Analysis of a Clostridium josui cellulase gene cluster containing the man5A gene and characterization of recombinant Man5A

A cellulase gene cluster of Clostridium josui was sequenced, and was found to encode 11 proteins responsible for cellulosome (cellulolytic complex) formation, viz., cipA, cel48A, cel8A, cel9A, cel9B, orfX, cel9C, cel9D, man5A, cel9E, and cel5B, in order from the upstream side. All the predicted enzymes had a dockerin module, suggesting that these proteins are members of the C. josui cellulosome. Among these genes, the man5A gene encoding β-mannanase was expressed in Escherichia coli and the recombinant enzyme (rMan5A) was characterized. rMan5A showed strong activity toward carob galactomannan and low activity toward guar gum, suggesting that it prefers non-galactosylated mannan to galactomannan. This enzyme hydrolyzed ivory nut mannan to produce mainly mannotriose and larger mannooligosaccharides, and was not active toward mannotriose. An antiserum raised against the recombinant enzyme detected Man5A in the culture supernatants of C. josui, which was grown on either ball-milled cellulose or glucose as a carbon source.     

Synthesis of β-D-fructopyranosyl-(2→6)-D-glucopyranose from D-glucose and D-fructose by a thermal treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Involvement of endotoxin in the mortality of mice with gut‐derived sepsis due to methicillin‐resistant Staphylococcus aureus

MRSA causes a wide diversity of diseases, ranging from benign skin infections to life‐threatening diseases, such as sepsis. However, there have been few reports of the pathophysiology and mechanisms of sepsis resulting from the gut‐derived origin of MRSA. Therefore, we established a murine model of gut‐derived sepsis with MRSA and factors of MRSA sepsis that cause deterioration. We separated mice into four groups according to antibiotic treatment as follows: (i) ABPC 40 mg/kg; (ii) CAZ 80 mg/kg; (iii) CAZ 80 mg/kg + endotoxin 10 μg/mouse; and (iv) saline‐treated control groups. Gut‐derived sepsis was induced by i.p. injection of cyclophosphamide after colonization of MRSA strain 334 in the intestine. After the induction of sepsis, significantly more CAZ‐treated mice survived compared with ABPC‐treated and control groups. MRSA were detected in the blood and liver among all groups. Endotoxin levels were significantly lower in the CAZ‐treated group compared to other groups. Inflammatory cytokine levels in the serum were lower in the CAZ‐treated group compared to other groups. Fecal culture showed a lower level of colonization of E. coli in the CAZ‐treated group compared to other groups. In conclusion, we found that CAZ‐treatment ameliorates infection and suppresses endotoxin level by the elimination of E. coli from the intestinal tract of mice. However, giving endotoxin in the CAZ‐treated group increased mortality to almost the same level as in the ABPC‐treated group. These results suggest endotoxin released from resident E. coli in the intestine is involved in clinical deterioration resulting from gut‐derived MRSA sepsis.     

Labeling study of avidin by modular method for affinity labeling (MoAL)

We studied the specific labeling of avidin with biotinylated modular ligand catalysts via MoAL, which we recently established. The labeling yield was found to depend on the linker length connecting the catalytic site to biotin in the modular ligand catalyst 1, and the maximum yield was obtained with 1d possessing octamethylene linker. The labeling reaction reached a maximum rate with only 4 equiv of the ligand catalyst. Presumably, all the subunits of avidin with homotetrameric structure formed a stable complex with 4 equiv of the catalyst because of the extremely high affinity. The ligand catalyst bound to avidin first catalyzed N-triazinylation of the ε-amino group of Lys111, and the resulting regenerated catalyst then catalyzed the reaction of Asp108 and CDMT.     

6,7-Dihydroxy-4-phenylcoumarin as inhibitor of aldose reductase 2

We report the structure–activity relationship of a series of coumarins as aldose reductase 2 (ALR2) inhibitors and their suppressive effect on the accumulation of galactitol in the rat lens. We evaluated their ALR2 selectivity profile against sorbitol dehydrogenase and aldehyde reductase (ALR1). Our study revealed that substitutions in the C7 OH group enhanced the potency toward ALR2, while the C6 OH group interferes with ALR1 inhibition activity. Having the phenyl moiety at C4 leads to improved potency and improved selectivity. A molecular docking study suggested that 6,7-dihydroxy-4-phenylcoumarin (15) binds to ALR2 in a different manner from epalrestat. Furthermore, compound 15 clearly suppressed galactitol accumulation in a dose-dependent manner. These results provide an insight into the structural requirements of coumarins for developing a new-type of selective ALR2 inhibitor.     

Investigation of Beckett–Casy model 3: Synthesis of novel naltrexone derivatives with contracted and expanded D-rings and their pharmacology

Novel naltrexone derivatives 7 and 8 with contracted and expanded D-rings were synthesized to investigate the importance of orientation of lone electron pair on the nitrogen for binding abilities to the opioid receptor. Compound 7 showed almost no binding affinity, whereas compound 8 was comparable to naltrexone (6) in binding affinity. Conformational analyses and NOE experiments in D₂O of compounds 6–8 suggested that the lone electron pairs of compounds 6 and 8 with respective six- and seven-membered D-rings would project in the pseudo-axial orientation, whereas compound 7 with five-membered D-ring would have the lone electron pair directing in pseudo-equatorial position. These results strongly supported the proposal that the axial orientation of the lone electron pair on nitrogen would provide sufficient binding abilities to the opioid receptor and that the 15–16 ethylene moiety in the morphine structure would play a role in fixation of the lone electron pair in the axial direction rather than interaction with the putative cavity in the Beckett–Casy model.     

Drug design and synthesis of a novel κ opioid receptor agonist with an oxabicyclo[2.2.2]octane skeleton and its pharmacology

A conformational analysis of κ opioid receptor agonists, TRK-820 and U-50,488H indicated an active conformation of TRK-820 in which the C-ring was in the boat form with the 14-OH interacting with the amide nitrogen. Based on the obtained active conformation of TRK-820, we designed and synthesized a novel κ agonist KNT-63 with oxabicyclo[2.2.2]octane skeleton. KNT-63 showed profound antinociceptive effects via the κ receptor which were as potent as that of TRK-820.     

Development of novel bisubstrate-type inhibitors of histone methyltransferase SET7/9

Histone modification, for example, by histone deacetylase (HDAC) and histone lysine methyltransferase (HMT), plays an important role in regulating gene expression. To obtain novel inhibitors as tools for investigating the physiological function of members of the HMT family, we designed and synthesized novel inhibitors, which are amine analogues of adenosylmethionine (AdoMet; the cofactor utilized in the methylation reaction) bearing various alkylamino groups coupled via an ethylene linker. The inhibitory activities of these compounds towards SET7/9, an HMT, were evaluated. It was found that introduction of an alkylamino group increased the inhibitory activity.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 2: Optimization of the side chains to improve in vitro and in vivo potencies

A series of 3-[2-{[(3-methyl-1-phenylbutyl)amino]carbonyl}-4-(phenoxymethyl)phenyl]propanoic acid analogs were synthesized and evaluated for their in vitro potency. In most cases, introduction of one or two substituents into the two phenyl moieties resulted in the tendency of an increase or retention of in vitro activities. Several compounds, which showed excellent subtype selectivity, were evaluated for their inhibitory effect against PGE₂-induced uterine contraction in pregnant rats, which is thought to be mediated by the EP3 receptor subtype. The structure–activity relationships (SARs) are also discussed.