Biomedical subjects

S Nagai

Publications and source records attributed to S Nagai.

At least 163 records · Page 9Linked to original sources

PubMed1996

[Idiopathic interstitial pneumonia: profiles of the subsets, and prognosis].

Idiopathic interstitial pneumonia (IIP) comprises diseases with different clinical courses and different histopathologic characteristics. Clinical differential diagnosis and differentiation based on histopathologic characteristics reflecting inflammatory lung injuries and fibrosis are used to classify the pneumonia and to decide on management. Corticosteroids and immunosuppressants are given, but knowing the correct indications, timing, and duration of therapy depends on knowledge of the natural course of the diseases and on long-term observation of the clinical courses. We compared the clinical profiles, prognoses, and histopathologic characteristics of subsets of idiopathic interstitial pneumonia, and therapeutic approaches based on their pathophysiological differences.

Anti-Inflammatory Agents↗

PubMed1995

Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice.

Our study investigates the recall of immunity in the mouse model of memory immunity to tuberculosis infection. The results provide evidence that recall of immunity is expressed as an accelerated accumulation of potent effector cells in the infected target organs. These effector cells were recruited from the resting pool of memory cells and were immediately triggered to exert their effector functions, leading to a massive release of Th1 cytokines detectable both in splenic extracts and in the serum within the first 24 h of infection. During a primary infection, in contrast, a 14-day delay was observed before significant cytokine levels were reached. After the initial effector phase, the cells blasted and entered into clonal expansion, resulting in a rapid increase in the total number of CD4 CD45RBlow cells in the spleen. The recall of memory immunity was highly efficient and controlled an infectious challenge within the first week. The molecules recognized by the memory effector subset were the proteins secreted from Mycobacterium tuberculosis during growth. By separating the CD4 population into CD45RBhigh and CD45RBlow subsets, the memory effector cells were demonstrated to reside predominantly in the activated population of CD45RBlow CD44high LFA-1high L-selectinlow cells. The key antigenic targets recognized by these cells were identified as Ag85B and a secreted 6-kDa protein (ESAT-6) that elicited the release of exceedingly high levels of IFN-gamma. ESAT-6 was biochemically purified, characterized, and the gene encoding the protein was cloned.

Amino Acid Sequence↗

PubMed1995

Pharmacological study of stem-cell-factor-induced mast cell histamine release with kinase inhibitors.

Stem cell factor (SCF) is a ligand for c-kit receptor and has a critical role in the development of mast cells. In this study, we investigated the effect of a panel of kinase inhibitors on SCF-induced histamine release from rat peritoneal mast cells. Genistein, an inhibitor of tyrosine kinases, inhibited SCF-induced histamine release with IC50 of 1.6 x 10(-5) M. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), inhibited histamine release stimulated with SCF dose-dependently with IC50 of 4 x 10(-9) M. KT5926, an inhibitor of myosin light chain (MLC) kinase, reduced histamine release with IC50 of 1.8 x 10(-7) M. Staurosporine, an inhibitor of protein kinases, also inhibited SCF-induced histamine release with IC50 of 6.5 x 10(-8) M. These results show the early involvement of tyrosine kinase and PI3 kinase and the possible role of MLC kinase in the late secretory phase in the signaling pathway used by SCF.

Alkaloids↗

PubMed1995

Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG.

Recombinant MPB64 (rMPB64), a mycobacterial antigen, was obtained from an Escherichia coli clone transformed with a recombinant expression vector, pMAL64c. The rMPB64 was examined for the activity to elicit delayed-type hypersensitivity (DTH) in guinea pigs injected with liver Mycobacterium tuberculosis H37Rv or live M. bovis BCG Tokyo. It was found that rMPB64 has the same reactivity as native MPB64 (nMPB64) or MPT64 (nMPT64) and the potency to elicit DTH was 13.4 times higher than that of PPD. Because MPB64 is secreted only by living M. tuberculosis and some strains of BCG, it is possible to use this antigen for the diagnosis of tuberculosis.

Amino Acid Sequence↗

PubMed1995

Establishment and characterization of human gastric and colonic xenograft lines resistant to CPT-11 (a new derivative of camptothecin).

CPT-11-resistant human gastric and colonic xenograft lines were established by direct intratumoral injection of CPT-11 into subcutaneous SC-1-NU and CC-2-NU tumors in nude mice once a week for 10 months. The resistance of these xenograft lines to CPT-11 was confirmed by growth inhibition rate, to be 36.3% and 45.4%, respectively, compared to each parent cell line. DNA topoisomerase I activity of the nuclear extracts of SC-1-NU/CPT-11 and CC-2-NU/CPT-11, as assayed by relaxation of supercoiled DNA Col-E1, was significantly less than those of the parent lines. The cellular levels of topoisomerase I in those resistant lines measured by Western blot analysis were 0.57- and 0.79-fold lower than those of the parental lines, respectively. However, the activity of DNA topoisomerase II of those resistant cell lines assayed by decatenation of kinetoplast DNA was higher than that of the parental lines and the cellular levels of topoisomerase II in the resistant lines measured by Western blot analysis were 10.8- and 8.1-fold higher than those of the parent lines. Intracellular accumulation of CPT-11 in CPT-11-resistant tumors was not changed as compared to that of the parental lines, but hydrolysis of CPT-11 to more active SN-38 was reduced in the resistant tumors.

Animals↗

PubMed1995

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Amino Acid Sequence↗

PubMed1995

CT and pathological correlation of pulmonary sarcoidosis.

In CT the presence of mediastinal or hilar lymphadenopathies and thickened bronchovascular bundles are landmarks for the diagnosis of pulmonary sarcoidosis. The major CT findings for parenchymal involvement are thickened bronchovascular bundles, large parenchymal nodules, pleural or subpleural nodules, ground-glass opacities, local lung volume loss (distortion of the lung parenchyma), and microscopic and macroscopic honeycombing. The thickened bronchovascular bundles correspond histologically to granulomas, either with or without perigranulomatous fibrosis in the connective tissue sheath around the pulmonary vessels and airways. Conglomerate granulomas are represented on CT by high-attenuation nodules, and the ground-glass opacities are caused by the summation of a number of small granulomas in the interstitium.

Adult↗

PubMed1995

Pulmonary sarcoidosis: population differences and pathophysiology.

Sarcoidosis is characterized as a hyperimmune response to an unknown agent(s) at the lesion sites. Regarding pulmonary lesions, macrophage-T lymphocyte alveolitis precedes an epithelioid cell granuloma formation. In the 35 years since the first International Conference on Sarcoidosis, the definition of sarcoidosis has been revised several times, and many new pathophysiologic mechanisms have been proposed. In this review, we focused on four important issues: (1) possible population differences, (2) possible causative agent(s) and the current problems of disease susceptibility or resistance, (3) recent data on pathogenesis (including a genetic approach) and pathophysiologic processes, and (4) monitoring of disease activity.

Adult↗

PubMed1995

Homology between the MPB70 and MPB83 proteins of Mycobacterium bovis BCG.

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.

Amino Acid Sequence↗

PubMed1995

Secretion of MPB64 antigen by a recombinant clone of Mycobacterium smegmatis: characterization and application for the diagnosis of tuberculosis.

MPB64, a specific antigen to Mycobacterium tuberculosis complex (TB complex), was produced and secreted by a clone of M. smegmatis-MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E. coli shuttle plasmid pNIS vector. Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly. RPLA tests were applied to the shock cultures and the clinical isolates of mycobacteria to identify TB complex. RPLA with anti-MPB64 antibody-coated latex beads completely distinguished TB complex from other mycobacteria. Thus, it is suggested that RPLA with anti-MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis.

Antigens, Bacterial↗

PubMed1995

Identification and functional characterization of thioredoxin of Mycobacterium tuberculosis.

We have previously described a Mycobacterium tuberculosis protein designated MPT46 that was present in culture filtrates. Here we report that the MPT46 protein is thioredoxin of M. tuberculosis. MPT46 is recognized by antibodies to thioredoxin (Trx) of Escherichia coli, and antibodies of MPT46 recognize Mycobacterium leprae Trx. Moreover, MPT46 was shown to have enzymatic activity identical to that of Trx of other species, such as its ability to reduce insulin. These findings identify MPT46 as a functionally active Trx.

Bacterial Proteins↗

PubMed1995

Guinea pig cellular immune responses to proteins secreted by Mycobacterium tuberculosis.

To study the immunological activity of proteins secreted by Mycobacterium tuberculosis, we carried out comparative studies in guinea pigs infected intravenously with 2.5 x 10(3) CFU of this organism or with 2.5 x 10(4) CFU of Mycobacterium bovis BCG. Groups of infected guinea pigs were skin tested with fractions of secreted proteins covering well-defined narrow-molecular-mass regions, or such fractions were used for lymphocyte stimulation experiments. The lymphocyte stimulation experiments showed that the fraction containing proteins with molecular masses below 10 kDa had a superior stimulating capacity in tuberculous guinea pigs whereas the 24- to 30-kDa fraction gave significantly higher skin reactions in this group compared with BCG-vaccinated guinea pigs. A precise mapping within the region from 23 to 35 kDa by using a combination of narrow overlapping fractions and purified proteins enabled the identification of the 24-kDa antigen MPT64 as a molecule specific for tuberculous infection. Thus, MPT64 is a promising candidate for a specific diagnostic skin test reagent for human tuberculosis.

Animals↗

PubMed1995

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Amino Acid Sequence↗

PubMed1995

T-cell receptor V gene expression in HLA-typed Japanese patients with pulmonary sarcoidosis.

The clinical features of sarcoidosis vary in different ethnic groups, suggesting that different genetic or environmental backgrounds influence the disease. In Scandinavian sarcoidosis patients, we have previously described a correlation between lung-accumulated CD4+ T cells expressing the T-cell receptor (TCR) V alpha 2.3 gene segment and a particular HLA type (DR3[17],DQ2). For purposes of comparison, we have in this study investigated TCR V gene usage and gamma delta TCR expression in CD4+ and CD8+ T cells in bronchoalveolar lavage (BAL) fluid and peripheral blood in an ethnically distinct and homogenous group of individuals consisting of Japanese sarcoidosis patients and healthy controls. We used a panel of 13 monoclonal antibodies (Mab) specific for different TCR V genes, which altogether stained approximately 50% of the T cells, and triple staining techniques with flow cytometry. The patients and controls were also HLA-typed. Our results show a high degree of expression of gamma delta TCR in peripheral blood T cells of close to half of the patients. Expansions of T-cell subsets were readily detected in the CD8+ T-cell population, while a more homogenous staining pattern was found in the CD4+ T-cell population. These findings show the importance of ethnic origin and environment in discussions of TCR V gene usage.

Adult↗

PubMed1995

Analysis of fused-membrane structures in bronchoalveolar lavage fluid from patients with alveolar proteinosis.

Alveolar macrophages of patients with alveolar proteinosis, obtained by lung lavage, contain a large number of intracytoplasmic inclusions with mutlilamellar membranous structures, called fused-membrane structures, having a periodicity of 4.7 nm. To analyze the composition of these structures, we concentrated them from the bronchoalveolar lavage fluid of patients by a combination of sucrose density-gradient ultracentrifugation and enzyme hydrolysis using proteinase K. Fused-membrane structures were most numerous (26.2%) in a fraction obtained at the interface between 1.0 and 1.1 M sucrose solutions separated after proteinase K hydrolysis. This fraction was rich in acidic phospholipids. Hydrophobic surfactant apoproteins constituted about 77% of the proteins in this fraction, and a remarkable increase in the content of surfactant-associated protein C (SP-C) was found. The phospholipid-to-protein ratio was 0.25:1. Based on these results, we tried to reconstitute fused-membrane structures from purified lipids and hydrophobic proteins isolated from the patients' lavage fluid or from pig lungs. Only in the presence of both phospholipids and SP-C were similar multilamellar structures, having a periodicity of 4.3 to 4.5 nm, formed. These results suggest that fused-membrane structures have a close relationship to a hydrophobic surfactant-associated protein, SP-C, which accumulates in alveolar macrophages, possibly by incomplete digestion.

Amino Acid Sequence↗

PubMed1995

Coronary artery disease in dermatomyositis. A case report.

A sixty-two-year-old woman with chronic dermatomyositis (DM) receiving steroid monotherapy developed coronary artery stenosis. She had lung fibrosis and complained of dyspnea, but no ischemia was suggested by electrocardiogram. Ateriographic findings and clinical symptoms of coronary artery disease in DM have not been previously reported.

Adult↗

PubMed1995

Gastrointestinal and skin lesions in piglets naturally infected with pseudorabies virus.

Pseudorabies virus (PRV) infection was diagnosed in 4 piglets from a litter by immunohistopathologic examination and virus isolation. Three piglets had moderate to severe neuronal degeneration, and PRV antigen was detected in Auerbach's myenteric plexus and Meissner's submucosal plexus of the gastrointestinal tract. One piglet had 2 types of skin lesions. One lesion appeared on the hip and ear and was characterized by ballooning degeneration, necrosis of epithelial cells, and intranuclear inclusion bodies. The other was found on the ear and hematoma-like lesion was composed of fibrinoid exudation and degenerative connective tissue. PRV antigen was clearly demonstrated in both skin lesions. These results suggested that degeneration of myenteric plexuses might be another characteristic of lesions in PRV-infected pigs and that the virus spreads by interaction between the skin and myenteric plexuses to the central nervous system.

Animals↗

PubMed1995

[Study on surfactin, a cyclic depsipeptide. I. Isolation and structure of eight surfuctin analogs produced by Bacillus natto KMD 2311].

Crude surfactin was simply prepared from the culture filtrate of Bacillus natto KMD 2311 twice by acidification of the filtrate and extraction of the precipitate with ethanol. Eight surfactin analogs were isolated from the crude surfactin by RP-HPLC and gel filtration. The structure of each analog was deduced by means of amino acid composition of the acid hydrolysate and FAB-MS measurement to be a cyclic depsipeptide containing a hydroxyfatty acid. The structure of the hydroxyfatty acid moieties was elucidated as n- or iso- or anteiso-3-hydroxyfatty acid composed of carbon number 13-16 by GC analysis and EI-MS after the methanolysis of the analogs. The amino acid sequence of the peptide portion was assigned as acyl-Glu-Leu-Leu-Val-Asp-Leu-Leu by EI-MS for eight analogs. The isolated four compounds were found to be identical with the known surfactin analogs, A1, B1, B2 and C1. Although surfactin A2 and C2 had not been isolated, their structures were deduced to be a surfactin analog. Surfactin A3 and D were novel analogs. The acyl groups of surfactin A2, A3, C2 and D were anteiso-3-hydroxytridecanoic acid, n-3-hydroxytridecanoic acid, anteiso-3-hydroxypentadecanoic acid and iso-3-hydroxyhexadecanoic acid, respectively.

Amino Acid Sequence↗