Loss of the mammal-specific tectorial membrane component CEA cell adhesion molecule 16 (CEACAM16) leads to hearing impairment at low and high frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16-/- mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal day 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16-/- mice tectorial membranes were significantly more often stretched out as compared to wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxy-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of membrane-bound CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 probably can form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea allowing hearing over an extended frequency range

Coupling of alpha(1)-Adrenoceptors to ERK1/2 in the Human Prostate

Introduction: alpha(1)-Adrenoceptors are considered critical for the regulation of prostatic smooth muscle tone. However, previous studies suggested further alpha(1)-adrenoceptor functions besides contraction. Here, we investigated whether alpha(1)-adrenoceptors in the human prostate may activate extracellular signal-regulated kinases (ERK1/2). Methods: Prostate tissues from patients undergoing radical prostatectomy were stimulated in vitro. Activation of ERK1/2 was assessed by Western blot analysis. Expression of ERK1/2 was studied by immunohistochemistry. The effect of ERK1/2 inhibition by U0126 on phenylephrine-induced contraction was studied in organ-bath experiments. Results: Stimulation of human prostate tissue with noradrenaline (30 mu M) or phenylephrine (10 mu M) resulted in ERK activation. This was reflected by increased levels of phosphorylated ERK1/2. Expression of ERK1/2 in the prostate was observed in smooth muscle cells. Incubation of prostate tissue with U0126 (30 mu M) resulted in ERK1/2 inhibition. Dose-dependent phenylephrine-induced contraction of prostate tissue was not modulated by U0126. Conclusions: alpha(1)-Adrenoceptors in the human prostate are coupled to ERK1/2. This may partially explain previous observations suggesting a role of alpha(1)-adrenoceptors in the regulation of prostate growth. Copyright (C) 2011 S. Karger AG, Base

Increased Liver Uptake and Reduced Hepatic Stellate Cell Activation with a Cell-Specific Conjugate of the Rho-kinase Inhibitor Y27632

Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC. Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl(4) model in mice. Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl(4)-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. We conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver

Spironolactone Lowers Portal Hypertension by Inhibiting Liver Fibrosis, ROCK-2 Activity and Activating NO/PKG Pathway in the Bile-Duct-Ligated Rat

OBJECTIVE: Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension. METHODS: Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson's trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo. RESULTS: Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector-protein kinase G (PKG) in the liver. CONCLUSION: Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension

Identification of potential areas for biomass production in China: Discussion of a recent approach and future challenges

A standard methodology is needed to recognize potentially suitable areas for sustainable bioenergy crop production. This facilitates better identification of promising crops and cropping systems, logistical and economic studies, and work needed to meet regulatory criteria. A possible approach is built upon three layers of internationally available spatial data: (1) degrading and abandoned areas, (2) potentially suitable land cover classes, (3) exclusion zones such as nature reserves and areas of high biodiversity. For China, areas identified as potentially suitable range from 1.2 to 6.0% of the national territory, depending on different levels of statistical confidence in degrading area status and allowable limits of terrestrial carbon. Verification on the ground showed that about 60% of points tested conformed to the remote suitability assessment in the scenario, which represents the results for the combination of all degrading areas and a terrestrial carbon stock limit of 200 t ha-1. A top-down approach is useful in framing potentially suitable locations, but a complementary bottom-up analysis is still required to ultimately identify areas for sustainable bio-fuel productio

Intrahepatic upregulation of RhoA and Rho‐kinase signalling contributes to increased hepatic vascular resistance in rats with secondary biliary cirrhosis

BACKGROUND AND AIMS: Portal hypertension in cirrhosis is mediated in part by increased intrahepatic resistance, reflecting an increased sensitivity of the hepatic microvasculature to vasoconstrictors. Activation of the RhoA/Rho‐kinase pathway is essential for contraction of vascular smooth muscle. The aim of this study was to investigate RhoA/Rho‐kinase mediated regulation of the intrahepatic vascular tone in cirrhotic rats. METHODS: Cirrhosis was induced by bile duct ligation (BDL). Hepatic RhoA and Rho‐kinase expressions were studied by real time reverse transcription polymerase chain reaction and western blot analysis. Hepatic Rho‐kinase activity in rat and human livers was assessed as phosphorylation of the Rho‐kinase substrate moesin. The effect of the Rho‐kinase inhibitor Y‐27632 on hepatic perfusion pressure was measured in livers perfused at constant flow. The in vivo effect of intravenous application of Y‐27632 was studied by haemodynamic measurements. RESULTS: Hepatic expressions of RhoA and Rho‐kinase were increased at mRNA and protein level in BDL rats. Intrahepatic moesin phosphorylation was increased in livers from cirrhotic rats and patients with alcohol induced cirrhosis. Y‐27632 reduced the basal perfusion pressure of in situ perfused livers in BDL rats but not in sham operated rats. Y‐27632 reduced the sensitivity to methoxamine in isolated perfused livers in sham operated rats more than in BDL rats. In vivo, Y‐27632 reduced portal pressure to a greater extent in BDL rats than in sham operated rats. Intrahepatic vascular resistance was decreased in response to bolus injection of Y‐27632 in BDL rats but not in sham operated rats. CONCLUSIONS: Upregulation of RhoA and Rho‐kinase contributes to increased intrahepatic resistance in cirrhotic rats and to an increased sensitivity of cirrhotic livers to vasoconstrictors

The Power of Bioenergy-Related Standards to Protect Biodiversity

The sustainable production of bioenergy is vital to avoiding negative impacts on environmental goods such as climate, soil, water, and especially biodiversity. We propose three key issues that should be addressed in any biodiversity risk-mitigation strategy: conservation of areas of significant biodiversity value; mitigation of negative effects related to indirect land-use change; and promotion of agricultural practices with few negative impacts on biodiversity. Focusing on biodiversity concerns, we compared principles and criteria set to address biodiversity and other environmental and social issues in seven standards (defined here as commodity-based standards or roundtables, or relevant European legislation): five voluntary initiatives related to bioenergy feedstocks, the Renewable Transport Fuel Obligation (United Kingdom), and the European Renewable Energy Source Directive. Conservation of areas of significant biodiversity value was fairly well covered by these standards. Nevertheless, mitigation of negative impacts related to indirect land-use change was underrepresented. Although the EU directive, with its bonus system for the use of degraded land and a subquota system for noncrop biofuels, offered the most robust standards to mitigate potential negative effects, all of the standards fell short in promoting agricultural practices with low negative impacts on biodiversity. We strongly recommend that each standard be benchmarked against related standards, as we have done here, and that efforts should be made to strengthen the elements that are weak or missing. This would be a significant step toward achieving a bioenergy industry that safeguards Earth's living heritage