Chronic cardiac resynchronization therapy and reverse ventricular remodeling in a model of nonischemic cardiomyopathy

While cardiac resynchronization therapy (CRT) has been shown to reduce morbidity and mortality in heart failure (HF) patients, the fundamental mechanisms for the efficacy of CRT are poorly understood. The lack of understanding of these basic mechanisms represents a significant barrier to our understanding of the pathogenesis of HF and potential recovery mechanisms. Our purpose was to determine cellular mechanisms for the observed improvement in chronic HF after CRT. We used a canine model of chronic nonischemic cardiomyopathy. After 15 months, dogs were randomized to continued RV tachypacing (untreated HF) or CRT for an additional 9 months. Six minute walk tests, echocardiograms, and electrocardiograms were done to assess the functional response to therapy. Left ventricular (LV) midmyocardial myocytes were isolated to study electrophysiology and intracellular calcium regulation. Compared to untreated HF, CRT improved HF-induced increases in LV volumes, diameters and mass (p<0.05). CRT reversed HF-induced prolongations in LV myocyte repolarization (p<0.05) and normalized HF-induced depolarization (p<0.03) of the resting membrane potential. CRT improved HF-induced reductions in calcium (p<0.05). CRT did not attenuate the HF-induced increases in LV interstitial fibrosis. Using a translational approach in a chronic HF model, CRT significantly improved LV structure; this was accompanied by improved LV myocyte electrophysiology and calcium regulation. The beneficial effects of CRT may be attributable, in part, to improved LV myocyte function.     

Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

Possible involvement of organic anion and cation transporters in renal excretion of xanthine derivatives, 3-methylxanthine and enprofylline

Whether organic anion and cation transporters are involved in the renal excretion of xanthine derivatives, 3-methylxanthie and enprofylline, remains unclear. In this study, we have investigated the effects of typically predominant substrates for organic anion and cation transporters on the tubular secretion of 3-methylxanthine and enprofylline in rats. In the renal clearance experiments using typical substrates for organic anion transporters, probenecid and p-aminohippurate, probenecid (20 mg/kg), but not p-aminohippurate (100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. The typical substrates for organic cation transport systems, tetraethylammonium (30.6 mg/kg) and cimetidine (50 or 100 mg/kg), significantly decreased the renal clearance and clearance ratio of 3-methylxanthine and enprofylline. These results suggest that the renal secretory transport of 3-methylxanthine and enprofylline are mediated by probenecid-, cimetidine- and tetraethylammonium-sensitive transport systems. Uric acid, an organic anion, significantly inhibited the renal secretion of 3-methylxanthine, but not enprofylline, suggesting that the renal tubular transport of 3-methylxanthine is also mediated via uric acid-sensitive transport system. These findings suggest the possibility that both organic anion and cation transporters are, at least, involved in the renal tubular transport of 3-methylxanthine and enprofylline in rats.     

Hydrogen Peroxide-Dependent Uptake of Iodine by Marine Flavobacteriaceae Bacterium Strain C-21

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I⁻). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, ¹²⁵I⁻. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.     

Crucial structural factors and mode of action of polyene amides as inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Natural antibiotic polyene amides such as myxalamides are potent inhibitors of mitochondrial complex I. Because of the significant instability of this series of compounds due to an extended pi-conjugation skeleton, a detailed characterization of their inhibitory action has not been performed. To elucidate the action mechanism as well as binding manner of polyene amides with complex I, identification of the roles of each functional group in the inhibitory action is needed. We here synthesized a series of amide analogues and carried out structure-activity studies with bovine heart mitochondrial complex I. With respect to the left-hand portion, the natural pi-conjugation skeleton common to many natural products is not required for the inhibition and can be substituted with a simpler substructure such as a conjugated diene. The geometry and shape of the left-hand portion were shown to be important for the inhibition, suggesting that this portion may bind to a narrow hydrophobic pocket in the enzyme rather than merely partitioning into the lipid membrane phase. Concerning the right-hand portion of the inhibitor, the presence of the 2-methyl, amide NH, and (S)-1'-methyl groups was crucial for the activity, suggesting that both methyl groups neighboring the amide group finely adjust the hydrogen-bonding ability of the amide group. In contrast, modifications of the 2'-OH group did not significantly influence the activity, suggesting that the role of this functional group is not to serve as a hydrogen bond donor to the enzyme but to act as a hydrophilic anchor directing the right-hand portion at or near the membrane surface. Detailed characterization of the action mechanism indicated that the polyene amides share a common binding domain with other complex I inhibitors, though their binding position (or manner) within the domain may differ considerably from that of other inhibitors.     

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

Release of the lipopolysaccharide deacylase PagL from latency compensates for a lack of lipopolysaccharide aminoarabinose modification-dependent resistance to the antimicrobial peptide polymyxin B in Salmonella enterica

Salmonella enterica modifies its lipopolysaccharide (LPS), including the lipid A portion, to adapt to its environments. The lipid A 3-O-deacylase PagL exhibits latency; deacylation of lipid A is not usually observed in vivo despite the expression of PagL, which is under the control of a two-component regulatory system, PhoP-PhoQ. In contrast, PagL is released from latency in pmrA and pmrE mutants, both of which are deficient in aminoarabinose-modified lipid A, although the biological significance of this is not clear. The attachment of aminoarabinose to lipid A decreases the net anionic charge at the membrane's surface and reduces electrostatic repulsion between neighboring LPS molecules, leading to increases in bacterial resistance to cationic antimicrobial peptides, including polymyxin B. Here we examined the effects of the release of PagL from latency on resistance to polymyxin B. The pmrA pagL and pmrE pagL double mutants were more susceptible to polymyxin B than were the parental pmrA and pmrE mutants, respectively. Furthermore, introduction of the PagL expression plasmid into the pmrA pagL double mutant increased the resistance to polymyxin B. In addition, PagL-dependent deacylation of lipid A was observed in a mutant in which lipid A could not be modified with phosphoethanolamine, which partly contributes to the PmrA-dependent resistance to polymyxin B. These results, taken together, suggest that the release of PagL from latency compensates for the loss of resistance to polymyxin B that is due to a lack of other modifications to LPS.     

Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation

Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.     

Electrochemical treatment of anaerobic digestion effluent using a Ti/Pt-IrO2 electrode

Electrochemical treatment of the anaerobic digestion effluents using a Ti/Pt-IrO(2) electrode was evaluated in this study. The effects of electric current, NaCl dosage, and initial pH on ammonia, nitrate, total organic carbon (TOC), inorganic carbon (IC), final pH, and turbidity variations were studied in a series of batch experiments. It was found that the electric current and NaCl dosage had a considerably larger effect on the oxidization of ammonia; this was less for the effect of the initial pH. In addition, electroflotation was the main mechanism for turbidity, TOC, and IC removals. Further, the IC removal was mainly affected by the pH of wastewater. The electrochemical treatment using Ti/Pt-IrO(2) electrode without pretreatment was feasible for the anaerobic digestion effluent.