Ōsaka, Japan
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Fujiwara N.,Tezukayama University | Fujiwara N.,Osaka City University | Ohara N.,Okayama University of Science | Ogawa M.,University of Occupational and Environmental Health Japan | And 6 more authors.
PLoS ONE | Year: 2015

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc2155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG-0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc2155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc2155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells. © 2015 Fujiwara et al.


Naka T.,Osaka City University | Naka T.,MBR Co. | Nakata N.,Japan National Institute of Infectious Diseases | Maeda S.,The Research Institute of Tuberculosis | And 10 more authors.
Journal of Bacteriology | Year: 2011

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2′- hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-L-rhamnose-(1→3)-α-L-rhamnose-(1→3)-α-L-rhamnose-(1→2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections. © 2011, American Society for Microbiology.


Fujiwara N.,Osaka City University | Naka T.,Osaka City University | Naka T.,MBR Co. | Ogawa M.,University of Occupational and Environmental Health Japan | And 4 more authors.
Tuberculosis | Year: 2012

Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc 2155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells. © 2011 Elsevier Ltd. All rights reserved.


Miyamoto Y.,Japan National Institute of Infectious Diseases | Mukai T.,Japan National Institute of Infectious Diseases | Naka T.,Osaka City University | Naka T.,MBR Co. | And 7 more authors.
Journal of Bacteriology | Year: 2010

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Tomida J.,Aichi Gakuin University | Sakamoto D.,Meiji Pharmaceutical University | Sugita T.,Meiji Pharmaceutical University | Fujiwara N.,Osaka City University | And 5 more authors.
Systematic and Applied Microbiology | Year: 2011

Novel actinobacterial strains, PAGU 1247 T, PAGU 1251 and PAGU 1252, were isolated from the skin of atopic dermatitis patients and were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were located within the family Dermacoccaceae. The most closely related species of PAGU 1247 T in phylogenetic terms was Branchiibius hedensis Mer 29717 T, with 16S rRNA gene sequence similarity of 99.6%, although the DNA-DNA relatedness value was less than 43.9%. Some biochemical traits, such as lipase (C14) and α-galactosidase activity, could distinguish these isolates from B. hedensis. Strain PAGU 1247 T contained iso-C 16:0 and brC 18:0 as the major fatty acids. The quinone system consisted of menaquinone MK-8(H 6 and H 4). The G+C content of the genomic DNA was 67.6mol%. On the basis of its phenotypic properties and genetic distinctiveness, strains PAGU 1247 T, PAGU 1251 and PAGU 1252 represents a novel species of the genus Branchiibius, for which the name Branchiibius cervicis sp. nov. is proposed. The type strain is PAGU 1247 T (=NBRC 106593 T=DSM 24166 T). © 2011 Elsevier GmbH.


Fujiwara N.,Osaka City University | Porcelli S.A.,Yeshiva University | Naka T.,Osaka City University | Naka T.,MBR Co. | And 9 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids | Year: 2013

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.


Uenishi Y.,Dainippon Sumitomo | Kusunose N.,Dainippon Sumitomo | Yano I.,Japan BCG Laboratory | Sunagawa M.,MBR Co.
Journal of Microbiological Methods | Year: 2010

A unique hydrolysis method using a two-layer solution, consisting of diluted hydrochloric acid and toluene was developed to isolate whole arabinose mycolates from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) in order to reveal its pivotal role in enhancing immune responses against tumors. © 2010 Elsevier B.V.


The object is to provide a stable SM-108 derivative, which is effective as a carcinostatic agent, particularly an SM-108 derivative having good storage stability. An SM-108 compound having good storage stability can be produced by producing an organic sulfonic acid salt compound of SM-108. Further, a crystalline SM-108 compound containing a trace amount of an organic carboxylic acid can be produced by using an aqueous solution of the organic sulfonic acid salt of SM-108, and by adding an alkali metal salt of an organic carboxylic acid to the aqueous solution to neutralize the aqueous solution and then causing the crystal precipitation in the solution.


Trademark
Mbr Llc | Date: 2011-02-22

Body and beauty care cosmetics, namely, lipstick, lip gloss, blush, eyeshadow, foundation, tinted moisturizer, concealer, soap, facial masks, facial scrubs, lotions, creams, serums, non-medicated anti aging creams for topical use.


Trademark
Mbr Llc | Date: 2014-02-04

Organic body and beauty care cosmetics, namely, lipstick, lip gloss, blush, eye shadow, foundation, tinted moisturizer, concealer, cosmetic preparations for lightening the skin, eyeliner, bronzer, lotions, creams, serums, non-medicated anti-aging creams for topical use.

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