Daily Rhythms of P‐glycoprotein Expression in Mice

Recent studies have shown the gene expression of several transporters to be circadian rhythmic. However, it remains to be elucidated whether the expression of P‐glycoprotein, which is involved in the transport of many medications, undergoes 24 h rhythmicity. To address this issue, we investigated daily profiles of P‐glycoprotein mRNA and protein levels in peripheral mouse tissues. In the liver and intestine, but not in the kidney, Abcb1a mRNA expression showed clear 24 h rhythmicity. On the other hand, Abcb1b and Abcb4, the other P‐glycoprotein genes, did not exhibit significant rhythmic expression in the studied tissues. In the intestine, levels of whole P‐glycoprotein also exhibited a daily rhythm, with a peak occurring in the latter half of the light phase and a trough at the onset of the light phase. Consistent with the day‐night change of P‐glycoprotein level, the ex vivo accumulation of digoxin, an Abcb1a P‐glycoprotein substrate, into the intestinal segments at the onset of dark phase was significantly lower than it was at the onset of the light phase. Thus, Abcb1a P‐glycoprotein expression, and apparently its function, are 24 h rhythmic at least in mouse intestine tissue. This circadian variation might be involved in various chronopharmacological phenomena.     

Effects of nitrates and calcium channel blockers on Ca2+-ATPase in the microsomal fraction of porcine coronary artery smooth muscle cells

The effects of the antianginal drugs nitroglycerin, nicorandil, diltiazem, verapamil and nicardipine on the activity of calcium-stimulated magnesium-dependent ATPase (Ca2+-ATPase) were investigated in the microsomal fraction from porcine coronary artery smooth muscle cells. Two discrete Ca2+-dependent ATPase components were observed: [1] a high affinity component, which was a specific Ca2+-ATPase, [with a half saturation constant for Ca2+ (Km) of 0.44 microM, and maximum velocity (Vmax) of 124.3 pmol of phosphate (Pi) released/micrograms of protein/30 min]: [2] a low affinity component in which Ca2+ could be replaced by Mg2+ without loss of its activity. Nitroglycerin and nicorandil (1 microM and 10 microM) both stimulated the activity of the Ca2+-ATPase significantly [142 +/- 12 (mean +/- standard error), and 137 +/- 10% of the control with nitroglycerin, and 152 +/- 17 and 135 +/- 20% with nicorandil] at a Ca2+ concentration of 0.3 microM. Diltiazem, verapamil and nicardipine did not cause significant stimulation. Nitroglycerin and nicorandil (1 microM), significantly decreased the Km for Ca2+ from the control value of 0.44 +/- 0.06 microM to 0.26 +/- 0.03 and 0.22 +/- 0.03 microM, respectively. Nitroglycerin and nicorandil may dilate coronary arteries by stimulating this Ca2+ extrusion pump enzyme through reduction of intracellular Ca2+ in smooth muscle cells.     

Genetic and Anatomical Basis of the Barrier Separating Wakefulness and Anesthetic-Induced Unresponsiveness

A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness. We previously provided experimental evidence for the existence of a behavioral barrier to transitions between these states of arousal, which we call neural inertia. Here we show that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, we demonstrate that increasing homeostatic sleep drive widens the neural inertial barrier. We propose that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.     

Cancer‐upregulated gene 2 (CUG2) overexpression induces apoptosis in SKOV‐3 cells

Cancer‐upregulated gene 2 (CUG2) was originally identified as a potential oncogene commonly up‐regulated in various human cancers. Recently, CUG2 was also identified as a new member of a centromere protein complex, important in the formation of a functional kinetochore complex. Presently, we report the pro‐apoptotic effect of CUG2 when this gene was overexpressed in the SKOV‐3 human ovarian cancer cell line. Apoptotic cell death mediated by CUG2 overexpression was independently demonstrated using cell viability determination, flow cytometry analysis, chromosome fragmentation assay, and the cleavage of the death substrate poly(ADP‐ribose) polymerase. Moreover, activation of caspase‐3 and ‐8 and the cytoplasmic translocation of mitochondrial cytochrome c were evident upon CUG2 expression. Apoptotic cell death was also observed during early development of zebrafish when CUG2 was overexpressed in zebrafish embryo. We propose that high expression of CUG2 induces apoptotic cell death. Copyright © 2010 John Wiley & Sons, Ltd.     

Clinical Benefit of Liver Stiffness Measurement at 3 Months after Kasai Hepatoportoenterostomy to Predict the Liver Related Events in Biliary Atresia

Background

The progression of hepatic fibrosis may result in decompensated hepatic failure with cirrhosis, liver related events (LRE) such as ascites, variceal bleeding, and death after successful and timely Kasai hepatoportoenterostomy (HPE) in biliary atresia. The aim of this study is to suggest clinical benefit of the liver stiffness measurement (LSM) using transient elastography at 3 months after the Kasai operation to predict LRE.

Methods

Between January 2007 and December 2011, 69 eligible biliary atresia patients who underwent Kasai HPE and performed transient elastography before and 3 months after HPE were included. The occurrences of LRE were analyzed for all patients. All patients were divided into 2 groups (with and without LRE) for comparison. Multivariate analysis was used to detect the independent risk factors of LRE. The area under the receiver operation characteristics curve (AUROC) was used to establish the LSM optimal cutoff value of 3 months after Kasai operation in predicting LRE.

Results

LSM value, aminotransferase, albumin, bilirubin, and PT-INR significantly differed among the two groups. Multivariate analysis demonstrated LSM value as the most powerful independent factor of the development of LRE. The cut-off value of 19.9 kPa was calculated to be optimal for predicting LRE development with total sensitivity and specificity of 1.804. AUROC resulted in 0.943, with sensitivity of 85.3% and specificity of 95.2%.

Conclusions

The LSM value of 3 months after Kasai HPE can be a useful predictor of LRE development.     

Adipose Vascular Endothelial Growth Factor Regulates Metabolic Homeostasis through Angiogenesis

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Highlights? Two-way modulations of adipose VEGF were generated with aP2-Cre transgene ? Adipose VEGF KO reduces vasculature, increases hypoxia and inflammation in fat ? Adipose VEGF KO accelerates the development of metabolic disease in high-fat diet ? Induced adipose VEGF has opposite effect on fat and restores metabolic homeostasis     

Adiponectin Provides Additional Information to Conventional Cardiovascular Risk Factors for Assessing the Risk of Atherosclerosis in Both Genders

Background

This study evaluated the relation between adiponectin and atherosclerosis in both genders, and investigated whether adiponectin provides useful additional information for assessing the risk of atherosclerosis.

Methods

We measured serum adiponectin levels and other cardiovascular risk factors in 1033 subjects (454 men, 579 women) from the Korean Genomic Rural Cohort study. Carotid intima–media-thickness (CIMT) was used as measure of atherosclerosis. Odds ratios (ORs) with 95% confidence intervals (95% CI) were calculated using multiple logistic regression, and receiver operating characteristic curves (ROC), the category-free net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were calculated.

Results

After adjustment for conventional cardiovascular risk factors, such as age, waist circumference, smoking history, low-density and high-density lipoprotein cholesterol, triglycerides, systolic blood pressure and insulin resistance, the ORs (95%CI) of the third tertile adiponectin group were 0.42 (0.25–0.72) in men and 0.47 (0.29–0.75) in women. The area under the curve (AUC) on the ROC analysis increased significantly by 0.025 in men and 0.022 in women when adiponectin was added to the logistic model of conventional cardiovascular risk factors (AUC in men: 0.655 to 0.680, p = 0.038; AUC in women: 0.654 to 0.676, p = 0.041). The NRI was 0.32 (95%CI: 0.13–0.50, p<0.001), and the IDI was 0.03 (95%CI: 0.01–0.04, p<0.001) for men. For women, the category-free NRI was 0.18 (95%CI: 0.02–0.34, p = 0.031) and the IDI was 0.003 (95%CI: −0.002–0.008, p = 0.189).

Conclusion

Adiponectin and atherosclerosis were significantly related in both genders, and these relationships were independent of conventional cardiovascular risk factors. Furthermore, adiponectin provided additional information to conventional cardiovascular risk factors regarding the risk of atherosclerosis.     

Characterization of store-operated Ca channels in pancreatic duct epithelia

Store-operated Ca²⁺ channels (SOCs) are activated by depletion of intracellular Ca²⁺ stores following agonist-mediated Ca²⁺ release. Previously we demonstrated that Ca²⁺ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca²⁺ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca²⁺ influx as Ca²⁺ was released from intracellular stores. Direct measurement of [Ca²⁺] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP₃ or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La³⁺ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca²⁺ entry through SOCs fills internal Ca²⁺ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca²⁺ such as salt and mucin secretion from the exocrine pancreatic ducts.