Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Combined association,segregation and aggregation analysis on case-control family data

Recently genetic epidemiologists have begun using case-control family study designs to investigate the role of genetic and environmental risk factors in disease etiology. The objective of these studies is to assess the association of environmental factors with the disease trait; to characterize the disease genes using segregation analysis; and to quantify the residual familial aggregation after controlling for environmental and genetic factors. Typically these objectives are achieved by conducting separate studies and analysis. This paper describes an estimating equation based approach for a combined association, segregation and aggregation analysis on data from case-control family studies. Simulations indicate that the method performs well in a variety of settings. The method is illustrated using simulated family history data made available to participants in a recent Genetic Analysis Workshop.     

Very low C-reactive protein in apparently healthy individuals: physiological status or just a reflection of an improved health profile

The objective of our study was to determine whether the very low concentrations of C-reactive protein (CRP) detected by high-sensitivity CRP (hs-CRP) assays that one encounters from time to time in apparently healthy individual represent a physiological status or are just a reflection of an improved general health profile. The concentration of hs-CRP was determined by using the Behring BN II nephelometer. The arbitrary cut-off point of hs-CRP (≤0.16mgl^-1) was determined at the lower detection level of the assay. A total of 6588 apparently healthy individuals were screened following exclusion of recent infection/inflammation by using a detailed questionnaire. One hundred and sixty (2.4%) individuals out of the above-mentioned cohort presented hs-CRP concentrations of ≤0.16mgl^-1. They were found to be significantly younger and lean, had an improved lipid profile and an attenuated acute-phase response in terms of lower erythrocyte sedimentation rate and fibrinogen concentration as well as white blood cell count. In addition, these individuals had less atherothrombotic risk factors, except for smoking habits which were as frequent as in those of individuals with a higher hs-CRP concentration. After calculating the concentration of this biomarker following multiple adjustments, the individuals with very low CRP remained with a very low value despite the multiplicity of the adjustments. We raise the possibility that this particular low concentration might represent a physiological status and is not necessarily a result of the improved general health profile per se.     

Internal Thoracic Impedance - A Useful Method for Expedient Detection and Convenient Monitoring of Pleural Effusion

Measurement of internal thoracic impedance (ITI) is sensitive and accurate in detecting acute pulmonary edema even at its preclinical stage. We evaluated the suitability of the highly sensitive and noninvasive RS-207 monitor for detecting pleural effusion and for demonstrating increased ITI during its resolution. This prospective controlled study was performed in a single department of internal medicine of a university-affiliated hospital between 2012-2013. One-hundred patients aged 25–96 years were included, of whom 50 had bilateral or right pleural effusion of any etiology (study group) and 50 had no pleural effusion (controls). ITI, the main component of which is lung impedance, was continuously measured by the RS-207 monitor. The predictive value of ITI monitoring was determined by 8 measurements taken every 8 hours. Pleural effusion was diagnosed according to well-accepted clinical and roentgenological criteria. During treatment, the ITI of the study group increased from 32.9±4.2 ohm to 42.8±3.8 ohm (p<0.0001) compared to non-significant changes in the control group (59.6±6.6 ohm, p = 0.24). Prominent changes were observed in the respiratory rate of the study group: there was a decrease from 31.2±4.0 to 19.5±2.4 ohm (35.2%) compared to no change for the controls, and a mean increase from 83.6±5.3%-92.5±1.6% (13.2%) in O2 saturation compared to 94.2±1.7% for the controls. Determination of ITI for the detection and monitoring of treatment of patients with pleural effusion enables earlier diagnosis and more effective therapy, and can prevent hospitalization and serious complications, such as respiratory distress, and the need for mechanical ventilation.

Trial Registration

The study is registered at ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01601444","term_id":"NCT01601444"}}NCT01601444     

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.