated groundwater led to a decrease in microporosity (V MI), defined as the pore class ranging from 0.2 to 50 μm diameter. On the other hand, a significant increase in cryptoporosity (V CRI) (< 0.2 μm) was identified throughout the soil profile. The presence of Na+ in both waters confirmed the role of this ion on pore size distribution and soil moisture (higher water retention).

Morphology, testes development and behaviour of unusual triploid males in microchromosome-carrying clones of Poecilia formosa.

In a microchromosome-carrying laboratory stock of the normally all-female Amazon molly Poecilia formosa triploid individuals were obtained, all of which spontaneously developed into males. A comparison of morphology of the external and internal insemination apparatus and the gonads, sperm ploidy and behaviour, to laboratory-bred F(1) hybrids revealed that the triploid P. formosa males, though producing mostly aneuploid sperm, are partly functional males that differ mainly in sperm maturation and sexual motivation from gonochoristic P. formosa males.

Drug-eluting beads loaded with antiangiogenic agents for chemoembolization: in vitro sunitinib loading and release and in vivo pharmacokinetics in an animal model.

PURPOSE: The combination of embolic beads with a multitargeted tyrosine kinase inhibitor that inhibits tumor vessel growth is suggested as an alternative and improvement to the current standard doxorubicin-eluting beads for use in transarterial chemoembolization. This study demonstrates the in vitro loading and release kinetics of sunitinib using commercially available embolization microspheres and evaluates the in vitro biologic efficacy on cell cultures and the resulting in vivo pharmacokinetics profiles in an animal model. MATERIALS AND METHODS: DC Bead microspheres, 70-150 µm and 100-300 µm (Biocompatibles Ltd., Farnham, United Kingdom), were loaded by immersion in sunitinib solution. Drug release was measured in saline in a USP-approved flow-through apparatus and quantified by spectrophotometry. Activity after release was confirmed in cell culture. For pharmacokinetics and in vivo toxicity evaluation, New Zealand white rabbits received sunitinib either by intraarterial injection of 100-300 µm sized beads or per os. Plasma and liver tissue drug concentrations were assessed by liquid chromatography-tandem mass spectroscopy. RESULTS: Sunitinib loading on beads was close to complete and homogeneous. A total release of 80% in saline was measured, with similar fast-release profiles for both sphere sizes. After embolization, drug plasma levels remained below the therapeutic threshold (< 50 ng/mL), but high concentrations at 6 hours (14.9 µg/g) and 24 hours (3.4 µg/g) were found in the liver tissue. CONCLUSIONS: DC Bead microspheres of two sizes were efficiently loaded with sunitinib and displayed a fast and almost complete release in saline. High liver drug concentrations and low systemic levels indicated the potential of sunitinib-eluting beads for use in embolization.

Determination of hydraulic properties of a tropical soil of Hawaii using column experiments and inverse modeling

A method for determining soil hydraulic properties of a weathered tropical soil (Oxisol) using a medium-sized column with undisturbed soil is presented. The method was used to determine fitting parameters of the water retention curve and hydraulic conductivity functions of a soil column in support of a pesticide leaching study. The soil column was extracted from a continuously-used research plot in Central Oahu (Hawaii, USA) and its internal structure was examined by computed tomography. The experiment was based on tension infiltration into the soil column with free outflow at the lower end. Water flow through the soil core was mathematically modeled using a computer code that numerically solves the one-dimensional Richards equation. Measured soil hydraulic parameters were used for direct simulation, and the retention and soil hydraulic parameters were estimated by inverse modeling. The inverse modeling produced very good agreement between model outputs and measured flux and pressure head data for the relatively homogeneous column. The moisture content at a given pressure from the retention curve measured directly in small soil samples was lower than that obtained through parameter optimization based on experiments using a medium-sized undisturbed soil column.

Electrical conductivity of soil irrigated with swine wastewater estimated by time-domain reflectometry¹

Wastewater application to soil is an alternative for fertilization and water reuse. However, particular care must be taken with this practice, since successive wastewater applications can cause soil salinization. Time-domain reflectometry (TDR) allows for the simultaneous and continuous monitoring of both soil water content and apparent electrical conductivity and thus for the indirect measurement of the electrical conductivity of the soil solution. This study aimed to evaluate the suitability of TDR for the indirect determination of the electrical conductivity (ECse) of the saturated soil extract by using an empirical equation for the apparatus TDR Trase 6050X1. Disturbed soil samples saturated with swine wastewater were used, at soil proportions of 0, 0.45, 0.90, 1.80, 2.70, and 3.60 m³ m-3. The probes were equipped with three handmade 0.20 cm long rods. The fit of the empirical model that associated the TDR measured values of electrical conductivity (EC TDR) to ECse was excellent, indicating this approach as suitable for the determination of electrical conductivity of the soil solution.

Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast.

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Performance of a reciprocal shaker in mechanical dispersion of soil samples for particle-size analysis

The dispersion of the samples in soil particle-size analysis is a fundamental step, which is commonly achieved with a combination of chemical agents and mechanical agitation. The purpose of this study was to evaluate the efficiency of a low-speed reciprocal shaker for the mechanical dispersion of soil samples of different textural classes. The particle size of 61 soil samples was analyzed in four replications, using the pipette method to determine the clay fraction and sieving to determine coarse, fine and total sand fractions. The silt content was obtained by difference. To evaluate the performance, the results of the reciprocal shaker (RSh) were compared with data of the same soil samples available in reports of the Proficiency testing for Soil Analysis Laboratories of the Agronomic Institute of Campinas (Prolab/IAC). The accuracy was analyzed based on the maximum and minimum values defining the confidence intervals for the particle-size fractions of each soil sample. Graphical indicators were also used for data comparison, based on dispersion and linear adjustment. The descriptive statistics indicated predominantly low variability in more than 90 % of the results for sand, medium-textured and clay samples, and for 68 % of the results for heavy clay samples, indicating satisfactory repeatability of measurements with the RSh. Medium variability was frequently associated with silt, followed by the fine sand fraction. The sensitivity analyses indicated an accuracy of 100 % for the three main separates (total sand, silt and clay), in all 52 samples of the textural classes heavy clay, clay and medium. For the nine sand soil samples, the average accuracy was 85.2 %; highest deviations were observed for the silt fraction. In relation to the linear adjustments, the correlation coefficients of 0.93 (silt) or > 0.93 (total sand and clay), as well as the differences between the angular coefficients and the unit < 0.16, indicated a high correlation between the reference data (Prolab/IAC) and results obtained with the RSh. In conclusion, the mechanical dispersion by the reciprocal shaker of soil samples of different textural classes was satisfactory. The results allowed recommending the use of the equipment at low agitation for particle size- analysis. The advantages of this Brazilian apparatus are its low cost, the possibility to simultaneously analyze a great number of samples using ordinary, easily replaceable glass or plastic bottles.

Device for simultaneous measurement of the Peltier and Seebeck coefficients verification of the Kelvin relation

We have designed and built an experimental device, which we called a "thermoelectric bridge." Its primary purpose is simultaneous measurement of the relative Peltier and Seebeck coefficients. The systematic errors for both coefficients are equal with this device and manipulation is not necessary between the measurement of one coefficient and the other. Thus, this device is especially suitable for verifying their linear relation postulated by Lord Kelvin. Also, simultaneous measurement of thermal conductivity is described in the text. A sample is made up of the couple nickel¿platinum, taking measurements in the range of ¿20¿60°C and establishing the dependence of each coefficient with temperature, with nearly equal random errors ±0.2%, and systematic errors estimated at ¿0.5%. The aforementioned Kelvin relation is verified in this range from these results, proving that the behavioral deviations are ¿0.3% contained in the uncertainty ±0.5% caused by the propagation of errors

Graphoepitaxy of CeO2 on MgO and its application to the fabrication of 45° grain boundary Josephson junctions of YBa2Cu3O7-x

We communicate a detailed study of the epitaxial growth of CeO2 on MgO. The key feature of the growth is the dependence of the in¿plane orientation of the CeO2 epitaxial layer on the MgO surface morphology. Atomic force microscopic (AFM) measurements, x¿ray analyses, as well as high¿resolution transmission electron microscopy (HRTEM) investigations reveal that on rough substrates a cube¿on¿cube growth of CeO2 on MgO occurs while on smooth substrates the CeO2 unit cell is rotated around the surface normal by 45° with respect to the MgO unit cell when the deposition rate is low (~0.3 Å/s) during the first stages of growth. This growth mechanism can be used for a defined fabrication of 45° grain boundaries in the CeO2 layer by controlling the surface roughness of the MgO substrate. This report demonstrates that these 45° grain boundaries may be used to fabricate YBa2Cu3O7¿x Josephson junctions.

Idarubicin-loaded ONCOZENE drug-eluting embolic agents for chemoembolization of hepatocellular carcinoma: in vitro loading and release and in vivo pharmacokinetics.

PURPOSE: To present in vitro loading and release characteristics of idarubicin with ONCOZENE (CeloNova BioSciences, Inc, San Antonio, Texas) drug-eluting embolic (DEE) agents and in vivo pharmacokinetics data after transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents in patients with hepatocellular carcinoma. MATERIALS AND METHODS: Loading efficacy of idarubicin with ONCOZENE DEE agents 100 µm and DC Bead (Biocompatibles UK Ltd, Farnham, United Kingdom) DEE agents 100-300 µm was monitored at 10, 20, and 30 minutes loading time by high-pressure liquid chromatography. A T-apparatus was used to monitor the release of idarubicin from the two types of DEE agents over 12 hours. Clinical and 24-hour pharmacokinetics data were recorded after transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents in four patients with unresectable hepatocellular carcinoma. RESULTS: Idarubicin loading in ONCOZENE DEE agents was > 99% at 10 minutes. Time to reach 75% of the release plateau level was 37 minutes ± 6 for DC Bead DEE agents and 170 minutes ± 19 for ONCOZENE DEE agents both loaded with idarubicin 10 mg/mL. After transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents, three partial responses and one complete response were observed with only two asymptomatic grade 3 biologic adverse events. Median time to maximum concentration for idarubicin in patients was 10 minutes, and mean maximum concentration was 4.9 µg/L ± 1.7. Mean area under the concentration-time curve from 0-24 hours was equal to 29.5 µg.h/L ± 20.5. CONCLUSIONS: ONCOZENE DEE agents show promising results with very fast loading ability, a favorable in vivo pharmacokinetics profile with a sustained release of idarubicin during the first 24 hours, and encouraging safety and responses. Histopathologic and clinical studies are needed to evaluate idarubicin release around the DEE agents in tumor tissue and to confirm safety and efficacy.

Urinary hexane diamine to assess respiratory exposure to hexamethylene diisocyanate aerosol: a human inhalation study

The use of urinary hexane diamine (HDA) as a biomarker to assess human respiratory exposure to hexamethylene diisocyanate (HDI) aerosol was evaluated. Twenty-three auto body shop workers were exposed to HDI biuret aerosol for two hours using a closed exposure apparatus. HDI exposures were quantified using both a direct-reading instrument and a treated-filter method. Urine samples collected at baseline, immediately post exposure, and every four to five hours for up to 20 hours were analyzed for HDA using gas chromatography and mass spectrometry. Mean urinary HDA (microg/g creatinine) sharply increased from the baseline value of 0.7 to 18.1 immediately post exposure and decreased rapidly to 4.7, 1.9 and 1.1, respectively, at 4, 9, and 18 hours post exposure. Considerable individual variability was found. Urinary HDA can assess acute respiratory exposure to HDI aerosol, but may have limited use as a biomarker of exposure in the workplace. [Authors]