Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis

Cytochrome P450_BSβ, a H₂O₂-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid–base residue around the heme, which is indispensable for the efficient generation of the active species using H₂O₂. On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450_BSβ, it was suggested that the role of the general acid–base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450_BSβ can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a “decoy molecule”. As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid–base catalyst. This result further confirms that the role of the acid–base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450_BSβ.     

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16^INK4a, p21^WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.     

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.     

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.     

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.     

Natural dental caries in molars of osteogenic disorder Shionogi rats

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.