Biomedical subjects

T Fujisawa

Publications and source records attributed to T Fujisawa.

At least 109 records · Page 6Linked to original sources

PubMed2000

Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells.

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and beta-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4(+)T cells.

Abatacept↗

PubMed2000

Ki-67 immunostaining and other prognostic factors including tobacco smoking in patients with resected nonsmall cell lung carcinoma.

BACKGROUND: To estimate the effectiveness of expression of the tumor proliferative marker Ki-67 antigen (Ki-67) as a postoperative prognostic marker, the authors analyzed Ki-67 expression and its correlation with postoperative survival and other clinicopathologic factors, including preoperative smoking habits, in patients with resected nonsmall cell lung carcinoma (NSCLC). METHODS: A total of 156 patients with resected NSCLC at the study institution were investigated. Postoperative survival rates were estimated based on demographic and clinicopathologic factors, including Ki-67 expression and preoperative tobacco smoking habits. RESULTS: The overall postoperative 5-year survival rate in patients with high Ki-67 labeling indices (>/= 20%) was 39.6% compared with 67.7% in patients with low Ki-67 labeling indices. This finding was significant for all resected cases and for each pathologic disease stage (P < 0.05). The postoperative 5-year survival rate in patients with a history of heavy smoking (>/= 30 pack-years) was 47.6% compared with 62.5% for other patients (P = 0.027). This result was especially significant in patients with International Union Against Cancer Stage I disease and in patients with nonsquamous cell carcinoma (P < 0.03). The authors also observed a positive correlation between the Ki-67 labeling index and preoperative smoking habits (P = 0.0002). Multivariate analysis demonstrated that lymph node involvement, tumor differentiation, and Ki-67 labeling index were significant prognostic factors in NSCLC (P < 0.01). CONCLUSIONS: Tumor Ki-67 expression is a strong prognostic factor in NSCLC, especially adenocarcinoma. It may be hypothesized that tobacco mutagenicity may play a role in the growth and extension of NSCLC, which is one of the major impediments to postoperative survival in patients with a history of heavy smoking.

Aged↗

PubMed2000

The kondo effect in the unitary limit

We observe a strong Kondo effect in a semiconductor quantum dot when a small magnetic field is applied. The Coulomb blockade for electron tunneling is overcome completely by the Kondo effect, and the conductance reaches the unitary limit value. We compare the experimental Kondo temperature with the theoretical predictions for the spin- 12 Anderson impurity model. Excellent agreement is found throughout the Kondo regime. Phase coherence is preserved when a Kondo quantum dot is included in one of the arms of an Aharonov-Bohm ring structure, and the phase behavior differs from previous results on a non-Kondo dot.

Journal Article↗

PubMed2000

Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells.

CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-alpha and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-gamma. The enhancement by IFN-gamma was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-alpha, IL-4, and IFN-gamma. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.

Adult↗

PubMed2000

Expression of CXCR4 in eosinophils: functional analyses and cytokine-mediated regulation.

We examined the expression of transcripts of a panel of chemokine receptors in human eosinophils and found intense constitutive expression of CXCR4 mRNA. Although surface CXCR4 protein was hardly detectable in the peripheral blood or freshly isolated eosinophils, surface expression of CXCR4 became gradually apparent during incubation at 37 degrees C. In contrast, the level of CCR3 expression was virtually unchanged during the incubation. Stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand of CXCR4, elicited an apparent Ca2+ influx in these cells and induced a strong migratory response comparable to that by eotaxin. The surface expression of CXCR4 in eosinophils was up-regulated by IFN-gamma, TNF-alpha, and TGF-beta while it was down-regulated by IL-4 and eosinophil-directed hemopoietins such as IL-5. The CXCR4 expression did not always parallel the apoptotic changes in cytokine-treated eosinophils. In contrast to IL-4 and IFN-gamma, IL-5 potently reduced the level of CXCR4 mRNA. It seems unlikely that CXCR4 is fundamentally involved in the pathogenesis of allergic disorders by inducing the migration of eosinophils toward inflammatory sites, because a Th2-dominant state down-regulates eosinophil CXCR4 expression. However, CXCR4 may affect the size of the mobilizable pool by holding eosinophils at noninflamed tissues. Th2-dominant state may favor the liberation of eosinophils by down-regulating CXCR4 expression. The interplay between CXCR4 and SDF-1alpha in eosinophils potentially plays an important role in the accumulation of these cells at the allergic inflammatory sites.

Cell Membrane↗

PubMed2000

Redesign of artificial globins: effects of residue replacements at hydrophobic sites on the structural properties.

Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability.

Amino Acid Sequence↗

PubMed2000

Occurrence of clostridia in glass bottled foods.

Forty-one marketed samples of imported and domestic glass bottled foods were tested for clostridia contamination. It was detected in nine (22%) samples. Clostridia were isolated from fish sauce (Nam pla, Nuoc-mam), dressing, mustard, hot and sour soup mix (Tom Yum), mysids boiled down in soy, and salmon flakes. The origin of all clostridia positive samples was Asia. Clostridium botulinum and C. perfringens were not detected. The frequency of occurrence was higher by enrichment broth culture detection methods than by agar plate or pouch methods. These findings suggest that the number of bacteria in most of these clostridia positive food samples is very low, and the use of enrichment methods for detection of clostridia is essential.

Animals↗

PubMed2000

Age-dependent changes in phenotypes and candidate gene analysis in a polygenic animal model of Type II diabetes mellitus; NSY mouse.

AIMS/HYPOTHESIS: The Nagoya-Shibata-Yasuda (NSY) mouse closely mimics human Type II (non-insulin-dependent) diabetes mellitus in that the onset is age-dependent, the animals are not severely obese, and both insulin resistance and impaired insulin response to glucose contribute to disease development. The aim of this study was to clarify the influence of age on the pathogenesis of diabetes and to analyse a candidate gene for Type II diabetes in this strain. METHODS: Several phenotypic characteristics related to diabetes mellitus were monitored longitudinally in male NSY and control C3H/He mice. The nucleotide sequence of Glut4, a candidate gene for Nidd1nsy (a susceptibility gene for Type II diabetes) on Chromosome 11, encoding insulin-sensitive glucose transporter, was determined in NSY and C3H mice. RESULTS: Glucose intolerance worsened with age, and fasting blood glucose and fasting plasma insulin concentration increased with age in NSY mice. Pancreatic insulin content increased until 24 weeks of age but then decreased at 48 weeks of age in NSY mice. The hypoglycaemic response to insulin was statistically significantly smaller in NSY than in C3H/He mice. The nucleotide sequence of GLUT4 cDNA was identical in NSY and C3H/He mice, but both were different from the sequence reported previously. CONCLUSION/INTERPRETATION: Insulin secretion and insulin resistance, as well as ageing possibly play an important part in the disease development in NSY mice. A decline of pancreatic insulin content in older age might cause the relative insulin deficiency in this strain. Nucleotide sequencing suggests that Glut4 is unlikely to be a candidate gene for Nidd1nsy.

3T3 Cells↗

PubMed2000

Nucleotide sequence of alpha-galactosidase MEL gene from Zygosaccharomyces mrakii.

The region encompassing the alpha-galactosidase MELr gene was amplified from Zygosaccharomyces mrakii IFO 1835(T) by inverse-PCR and then sequenced. The nucleotide sequence of this region revealed a single open reading frame of 1410 bp encoding a 470 amino acid protein with a molecular weight of 51,909. The similarity of the deduced mature protein to other yeast alpha-galactosidases was 63.3% to Zygosaccharomyces cidri, 71.5% to Torulaspora delbrueckii, and 70. 7-73.9% to Saccharomyces species. The nucleotide and amino acid sequences are deposited in the DDBJ/EMBL/GenBank database under Accession Number AB030209.

Fungal Proteins↗

PubMed2000

Time-resolved two-dimensional X-ray diffraction study of the effect of shortening on activation of contracting skeletal muscle.

In twitch contractions of frog skeletal muscle, the isometric tension peaks when intracellular calcium has fallen to near-resting levels. To understand the mechanism of this delayed tension maintenance in the context of calcium regulation, the time course of the tropomyosin movement on actin was monitored by recording the intensity of the 2nd actin layer lines in a time-resolved two-dimensional X-ray diffraction study. The intensity rose ahead of tension, reflecting the tropomyosin movement from its "off" to "on" positions, but it fell with a time course similar to that of tension. Muscle shortening applied at the tension peak was followed by a poor recovery of tension, and accelerated the fall of the reflection intensity. The results suggest that the force-generating myosin heads retain the tropomyosin in its "on" position after the fall of intracellular calcium, and their shortening-induced detachment makes the tropomyosin return to its "off" position, thereby preventing myosin reattachment to actin.

Animals↗

PubMed2000

Expression and evolutionary conservation of nanos-related genes in Hydra.

The Drosophila gene nanos encodes two particular zinc finger motifs which are also found in germline-associated factors from nematodes to vertebrates. We cloned two nanos (nos)-related genes, Cnnos1 and Cnnos2 from Hydra magnipapillata. Using whole-mount in situ hybridization, the expression of Cnnos1 and Cnnos2 was examined. Cnnos1 was specifically expressed in multipotent stem cells and germline cells, but not in somatic cells. Cnnos2 was weakly expressed in germline cells and more specifically in the endoderm of the hypostome where it appears to be involved in head morphogenesis. In addition to structural conservation in the zinc finger domain of nanos-related genes, functional conservation of Cnnos1 was also demonstrated by the finding that a Cnnos1 transgene can partially rescue the nosRC phenotype that is defective in the egg production of Drosophila. Thus, the function of nanos-related genes in the germline appears to be well conserved from primitive to highly evolved metazoans.

Amino Acid Sequence↗

PubMed2000

Castleman's disease in the posterior mediastinum: report of a case.

We report a case of Castleman's disease which developed in the posterior mediastinum, with a review of the Japanese literature. A 19-year-old female patient with asthma was pointed out to have an abnormal shadow in the right posterior mediastinum on chest X-ray. We had tried to perform thoracoscopic surgery for this tumor, but we had to convert the surgical approach from thoracoscopy to a thoracotomy because of both tight adhesion of the tumor and muscle and profuse bleeding from the tumor. The tumor was diagnosed to be the hyaline vascular type of Castleman's disease histopathologically. In the Japanese literature, Castleman's disease, which develops in the posterior mediastinum, has been reported to often accompany tight adhesion between the tumor and surrounding tissue, and profuse bleeding thus cannot be avoided at surgery. The large amount of bleeding observed during the surgery of a patient with Castleman's disease in the posterior mediastinum may be due to tight adhesion and hypervascularity of the tumor. Therefore, care should be exercised in choosing the surgical approach if Castleman's disease is suspected in cases of posterior mediastinal tumor.

Adult↗

PubMed2000

Long-term survival achieved by resection of metastases in the liver and lung in a patient with recurrent colonic cancer: report of a case.

A 58-year-old man who underwent a potentially curative resection of cancer of the sigmoid colon at another hospital was subsequently followed up at our hospital. A lateral segmentectomy was performed for a solitary hepatic metastasis, and partial resection of right S1 was later carried out for a pulmonary metastasis. Another pulmonary metastasis was found 6 years after his third operation and to minimize the area to be resected, bronchial arterial infusion chemotherapy was performed twice. A 51% reduction in the size of the tumor was achieved, so a right upper lobectomy and wedge resection of the bronchus were performed. The patient remains alive 14 years after the initial resection of colonic cancer. This case is considered noteworthy because it demonstrates the potential effectiveness of local adjuvant chemotherapy and the possibility of extended survival in a patient who has undergone resection of both hepatic and pulmonary metastases from colonic cancer.

Adenocarcinoma↗

PubMed2000

Undifferentiated spindle-cell sarcoma of the chest wall with vascular endothelial growth factor expression: report of a case.

The expressions of some growth factors have been immunohistochemically confirmed in several kinds of tumors, and in particular the expression of vascular endothelial growth factor (VEGF) has been reported to be closely related to tumor cell proliferation. We report herein a case of undifferentiated spindle-cell sarcoma arising in the chest wall with VEGF expression. A 67-year-old man, who presented with coughing, was found to have an abnormal shadow on his right chest wall. He was admitted to Chiba Rosai Hospital and preoperative diagnosis of the tumor was sarcoma. The tumor was thus resected along with the right chest wall and right lower lobe of the lung. Histopathologically, the tumor cells were spindle-shaped and showed severe atypism. The tumor cells were positive for vimentin and VEGF antibody with immunohistochemical staining, but they did not show differentiation to any special type of sarcoma. The tumor was diagnosed to be undifferentiated spindle-cell sarcoma. The microvessel density of the tumor was measured using CD34 and it was found to be higher than the average density of usual sarcomas. The prognosis of this case was poor. The patient died of tumor metastasis to the lung and bone 1 year later in spite of the fact that the tumor was resected.

Aged↗

PubMed2000

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Amino Acid Sequence↗

PubMed2000

Paternal-maternal effects on phenotypic characteristics in spontaneously diabetic Nagoya-Shibata-Yasuda mice.

The Nagoya-Shibata-Yasuda (NSY) mouse is an inbred strain with spontaneous development of type 2 (non-insulin-dependent) diabetes mellitus. The purpose of this study was to determine the mode of inheritance of various phenotypes related to diabetes in this strain. Two reciprocal outcrosses, female C3H/He x male NSY F1 (C3NF1) and female NSY x male C3H/He F1 (NC3F1) mice, were performed. The phenotypic characteristics in both F1 mice were investigated. The cumulative incidence of diabetes was 100% (25 of 25) in male C3NF1 mice and 97% (29 of 30) in male NC3F1 mice at 48 weeks of age, indicating that diabetes in NSY mice was transmitted to male F1 hybrids in an autosomal dominant manner. Fatty liver also showed an autosomal dominant mode of inheritance. In contrast, epididymal fat accumulation and impaired insulin secretion showed an autosomal recessive mode of inheritance. The body mass index (BMI) showed a codominant mode of inheritance. Paternal-maternal effects associated with the severity of diabetes were observed. Insulin resistance was much more severe in male F1 mice than in the parental NSY strain. These data indicate different modes of inheritance among phenotypes related to type 2 diabetes. The presence of more severe insulin resistance in F1 mice versus the parental strains suggests the interaction of both parental genomes in the development of insulin resistance. The F1 mouse is expected to be useful for studies of the pathogenesis and genetic synergism of the insulin resistance syndrome.

Animals↗

PubMed2000

Chemokine production by the BEAS-2B human bronchial epithelial cells: differential regulation of eotaxin, IL-8, and RANTES by TH2- and TH1-derived cytokines.

BACKGROUND: Bronchial epithelial cells produce many types of chemokines and may contribute to lung inflammation by recruiting inflammatory cells. The CC chemokine eotaxin is a potent, eosinophil-specific chemoattractant that has been detected in the bronchial epithelium of patients with asthma. OBJECTIVES: The aim of this study was to investigate the regulatory mechanisms of chemokine production from bronchial epithelium by inflammatory cytokines, especially T(H)2- and T(H)1-derived cytokines, in bronchial asthma. METHODS: BEAS-2B human bronchial epithelial cells were cultured with TNF-alpha, IL-4, IL-13, and IFN-gamma alone or in combination, after which supernatants were assayed for eotaxin, IL-8, and RANTES proteins with ELISA. Reverse transcription-PCR was also performed. RESULTS: TNF-alpha induced production of eotaxin, IL-8, and RANTES in a concentration-dependent manner. Both IL-4 and IL-13 synergistically enhanced TNF-alpha-induced eotaxin production, whereas IL-8 production induced by TNF-alpha was significantly down-regulated by the T(H)2-derived cytokines. IFN-gamma, a T(H)1 cytokine, counteracted the enhancing effects of IL-4 and IL-13 on eotaxin production. RANTES production by TNF-alpha was not affected by IL-4 and IL-13 but was markedly enhanced by IFN-gamma. CONCLUSIONS: These results suggest that T(H)2 cytokines are involved in preferential recruitment of eosinophils in bronchial asthma by enhancing eotaxin and reducing IL-8 production from bronchial epithelial cells and that T(H)1 cytokines counteract the effects of T(H)2 cytokines by reducing eotaxin production.

Bronchi↗

PubMed2000

Age-related association of MHC class I chain-related gene A (MICA) with type 1 (insulin-dependent) diabetes mellitus.

To assess the contribution of the HLA class I region to susceptibility to and heterogeneity of type 1 diabetes, we investigated the association of polymorphism of MHC class I chain-related gene A (MICA) with age-at-onset as well as susceptibility to type 1 diabetes. One hundred one Japanese patients and 110 healthy control subjects were studied. The frequency of A4 allele was significantly higher and that of A6 allele was significantly lower in patients than in control subjects. The frequency of A5.1 allele was highest in early-onset patients (23.0%), intermediate in intermediate-onset patients (9.2%) and lowest in late-onset patients (7.7%) (trend chi-squared test, p = 0.0098). A5. 1 allele was strongly associated with HLA-B7 and Cw7, suggesting that MICA*A5.1-B7-Cw7 haplotype contains a gene responsible for age-at-onset. A4 allele was associated with a susceptible haplotype, DR4-DQB1*0401, and A6 allele was associated with a protective haplotype, DR2-DQB1*0601, suggesting that the association of MICA with type 1 diabetes susceptibility may be due to linkage disequilibrium with class II haplotypes. These data suggest that MICA gene is associated with age-at-onset and that a gene (or genes) responsible for age-at-onset of type 1 diabetes is located in the HLA class I region, probably near the region of MICA-B-C.

Adolescent↗