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Aims: To investigate the participation of K(ATP) channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis., Main Methods: Eight-week-old male Sprague-Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300 μg/kg intraperitoneally), 30 min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24h period of reperfusion followed. Then, expressions of K(IR)6.1, K(IR)6.2, caspase-3, PARP, Fas, FasL, and K(IR)6.1 and K(IR)6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining., Key Findings: Expressions of K(IR)6.1 protein and mRNA were more than 10-fold of those of K(IR)6.2 protein and mRNA in the testis. IR significantly increased the expressions of K(IR)6.1 protein and mRNA as well as K(IR)6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of K(IR)6.2 expressions between any of the groups., Significance: Treatment with cromakalim has a protective effect against IR-induced testicular damage via activating K(ATP) channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.

Diabetic neuropathy, a major complication of diabetes mellitus, is associated with the development of vascular dysfunction and autonomic neuropathy. We studied the effects of cyclohexenonic long-chain fatty alcohol (FA) on streptozotocin-diabetic hyperreactivity in the rat aorta smooth muscle. The rats were divided randomly into four groups and were maintained for 4 weeks: age-matched control rats, diabetic rats without treatment with FA, and diabetic rats treated with FA (2 and 8 mg/kg, i.p. everyday). The serum glucose and insulin levels were determined, and the contractile responses of the aorta induced by a thromboxane A2 agonist, U46619 and KCl were investigated. Treatment with FA did not alter rats' diabetic status, i.e., body weight, thickness of the aorta, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyperreactivity of the rat aorta in a dose-dependent manner. Removal of endothelium did not change contractile force between groups. In histological examinations, thinning of smooth muscle bundle in the wall of aorta was observed in the diabetic rat, which was not significantly improved by treatment with FA. Our data indicate that FA can prevent hyperreactivity in the diabetic aorta.

In order to investigate the diabetes-associated neuropathy and prevent effects of cyclohexenonic long-chain fatty alcohol, a neurotrophic substance, in trachea, we studied its effect on streptozotocin-diabetic hyper-reactivity in the rat trachea. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of streptozotocin (50 mg/kg). The rats were divided randomly into four groups and were maintained for four weeks: age-matched control rats, diabetic rats without treatment with cyclohexenonic long-chain fatty alcohol, and diabetic rats treated with cyclohexenonic long-chain fatty alcohol (2 and 8 mg/kg, i.p. every day). The serum glucose and insulin levels were determined, and the contractile responses of the trachea induced by carbachol and KCl were investigated. Treatment with cyclohexenonic long-chain fatty alcohol did not alter the rats' diabetic status, i.e., body weight, thickness of the trachea, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyper-reactivity of the rat trachea in a dose-dependent manner. There was no significant difference in either the carbachol- or KCl-induced contractile forces between groups with or without mucosa in the functional studies. In histological examinations, thinning of cricoid cartilage, thickness of basal membrane, and degeneration, fragmentation of elastic fibers in the submucosal layer, and hypertrophy of smooth muscle bundle in the membranous wall of trachea were observed in the diabetic rat trachea, which were improved by treatment with cyclohexenonic long-chain fatty alcohol. Our data indicate that this drug can prevent hyper-reactivity in the diabetic trachea.

We cloned a novel ankyrin repeat protein, Arpp, by immunoscreening a cDNA library constructed from a human esophageal carcinoma cell line, TE1, with an antibody directed to a hypothetical protein encoded by antisense p53 mRNA. Arpp protein is composed of 333 amino acids and contains four ankyrin-like repeat motifs in the middle portion of the protein, a PEST-like sequence and a lysine-rich sequence similar to a nuclear localization signal in the N-terminal region, and a proline-rich region containing consensus phosphorylation sites in the C-terminal region. Protein sequence analysis revealed that Arpp is homologous (52.7% identity) to Carp which is shown to be involved in the regulation of the transcription of the cardiac ventricular myosin light chain 2 gene. Arpp mRNA was found to be expressed in normal skeletal and cardiac muscle. Interestingly, Arpp expression was detectable in bilateral ventricles, but undetectable in bilateral atria and large vessels, suggesting that Arpp may play a specific function in cardiac ventricles as well as skeletal muscles., (Copyright 2001 Academic Press.)