Biomedical subjects

Yukiko Fujita

Publications and source records attributed to Yukiko Fujita.

14 recordsLinked to original sources

PubMed2006

Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-alpha release.

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6'-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6'-tetraacyl trehalose 2'-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-alpha) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-alpha release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-alpha release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.

Animals↗

PubMed2006

Differences in serological responses to specific glycopeptidolipid-core and common lipid antigens in patients with pulmonary disease due to Mycobacterium tuberculosis and Mycobacterium avium complex.

Disease due to the Mycobacterium avium complex (MAC) is one of the most important opportunistic pulmonary infections. Since the clinical features of MAC pulmonary disease and tuberculosis (TB) resemble each other, and the former is often difficult to treat with chemotherapy, early differential diagnosis is desirable. The humoral immune responses to both diseases were compared by a unique multiple-antigen ELISA using mycobacterial species-common and species-specific lipid antigens, including glycopeptidolipid (GPL)-core. The results were assessed for two patient groups hospitalized and diagnosed clinically as having TB or MAC pulmonary disease. Diverse IgG antibody responsiveness was demonstrated against five lipid antigens: (1) monoacyl phosphatidylinositol dimannoside (Ac-PIM2), (2) cord factor (trehalose 6,6'-dimycolate) (TDM-T) and (3) trehalose monomycolate from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) (TMM-T), and (4) trehalose monomycolate (TMM-M) and (5) GPL-core from MAC. Anti-GPL-core IgG antibody was critical, and detected only in the primary and the secondary MAC diseases with high positivity, up to 88.4 %. However, IgG antibodies against Ac-PIM2, TDM-T and TMM-T were elevated in both TB and MAC patients. Anti-TMM-M IgG antibody was also elevated in MAC disease preferentially, with a positive rate of 89.9 %, and therefore, it was also useful for the diagnosis of the disease. IgG antibody levels were increased at the early stages of the disease and declined in parallel to the decrease of bacterial burden to near the normal healthy control level, when the anti-mycobacterial chemotherapy was completed successfully. Unexpectedly, about 25 % of hospitalized TB patient sera were anti-GPL-core IgG antibody positive, although the specificity of GPL-core was sufficiently high (95.8 % negative in healthy controls), suggesting that a considerable number of cases of latent co-infection with MAC may exist in TB patients. Taken together, the combination of multiple-antigen ELISA using mycobacterial lipids, including GPL-core and TMM-M, gives good discrimination between healthy controls and sera from patients with TB or MAC disease, although for accurate diagnosis of TB more specific antigen(s) are needed.

Antibodies, Bacterial↗

PubMed2005

Diverse humoral immune responses and changes in IgG antibody levels against mycobacterial lipid antigens in active tuberculosis.

Humoral immune responses of active TB patients against six mycobacterial lipid antigens [trehalose 6,6'-dimycolate (TDM) from Mycobacterium bovis BCG (TDM-T) and Mycobacterium avium complex (TDM-M), trehalose 6-monomycolate (TMM) from M. bovis BCG (TMM-T) and M. avium complex (TMM-M), triacyl (PL-2) and tetraacyl (PL-1) phosphatidylinositol dimannosides] were examined by ELISA. IgG antibodies of TB patients with active disease reacted against the six lipid antigens distinctively, but heterogeneously. If tests were combined and an overall positive was scored cumulatively when any one of the six tests was positive, a good discrimination between patient and normal subject was obtained. A positive result in any one of the six tests was obtained in 91.5% of all 924 hospitalized patients and 93.3% of 210 patients at their first visit to the outpatient clinic. The IgG antibody response differed considerably from patient to patient, and the response patterns were grouped into several types. IgG antibody levels paralleled the bacterial burden; however, the smear-negative (culture-positive) patient group also showed high positive rates and mean ELISA DeltaA values against the six lipid antigens. There were also marked differences in positive rate and mean DeltaA values between cavity-positive and -negative groups, the former being higher than the latter. After anti-TB chemotherapy was initiated, IgG antibody levels decreased dramatically, paralleling the decrease in the amount of excretion of bacteria. Since multiple-antigen ELISA using particular lipid antigens was highly sensitive, and IgG antibody levels vary greatly at different stages of the disease, this technique is applicable for early diagnosis of smear-negative (and -positive) active TB and the prognosis for completion of anti-TB chemotherapy.

Adult↗

PubMed2005

Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry.

Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 microg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (alpha, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) alpha-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic alpha-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium avium-intracellulare group, M. phlei and M. flavescens. The M. avium-intracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester-mycoloyl TMM. This technique has marked advantages in the rapid analysis of not only intact glycolipid TMM, but also the mycolic acid composition of each mycobacterial species, since it does not require any degradation process.

Animals↗

PubMed2005

Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Animals↗

PubMed2005

Serotype-specific modulation of human monocyte functions by glycopeptidolipid (GPL) isolated from Mycobacterium avium complex.

Immunomodulating activity of glycopeptidolipids (GPL), separated from different serovars of Mycobacterium avium complex (MAC), on macrophage functions was compared. When peripheral blood mononuclear cells (PBMC), from healthy donors showing strongly positive reactions to mycobacterial purified protein derivatives (PPD), were incubated with heat-killed Staphylococcus aureus coated with GPL serovar 4, phagocytosis of monocytes increased in dose-dependent manner. However, coating with GPL serovar 9 did not show any effects. After phagocytosis of heat-killed S. aureus, the phagosome-lysosome (P-L) fusion in monocytes was inhibited dose-dependently by coating of S. aureus with GPL serovar 4, but not serovar 9. These results indicate that GPL serovar 4 facilitates invasion of MAC into monocytes and renders resistance to bactericidal reactions due to the inhibition of P-L fusion. Regarding accessory function of macrophages in proliferative responses of T cells, the addition of GPL serovar 4 to cultures resulted in significant inhibition of anti-CD3 monoclonal antibody (mAb)-induced proliferation, whereas both serovar GPLs did not cause reduction of cell viability. Furthermore, the PPD-specific T cell proliferative response was downregulated markedly by GPL serovar 4, but weakly suppressed by GPL serovar 9. These results indicated that the immunomodulating activity of GPL on macrophage functions is serovar-dependent.

Dose-Response Relationship, Drug↗

PubMed2005

Effect of inoculum conditioning on hydrogen fermentation and pH effect on bacterial community relevant to hydrogen production.

The effect of conditioning for a variety of inoculums on fermentative hydrogen production was investigated. In addition, the effects of pH condition on hydrogen fermentation and bacterial community were investigated. The effect of conditioning on hydrogen production was different depending on the inoculum types. An appreciable hydrogen production was shown with anaerobic digested sludge and lake sediment without conditioning, however, no hydrogen was produced when refuse compost and kiwi grove soil were used as inoculums without conditioning. The highest hydrogen production was obtained with heat-conditioned anaerobic digested sludge, almost the same production was also obtained with unconditioned digested sludge. The pH condition considerably affected hydrogen fermentation, hydrogen gas was efficiently produced with unconditioned anaerobic sludge when the pH was controlled at 6.0 throughout the culture period and not when only the initial pH was adjusted to 6.0 and 7.0. Hydrogen production decreased when the culture pH was only adjusted at the beginning of each batch in continuous batch culture, and additionally, bacterial community varied with the change in hydrogen production. It was suggested that Clostridium and Coprothermobacter species played important role in hydrogen fermentation, and Lactobacillus species had an adverse effect on hydrogen production.

Anaerobiosis↗

PubMed2005

Clinical evaluation of serodiagnosis of active tuberculosis by multiple-antigen ELISA using lipids from Mycobacterium bovis BCG Tokyo 172.

The humoral immune responses of 69 active tuberculosis (TB) patients against three major mycobacterial lipid antigens, monoacyl phosphatidylinositol dimannoside (Ac-PIM2), trehalose 6,6'-dimycolate (TDM-T) and trehalose 6-monomycolate (TMM-T) from Mycobacterium bovis BCG Tokyo 172, were examined by ELISA. IgG antibodies from active TB patients were reactive against each of the three lipid antigens (Ac-PIM2, TDM-T and TMM-T), giving positive results of 42.0-59.4%. If tests were combined and an overall positive was scored when any of the three tests was positive, sensitivity was 71.0%, showing better discrimination between patients and normal subjects. Although this value is not satisfactory for the clinical diagnosis of active TB, it is still higher than values for Determinar TBGL (56.5%) and MycoDot (31.9%) test results, both of which are commercially available. IgG antibody responses to particular lipid antigens in an individual patient differed diversely between patients. Positive IgG antibody rates and IgG antibody levels to lipid antigens were mostly paralleled by the amount of mycobacteria excreted and by the severity of pathological lesions (size and cavity formation). Serologically positive responsiveness was shown in 60.0% of tuberculin skin test (TST)-negative TB patients. Furthermore, seropositivity for multiple-antigen ELISA in active TB patients was demonstrated in other possible immune-suppressed cases, such as senile, diabetes mellitus and fulminant TB patients. Therefore, in contrast to tests based on cellular immune responses such as the TST, the humoral immune responses of TB patients against mycobacterial lipid antigens were disease-specific and were shown to be useful for the early diagnosis of active TB disease in conjunction with smear and cultivation tests, even if cellular immune responses are decreased.

Adult↗

PubMed2004

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes.

Animals↗

PubMed2003

Estrogenic chemicals and estrogenic activity in leachate from municipal waste landfill determined by yeast two-hybrid assay.

Estrogenic activity and estrogenic chemicals in landfill leachate were investigated by yeast two-hybrid assay and chemical analysis. Leachate sample extracted by liquid-liquid extraction with dichloromethane at pH 7.0 showed a higher dose-response curve than sample extracted at pH 3.0. or than sample extracted by solid phase extraction at either pH 7.0 or 3.0. The fraction extracted at pH 3.0 specifically inhibited not only growth of yeast but also estrogenic activity in this assay, suggesting that it contained anti-estrogenic chemicals. The greatest contributor to estrogenic activity among the chemicals identified in leachate extract was bisphenol A, with an estimated contribution ratio of 84%. The contribution ratios of 4-nonyl phenol (4-np) and 4-tert-octyl phenol (4-t-op) were estimated at 1.0%, and 0.1%, respectively, while natural estrogens such as 17beta-estradiol or estrone were below detection limit, so that their contribution ratio was estimated at no more than 10%. The estrogenic activity of leachate was decreased by aeration treatment alone after 7 days, and was no longer detected after 22 days. Concentrations of bisphenol A, 4-np and 4-t-op likewise decreased with aeration.

Biological Assay↗

PubMed2003

Mouse germ cell-less as an essential component for nuclear integrity.

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

Animals↗

PubMed2003

Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production.

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.

Animals↗

PubMed2002

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells.

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.

Amino Acid Sequence↗

PubMed2002

[Evaluation before and after pranlukast administration with the QOL questionnaire (revised version 2001) for pediatric patients with bronchial asthma and their parents or caregivers].

We conducted a longitudinal investigation with the QOL questionnaire (revised version 2001) before and after the 4-week-administration of a leukotriene receptor antagonist pranlukast. A significant improvement in the < 4 yrs group was observed at week 1, and that in > or = 4 yrs group at week 2. Under these conditions, the overall QOL score, physical domains and mental domains, significantly improved in both the < 4 yrs group and the > or = 4 yrs group. Overall, a slight correlation was observed between ratio changes in QOL scores and differences in symptom scores. However, no correlation was found in part of patients, suggesting that the QOL questionnaire allows measurement of mental changes in the patients themselves and their parents or caregivers for therapeutic effects which cannot be determined with ordinary physical findings only. In "event present" group, a significant difference in physical and mental domains was revealed by the comparison of QOL scores before and after administration. And furthermore in "event absence" group, the p-value for physical domain and mental domain was 0.0505 and 0.0912 in the < 4 yrs group, respectively, 0.0101 and 0.0446 in the > or = 4 yrs group, respectively. The above results led us to consider the QOL questionnaire (revised version 2001) useful for routine medical care. Furthermore, pranlukast was considered useful for improvement not only of physical symptoms of bronchial asthma but also of the patient's QOL, although the placebo effects in this open trial must be considered.

Anti-Asthmatic Agents↗