Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core,Catalyzed by the GlcNAc-1-phosphate Transferase WecA,by the Novel Caprazamycin Derivative CPZEN-45

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate–GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.     

FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn’s disease through affecting the ADCC activity

An association between FCGR3A-158 V/F polymorphism and biological responses to infliximab has been reported in Crohn’s disease (CD) in Western countries. However, little is known about the mechanism by which gene polymorphism affects the responses to infliximab. The aims of this study were to confirm the association in Japanese CD patients and to reveal the effect of gene polymorphism on biological responses to infliximab. Japanese CD patients were examined retrospectively at weeks 8 and 30. Clinical and biological responses were assessed by the Crohn’s disease activity index and C-reactive protein levels, respectively. The infliximab-binding affinity of natural killer (NK) cells from FCGR3A-158 V/V, V/F and F/F donors was examined. Infliximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activities were also determined using transmembrane TNF-α-expressing Jurkat T cells as target cells and peripheral blood mononuclear cells (PBMCs) from V/V, V/F and F/F donors as effector cells. Biological responses at week 8 were statistically higher in V/V patients, whereas no significant differences were observed in either clinical responses at weeks 8 and 30 or biological responses at week 30 among the three genotypes. NK cells and PBMCs from V/V patients also showed higher infliximab-binding affinity and infliximab-mediated ADCC activity, respectively. Our results suggest that FCGR3A-158 polymorphism is a predicting factor of biological responses to infliximab in the early phases. FCGR3A-158 polymorphism was also found to affect the infliximab-binding affinity of NK cells and infliximab-mediated ADCC activity in vitro, suggesting that an effect on ADCC activity influences biological responses to infliximab in CD patients.     

Identification of the redox partners of ERdj5/JPDI,a PDI family member,from an animal tissue

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.     

Exercise-induced enhancement of insulin sensitivity is associated with accumulation of M2-polarized macrophages in mouse skeletal muscle

Exercise enhances insulin sensitivity in skeletal muscle, but the underlying mechanism remains obscure. Recent data suggest that alternatively activated M2 macrophages enhance insulin sensitivity in insulin target organs such as adipose tissue and liver. Therefore, the aim of this study was to determine the role of anti-inflammatory M2 macrophages in exercise-induced enhancement of insulin sensitivity in skeletal muscle. C57BL6J mice underwent a single bout of treadmill running (20 m/min, 90 min). Twenty-four hours later, ex vivo insulin-stimulated 2-deoxy glucose uptake was found to be increased in plantaris muscle. This change was associated with increased number of CD163-expressing macrophages (i.e. M2-polarized macrophages) in skeletal muscle. Systemic depletion of macrophages by pretreatment of mice with clodronate-containing liposome abrogated both CD163-positive macrophage accumulation in skeletal muscle as well as the enhancement of insulin sensitivity after exercise, without affecting insulin-induced phosphorylation of Akt and AS160 or exercise-induced GLUT4 expression. These results suggest that accumulation of M2-polarized macrophages is involved in exercise-induced enhancement of insulin sensitivity in mouse skeletal muscle, independently of the phosphorylation of Akt and AS160 and expression of GLUT4.     

Aroma Components of Baelfruit (Aegle marmelos CORREA)

The attractive and characteristically sweet aroma components of baelfruit—a tropical fruit— were investigated. The aroma concentrates possessing the sweet floral and somewhat terpene-like aroma were obtained from both the pulp and peel of fresh baelfruits by means of lyophilization and ether extraction, being analyzed mainly by GC-MS, A total of 39 components were identified. Among these components, terpene alcohols and β-βonone were considered to contribute to the aroma of baelfruit. At optimum ripeness, the fruit with excellent flavor contained a large quantity of an isomeric compound of 3,7-dimethyl-1,5,7-octatrien-3-ol. This compound couldn’t be found in unripe fruit, and seems to be ?mportant in making the baelfruit flavor attractive.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

Syntheses and Biological Activities of 1-O-Glucopyranosyl Fatty Acid Esters

1-O-Palmitoyl-d-glucopyranose was prepared by the selective 1-O-acylation of 4,6-O-benzylideneglucose followed by hydrogenolysis of the protecting group. 1-O-Oleoyl-d-glucopyranose was synthesized from the corresponding benzylidene derivative by selective hydrolysis in acetic acid. This procedure constitutes a useful method for the synthesis of 1-O-acyl-d-glucopyranoses containing unsaturated carboxylic acids. However, 4,6-O-benzylidene-l-O-linolenoyl-d-glucopyranose was converted to 3-O-linolenoyl-d-glucopyranose by the acidic hydrolysis due to acyl migration.

Synthesized glucosyl esters were inactive in the bean second-internode bioassay. However, it was found that 3-O-linolenoyl-d-glucopyranose had a promoting activity on germination of pollen and growth of pollen tube.     

Candidusin A and B: New p-Terphenyls with Cytotoxic Effects on Sea Urchin Embryos

Three fungal trichothecenes, verrucarin A, roridin A and 8-β-hydroxyroridin E, were isolated as callus-initiating promoters from Myrothecium sp. 301. These trichothecenes promoted callus induction, synergistically coupled with a low concentration of 2,4-dichlorophenoxyacetic acid.     

X-Ray Crystal Structure of Pyrenocine A,a Phytotoxin from Pyrenochaeta terrestris

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.