Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Intermittent hypoxia reduces upper airway stability in lean but not obese Zucker rats

Obstructive sleep apnea involves intermittent periods of airway occlusions that lead to repetitive oxygen desaturations. Exposure to chronic intermittent hypoxia (IH) in rats increases diurnal blood pressure and alters skeletal muscle physiology. The impact of IH on upper airway muscle function is unknown. We hypothesize that IH exposure increases upper airway collapsibility in rats due to alterations of the muscles surrounding the upper airway. Lean and obese rats were exposed to cyclic alterations in O(2) levels (20.6%-5%) every 90 s, 8 h/day for 6 days/wk for 12 wk. Following the exposure period, arterial pressure was recorded via the tail artery in conscious unrestrained rats. Mean arterial pressure was increased in lean IH but not in obese IH-exposed Zucker rats (P < 0.05). The pharyngeal pressure associated with airway collapse (P(crit)) was measured under anesthesia during baseline conditions and then during supramaximal stimulation of the hypoglossal nerve (cnXII). Baseline P(crit) was more positive (more collapsible) in lean but not obese rats following 12 wk of IH (P < 0.05), while supramaximal stimulation of cnXII increased airway stability (decreased P(crit)) in both lean and obese Zucker rats following IH to levels that were similar to their respective room air controls. The in vitro peak tension and the expression of the individual myosin heavy chain isoforms from the upper airway muscles were unaltered following IH. We conclude that IH leads to increases in baseline collapsibility in lean Zucker rats exposed to IH by nonmyogenic mechanisms.     

Detection of single-base mismatch at distal end of DNA duplex by electrochemical impedance spectroscopy

Herein, we report an anomalous electrochemical behavior of surface-bound DNA duplex that has single-base mismatches at its distal end. Single-stranded 15-base DNA was immobilized at its 5'end onto gold electrode surfaces. After hybridization with complementary or mismatched DNA, electrochemical impedance spectra were obtained using [Fe(CN)(6)]3-/4- as redox marker ions. Hybridization with the complementary DNA reduced the charge-transfer resistance (R(CT)), whereas single-base mismatches at the distal end of the duplex largely increased the R(CT). This anomaly was found only with the distal end: the increase in R(CT) was not observed for mismatches at either the middle or the proximal end. These results indicate that electrochemical detection of single-base alterations at an end of sample DNA is exceptionally easy because of the diametrically opposite responses. This detection principle is promising for the typing of single-nucleotide polymorphisms in combination with the single-base primer extension protocol.     

Development of multidrug resistance type I Cmdr1 expression in chicken embryonic gonads

A primary role of P-glycoprotein (P-gp), encoded by the multidrug resistance type I gene, is to protect against naturally occurring xenotoxics. Recently, the preferential expression of chicken multidrug resistance type I (Cmdr1) was identified in the embryonic gonads during the early periods of development. Here we investigated the expression of Cmdr1 and P-gp in the gonads during embryogenesis, and compared to that in the ovarian follicles of domestic hens (Gallus gallus). As revealed by immunohistochemistry, P-gp was highly expressed in theca cells of mature follicles, whereas the expression was low in immature follicles. Immunohistochemical analysis showed that expression of Cmdr1-type P-gp was very low in embryonic gonads. Cmdr1 mRNA was undetectable in the gonads of 5-day embryos (E5) by RT-PCR, whereas Cmdr1 mRNA was significantly detectable in the developing gonads at E9 and E21. In the testicular tissues, germ cells were distributed along developing seminiferous cords as identified by a specific marker gene, whereas Cmdr1-type P-gp positive cells were observed evenly on testicular tissues. Collectively, it is concluded that Cmdr1 expression is initiated in the chicken ovary and testis after sexual differentiation, but expression of Cmdr1-type P-gp is very low through embryogenesis.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.     

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

Background  

Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.     

Redox-active cellulose Langmuir-Blodgett films containing beta-carotene as a molecular wire

Redox-active Langmuir-Blodgett (LB) films containing dihydrophytyl ferrocenoate (DFc) and beta-carotene (betaC) were fabricated by use of 6-O-dihydrophytylcellulose (DHPC) as a matrix. A mixture of DFc-DHPC formed a stable monolayer. Atomic force microscopy images revealed that the DFc molecules were dispersed uniformly throughout the surface in the ratio DFc:DHPC = 2:8 at 30 mN m-1. The DFc-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films. Cyclic voltammograms for the DFc-DHPC LB films on an indium tin oxide (ITO) electrode exhibited a well-defined surface wave. The voltammograms of the DFc-DHPC LB films exhibited 60-40% redox-active ferrocene moieties, whereas those of the DFc-DHPC-betaC LB films exhibited 90-70%. X-ray diffraction patterns indicated that the distance between layers was independent of betaC molecules incorporated into the LB films. Consequently, these results suggested that betaC can function as a molecular wire.