Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-gamma-ligand, in insulin-deficient diabetic rats

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-γ in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-γ. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-α. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway.     

Mild Electrical Stimulation with Heat Shock Ameliorates Insulin Resistance via Enhanced Insulin Signaling

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.     

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.     

Escherichia coli cytosolic glycerophosphodiester phosphodiesterase (UgpQ) requires Mg2+, Co2+, or Mn2+ for its enzyme activity

Escherichia coli cytosolic glycerophosphodiester phosphodiesterase, UgpQ, functions in the absence of other proteins encoded by the ugp operon and requires Mg²⁺, Mn²⁺, or Co²⁺, in contrast to Ca²⁺-dependent periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophosphodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ.     

Mechanisms of atrial fibrillation termination by rapidly unbinding Na+ channel blockers: insights from mathematical models and experimental correlates

Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is a problem of growing proportions. Recent studies have increased interest in fast-unbinding Na(+) channel blockers like vernakalant (RSD1235) and ranolazine for AF therapy, but the mechanism of efficacy is poorly understood. To study how fast-unbinding I(Na) blockers affect AF, we developed realistic mathematical models of state-dependent Na(+) channel block, using a lidocaine model as a prototype, and studied the effects on simulated cholinergic AF in two- and three-dimensional atrial substrates. We then compared the results with in vivo effects of lidocaine on vagotonic AF in dogs. Lidocaine action was modeled with the Hondeghem-Katzung modulated-receptor theory and maximum affinity for activated Na(+) channels. Lidocaine produced frequency-dependent Na(+) channel blocking and conduction slowing effects and terminated AF in both two- and three-dimensional models with concentration-dependent efficacy (maximum approximately 89% at 60 microM). AF termination was not related to increases in wavelength, which tended to decrease with the drug, but rather to decreased source Na(+) current in the face of large ACh-sensitive K(+) current-related sinks, leading to the destabilization of primary generator rotors and a great reduction in wavebreak, which caused primary rotor annihilations in the absence of secondary rotors to resume generator activity. Lidocaine also reduced the variability and maximum values of the dominant frequency distribution during AF. Qualitatively similar results were obtained in vivo for lidocaine effects on vagal AF in dogs, with an efficacy of 86% at 2 mg/kg iv, as well as with simulations using the guarded-receptor model of lidocaine action. These results provide new insights into the mechanisms by which rapidly unbinding class I antiarrhythmic agents, a class including several novel compounds of considerable promise, terminate AF.     

Glycosylation of the OMP85 homolog of Porphyromonas gingivalis and its involvement in biofilm formation

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.     

A case of infanticide in the Spot-billed Duck in circumstances of high breeding density

The first observations of infanticide by a precocial avian species, Spot-billed Duck (Anas poecilorhyncha), were recorded on a small pond at Yatsu tidal flat, central Japan, from May to June 2000, where six families of conspecific and one Gadwall A. strepera family, respectively, occupied territories. A female Spot-billed Duck with 11 ducklings attacked ducklings of all the other families killing eight conspecifics and three of the Gadwall. High breeding density (15.0 families/ha) may have increased the aggression of the female. Electronic Publication