Biomedical subjects

Nagatoshi Fujiwara

Publications and source records attributed to Nagatoshi Fujiwara.

16 recordsLinked to original sources

PubMed2006

Structural characterization of a specific glycopeptidolipid containing a novel N-acyl-deoxy sugar from mycobacterium intracellulare serotype 7 and genetic analysis of its glycosylation pathway.

The nontuberculous Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is distributed ubiquitously in the environment and is an important cause of respiratory and lymphatic disease in humans and animals. These species produce polar glycopeptidolipids (GPLs), and of particular interest is their serotype-specific antigenicity. Structurally, GPLs contain an N-acylated tetrapeptide-amino alcohol core that is glycosylated at the C terminal with 3,4-di-O-methyl rhamnose and at the d-allo-threonine with a 6-deoxy-talose. This serotype nonspecific GPL is found in all MAC species. The serotype-specific GPLs are further glycosylated with a variable haptenic oligosaccharide at 6-deoxy-talose. At present, 31 distinct serotype-specific GPLs have been identified on the basis of oligosaccharide composition, and the complete structures of 14 serotype-specific GPLs have been defined. It is considered that the modification of the GPL structure plays an important role in bacterial physiology, pathogenesis, and host immune responses. In this study, we defined the complete structure of a novel serotype 7 GPL that has a unique terminal amido sugar. The main molecular mass is 1,874, and attached to the tetrapeptide-amino alcohol core is the serotype 7-specific oligosaccharide unit of 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-2-O-methyl-beta-hexose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->3)-alpha-l-rhamnose-(1-->2)-alpha-l-6-deoxy-talose. Moreover, we isolated and characterized the serotype 7-specific gene cluster involved in glycosylation of the oligosaccharide. Nine open reading frames (ORFs) were observed in the cluster. Based on the sequence homology, the ORFs are thought to participate in the biosynthesis of the serotype 7 GPL.

Carbohydrate Sequence↗

PubMed2006

Neutrophil-activating activity and platelet-activating factor synthesis in cytokine-stimulated endothelial cells: reduced activity in growth-arrested cells.

The reactivity of endothelial cells (ECs) to proinflammatory cytokines is critically important for the pathogenesis of vascular diseases. Here, we studied functional alterations of human ECs during culture under a confluent condition; i.e., the alterations of neutrophil-activating activity, platelet-activating factor (PAF) synthesis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in cytokine-stimulated ECs. Human umbilical vein-derived ECs exhibited the increased activity in neutrophil activation, PAF synthesis, and GM-CSF production when stimulated by proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). The activity of cytokine-stimulated ECs to stimulate superoxide release in human neutrophils and to produce PAF declined markedly in parallel as ECs became growth-arrested during culture under a confluent condition. By contrast, GM-CSF production induced by cytokine stimulation was modestly increased, and up-regulation of intercellular adhesion molecule-1 (ICAM-1) and activation of mitogen-activated protein kinases were not altered. The neutrophil-activating activity of cytokine-stimulated ECs was dependent on PAF synthesis and GM-CSF production from ECs. These findings indicate that the reduced neutrophil-activating activity in growth-arrested ECs may be, at least in part, ascribed to down-regulation of PAF synthesis.

Antibodies↗

PubMed2006

Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex.

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).

Bacterial Proteins↗

PubMed2005

Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides.

A form of alpha-galactosylceramide, KRN7000, activates CD1d-restricted Valpha14-invariant (Valpha14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0 N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of Valpha14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-gamma production and reduced Valpha14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of Valpha14i NKT cell agonists may prove to be useful in immunotherapeutic applications.

Animals↗

PubMed2005

Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule.

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

Animals↗

PubMed2005

Use of glycopeptidolipid core antigen for serodiagnosis of mycobacterium avium complex pulmonary disease in immunocompetent patients.

We report the development of a serodiagnostic method for Mycobacterium avium complex (MAC) disease with an enzyme immunoassay (EIA) with the MAC-specific glycopeptidolipid (GPL) core as the antigen. In this study, we confirmed by EIA that the GPL core antibody was in the sera of immunocompetent patients with MAC disease. The EIA for quantifying the GPL core antibody was evaluated as a clinical tool for serodiagnosis of pulmonary MAC disease. A significant increase in GPL core antibodies (immunoglobulins G, A, and M) was detected in sera of patients with MAC pulmonary diseases when they were compared to patients who were colonized with MAC, patients with Mycobacterium kansasii disease or tuberculosis, and healthy subjects. The sensitivities and specificities of the GPL core-based EIA for diagnosis of MAC pulmonary disease were 72.6% and 92.2%, respectively, for IgG, 92.5% and 95.1%, respectively, for IgA, and 78.3% and 91.0%, respectively, for IgM. The best sensitivity and specificity were obtained by measuring immunoglobulin A antibodies against GPL core antigen. The level of GPL core antibodies reflected disease activity, since it decreased in cured MAC patients who had responded to chemotherapy. Measurement of serum antibodies against GPL core is useful for both diagnosis and assessment of disease activity in MAC disease of the lung.

Adult↗

PubMed2005

Macrophages in inflammation.

The inflammatory process is usually tightly regulated, involving both signals that initiate and maintain inflammation and signals that shut the process down. An imbalance between the two signals leaves inflammation unchecked, resulting in cellular and tissue damage. Macrophages are a major component of the mononuclear phagocyte system that consists of closely related cells of bone marrow origin, including blood monocytes, and tissue macrophages. From the blood, monocytes migrate into various tissues and transform macrophages. In inflammation, macrophages have three major function; antigen presentation, phagocytosis, and immunomodulation through production of various cytokines and growth factors. Macrophages play a critical role in the initiation, maintenance, and resolution of inflammation. They are activated and deactivated in the inflammatory process. Activation signals include cytokines (interferon gamma, granulocyte-monocyte colony stimulating factor, and tumor necrosis factor alpha), bacterial lipopolysaccharide, extracellular matrix proteins, and other chemical mediators. Inhibition of inflammation by removal or deactivation of mediators and inflammatory effector cells permits the host to repair damages tissues. Activated macrophages are deactivated by anti-inflammatory cytokines (interleukin 10 and transforming growth factor beta) and cytokine antagonists that are mainly produced by macrophages. Macrophages participate in the autoregulatory loop in the inflammatory process. Because macrophages produce a wide range of biologically active molecules participated in both beneficial and detrimental outcomes in inflammation, therapeutic interventions targeted macrophages and their products may open new avenues for controlling inflammatory diseases.

Animals↗

PubMed2004

Rothia aeria sp. nov., Rhodococcus baikonurensis sp. nov. and Arthrobacter russicus sp. nov., isolated from air in the Russian space laboratory Mir.

Four Gram-positive bacteria, strains A1-17B(T), A1-22(T), A1-3(T) and A1-8, isolated from the air in the Russian space laboratory Mir, were subjected to a polyphasic taxonomic study. Phylogenetic analysis of the bacteria based on their 16S rDNA sequence showed that they belong to the genera Rothia (A1-17B(T)), Rhodococcus (A1-22(T)) and Arthrobacter (A1-3(T) and A1-8). Morphological, physiological, chemotaxonomic and genomic characteristics supported the assignments of these strains to these genera, but they could not be classified as any existing species within each respective genus. 16S rDNA similarity values between strain A1-17B(T) and its neighbours, Rothia dentocariosa genomovar II, Rothia dentocariosa, Rothia mucilaginosa and Rothia nasimurium, were respectively 99.8, 98.0, 96.4 and 95.4 %. Polyphasic taxonomic evidence indicated that strain A1-17B(T) should be categorized together with the unofficially named Rothia dentocariosa genomovar II, but clearly differentiated them from the established species of the genus ROTHIA: Strain A1-22(T) formed a coherent cluster with Rhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus marinonascens and Rhodococcus percolatus in 16S rDNA sequence analysis, but DNA-DNA relatedness values were only 45.5, 35.3, 18.9 and 21.9 %. Strains A1-3(T) and A1-8 shared 99.9 % 16S rDNA sequence similarity, and strain A1-3(T) showed the highest level of 16S rDNA similarity, 96.6 %, to Arthrobacter polychromogenes. Contrasting biochemical characteristics were also identified. Finally, as a result of the polyphasic taxonomic study, three of the strains are proposed as type strains of novel species: Rothia aeria sp. nov. (A1-17B(T)=GTC 867(T)=JCM 11412(T)=DSM 14556(T)), Rhodococcus baikonurensis sp. nov. (A1-22(T)=GTC 1041(T)=JCM 11411(T)=DSM 44587(T)) and Arthrobacter russicus sp. nov. (A1-3(T)=GTC 863(T)=JCM 11414(T)=DSM 14555(T)).

Air Microbiology↗

PubMed2004

Sphingomonas yabuuchiae sp. nov. and Brevundimonas nasdae sp. nov., isolated from the Russian space laboratory Mir.

On the basis of phenotypic and genotypic characteristics and 16S rRNA gene sequence analysis, novel species belonging to the genera Sphingomonas and Brevundimonas were identified from samples taken from the Russian space laboratory Mir. Strain A1-18(T) was isolated from the air. 16S rDNA sequence analysis showed that strain A1-18(T) formed a coherent cluster with Sphingomonas sanguinis, Sphingomonas parapaucimobilis, Sphingomonas paucimobilis and Sphingomonas roseiflava with sequence similarity of 97.5-98.6 %. Similar to other Sphingomonas species, the G+C content was 66.1 mol%, but DNA-DNA hybridization rates at optimal temperatures among these related species were only 24.7-51.7 %. Strain A1-18(T) can be differentiated biochemically from related species. Strain W1-2B(T) was isolated from condensation water. It forms a distinct lineage within the genus Brevundimonas, forming a coherent cluster with Brevundimonas vesicularis, Brevundimonas aurantiaca and Brevundimonas intermedia. 16S rDNA sequence similarities were 98.6-99.5 % and the G+C content was 66.5 mol%, similar to other Brevundimonas species, but DNA-DNA relatedness was only 50.2-54.8 %. Strain W1-2B(T) also showed some differential biochemical properties from its related species. A series of polyphasic taxonomic studies led to the proposal of two novel species, Sphingomonas yabuuchiae sp. nov. (type strain A1-18(T)=GTC 868(T)=JCM 11416(T)=DSM 14562(T)) and Brevundimonas nasdae sp. nov. (type strain W1-2B(T)=GTC 1043(T)=JCM 11415(T)=DSM 14572(T)).

Air Microbiology↗

PubMed2003

Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium.

The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T). To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells.

Ceramides↗

PubMed2003

Chryseobacterium miricola sp. nov., a novel species isolated from condensation water of space station Mir.

Classification of strain W3-B1, which was isolated from condensation water in the Russian space laboratory Mir, was investigated by a polyphasic taxonomic approach. Cells of strain W3-B1 were nonmotile, asporogenous, gram-negative slender rods with rounded ends. 16S rRNA gene sequence analysis indicated that organism should be placed in the genus Chryseobacterium. This organism contains menaquinone MK-6 as the predominent isoprenoid quinone and 3-OH iso 17:0 (40%), iso 15:0 (33%) as the major fatty acids. Phylogenetically, the nearest relative of strain W3-B1 is Chryseobacterium meningosepticum with sequence similarity of 98.4%, but DNA-DNA hybridization resulted in similarity values of only 52.3%. The G+C mol% is 34.6 mol%. Based upon results obtained by morphological, biochemical, chemotaxonomic, and molecular methods, strain W3-B1 was clearly distinguishable from other Chryseobacterium species. For these reasons, a novel species of family Flavobacteriaceae is proposed; strain W3-B1(T) (= GTC 862(T) = JCM 11413(T) = DSM 14571(T)) is the type strain.

Base Composition↗

PubMed2002

Serodiagnosis of pulmonary disease due to Mycobacterium avium complex with an enzyme immunoassay that uses a mixture of glycopeptidolipid antigens.

It is difficult to distinguish pulmonary disease due to Mycobacterium avium complex (MAC) from that due to other mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium kansasii. We developed an enzyme immunoassay (EIA) for diagnosis of MAC pulmonary diseases that uses glycopeptidolipid (GPL) antigens specific for MAC, and we used it for diagnosis in immunocompetent patients. The mean optical densities (+/- standard deviation) of serum immunoglobulin G antibodies to GPLs in patients with MAC disease, MAC colonization, M. kansasii disease, and tuberculosis and in healthy subjects were 0.778+/-0.784, 0.042+/-0.035, 0.059+/-0.035, 0.071+/-0.035, and 0.030+/-0.027, respectively. A significant increase in the level of anti-GPL antibodies was detected in patients with MAC disease. The level of anti-GPL antibodies reflected disease activity, because the level was decreased in culture-negative patients who had conversion of culture results. When a cutoff level of seropositivity (0.119) was defined, the sensitivity of EIA for diagnosis of MAC disease was 92.3%, and the specificity was 96.7%. Measurement of serum anti-GPL antibodies is useful for both the diagnosis of and assessment of activity in MAC disease.

Adolescent↗

PubMed2002

Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy.

Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid). P450SP(alpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450BS(beta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively. Using deuterium-substituted fatty acids ([2,2-d2]-myristic acid and d27-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated. In the P450SP(alpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on Vmax (DV = 4.1) when [2,2-d2]-myristic acid was used, suggesting that an isotopically sensitive step involving the alpha-hydrogen of the fatty acid is present in the catalytic cycle. On the other hand, D(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450. The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation. A similar kinetic isotope effect was observed [DV = 4.9, D(V/K) approximately 1] for the P450BS(beta) reaction, when d27-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases. The resonance Raman spectral data of P450BS(beta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed. On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450SP(alpha) and P450BS(beta).

Bacillus subtilis↗

PubMed2002

Emendation of the genus Sphingomonas Yabuuchi et al. 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola.

The 16S rDNA sequence similarities between the type strains of Sphingomonas paucimobilis and 32 other Sphingomonas species range from 90.2 to 99.6%. It might be possible to divide the genus into several new genera according to a dendrogram drawn from 16S rDNA sequence similarity. However, the phenotypic and biochemical information needed to define clusters of strains representing distinct genera within this group of organisms was not previously available. Although the cellular lipids of type strains of all 28 Sphingomonas species tested contained glucuronosyl-(1 --> 1)-ceramide together with 2-hydroxymyristic acid, other molecular species of sphingoglycolipids were distributed randomly. Sphingomonas natatoria and Sphingomonas ursincola, bacteriochlorophyll a-containing, gram-negative facultative phototrophs, belong to the cluster of the genus Sphingomonas. Other phototrophic Porphyrobacter and Erythrobacter species in the Sphingomonadaceae were classified into a cluster different from the genus Sphingomonas, as reported previously. None of the physiological and biochemical characteristics considered, including cellular lipids and fatty acid composition, provided evidence for the division of the current genus Sphingomonas. It is therefore concluded that the genus Sphingomonas should remain undivided at this time. The species of three recently proposed genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastobacter ursincola, are junior objective synonyms of species of the genus Sphingomonas.

Alphaproteobacteria↗

PubMed2002

Sphingomonas melonis sp. nov., a novel pathogen that causes brown spots on yellow Spanish melon fruits.

A polyphasic taxonomic study was performed on the phytopathogenic bacterial strains DAPP-PG 224(T) and DAPP-PG 228, which cause brown spot on yellow Spanish melon (Cucumis melo var. inodorus) fruits. Based on the presence of glucuronosyl ceramide (SGL-1) in cellular lipids, the results of fatty acid analysis and 16S rDNA sequence comparison, the strains had been identified as belonging to the genus Sphingomonas and as phylogenetically related to Sphingomonas mali, Sphingomonas pruni and Sphingomonas asaccharolytica. The levels of 16S rDNA sequence similarity of these three species to strain DAPP-PG 224(T) were respectively 98.0, 98.0 and 97.4%. DNA-DNA hybridization experiments between strains pathogenic on melon fruit and S. mali, S. pruni and S. asaccharolytica revealed < or = 16% relatedness. Based on these results, the two isolates studied are regarded as independent from the type strains of the three species mentioned above. Sphingomonas strains from melon fruits are recognized as forming a genetically and phenotypically discrete species and to be differentiated by phenotypic characteristics from all 29 named species of the genus. Thus, the name Sphingomonas melonis sp. nov. is proposed for the isolates from diseased melon fruits. The type strain is DAPP-PG 224(T) (= LMG 19484(T) = DSM 14444(T)). The G+C content of DNA of the type strain is 65.0 mol%.

Cucumis melo↗

PubMedDate not supplied

Mycobacterial trehalose 6,6'-dimycolate preferentially induces type 1 helper T cell responses through signal transducer and activator of transcription 4 protein.

Mycobacterium tuberculosis is an intracellular pathogen of tuberculosis and its pathogenicity is related to the ability to escape killing by ingested macrophages and induce delayed-type hypersensitivity (DTH). A major component of the cell wall of M. tuberculosis is trehalose 6,6'-dimycolate (TDM), which has been implicated as a pathogenetic factor. The expression of DTH and cell-mediated immunity is dependent on the macrophage-cytokine-type 1 helper T (Th1) lymphocyte axis. Cytokines, interleukin-12 (IL-12) and interferon-gamma (IFN-gamma), play a critical role in the process and IL-12-activated signal transducer and activator of transcription (STAT) 4 is required for the development of fully functional Th1 cells. To clarify host responses to mycobacterial TDM, we have analyzed footpad reaction, histopathology and cytokine profile of experimental granulomatous lesions using STAT4-deficient mice. In the present study, we have demonstrated that mycobacterial TDM selectively induces the Th1 response through the STAT4 signaling pathway, because mice lacking STAT4 protein significantly reduced to develop DTH, hypersensitivity granulomas, and Th1 cytokine responses, when compared to BALB/c mice. These results shed light on the molecular pathogenesis of mycobacterial disease. Taken together with previous studies, TDM is a pleiotropic molecule against the host and participates in the pathogenesis.

Animals↗