Enhancement of 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3 fed-batch culture

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.     

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.     

Structural basis for recognition of the matrix attachment region of DNA by transcription factor SATB1

Special AT-rich sequence binding protein 1 (SATB1) regulates gene expression essential in immune T-cell maturation and switching of fetal globin species, by binding to matrix attachment regions (MARs) of DNA and inducing a local chromatin remodeling. Previously we have revealed a five-helix structure of the N-terminal CUT domain, which is essentially the folded region in the MAR-binding domain, of human SATB1 by NMR. Here we determined crystal structure of the complex of the CUT domain and a MAR DNA, in which the third helix of the CUT domain deeply enters the major groove of DNA in the B-form. Bases of 5'-CTAATA-3' sequence are contacted by this helix, through direct and water-mediated hydrogen bonds and apolar and van der Waals contacts. Mutations at conserved base-contacting residues, Gln402 and Gly403, reduced the DNA-binding activity, which confirmed the importance of the observed interactions involving these residues. A significant number of equivalent contacts are observed also for typically four-helix POU-specific domains of POU-homologous proteins, indicating that these domains share a common framework of the DNA-binding mode, recognizing partially similar DNA sequences.     

Reconstruction of paleodemographic characteristics from skeletal age at death distributions: perspectives from Hitotsubashi, Japan

This is a demographic exploration of the city of Edo, which reveals the changes that accompanied its urbanization and analyzes the skeletal remains of 207 individuals from a specific site in Tokyo (Hitotsubashi), using several paleodemographic approaches. A comparison of the three methods employed herein suggests that the Bayesian and maximum likelihood estimation techniques provide more plausible mortality patterns than the direct method of age estimation because the direct method of age estimation relies on published age intervals for the auricular surface and that would account for the underestimation of old people relative to the other two methods. Analyses using these new approaches indicate a short life span tendency for the people of Hitotsubashi. Although we cannot rule out methodological problems of adult-age estimation, one plausible interpretation of that life expectancy is an inadequate food supply and a poor public health situation. This study suggests that, in Tokugawa Japan, urbanization might have imposed health risks, increasing the risk of mortality. Analysis of demographic data from Hitotsubashi has refined our understanding on the impact of urbanization on the Edo period, and presents new perspectives on paleodemography in Japan.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by Propionibacterium freudenreichii ET-3 Fed-Batch Culture

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.     

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.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.