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Biomedical subjects

N Kawaguchi

Publications and source records attributed to N Kawaguchi.

At least 19 recordsLinked to original sources
PubMed2001

Establishment and characterization of a cell line from a malignant fibrous histiocytoma of bone developing in a patient with multiple fibrous dysplasia.

PURPOSE AND METHODS: In order to provide material for genetic analysis of fibrous dysplasia (FD), a cell line designated GBS-1 was established from a secondary bone malignant fibrous histiocytoma (MFH) developing in a rib of a 44-year-old male polyostotic FD patient. RESULTS: The GBS-1 cells are characterized by a pleomorphic spindle cell morphology with abundant mucus production. On transplantation to nude mouse subcutis the cell line forms myxoid-spindle cell sarcomas with giant cells, the myxoid product being positive for periodic acid-Schiff (PAS) and alcian blue (Al-B) stains and completely digested by hyaluronidase, mimicking the original tumor. Chromosome and genetic analyses revealed multiple structural and numerical abnormalities of chromosomes with a large number of unidentifiable chromosomes and p53 mutation in exon 7 with LOH in the counterpart. CONCLUSIONS: Since cell lines for FD have hitherto not been available, the GBS-1 cells should prove useful for genetic analyses of FD and also MFH of bone origin.

Adult↗
PubMed2001

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts.

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

3T3 Cells↗
PubMed2001

Serum levels of tumor necrosis factor-alpha in chronic inflammatory demyelinating polyneuropathy.

BACKGROUND: Activated macrophages and T lymphocytes may play a role in the pathogenesis of chronic inflammatory demyelinating polyneuropathy (CIDP). Both cell types secrete tumor necrosis factor-alpha (TNFalpha), which has toxic effects on myelin and endothelial cells. METHODS: The serum concentration of TNFalpha was measured by ELISA and compared with clinical and electrophysiological profiles in 20 patients with CIDP. RESULTS: An increased serum level of TNFalpha was detected in 5 (25%) patients and was associated with subacute progression, severe neurologic disabilities, and symmetric weakness involving proximal as well as distal muscles. TNFalpha levels increased during the active phase in this subgroup of patients. The levels of TNFalpha correlated with the severity of demyelinating conduction abnormalities in the intermediate as well as distal nerve segments, suggesting demyelination diffusely distributed along the nerves. CONCLUSION: Circulating TNFalpha increases during the active phase in a subgroup of CIDP patients and may play a role in the pathogenesis of demyelination and the breakdown of the blood-nerve barrier in CIDP.

Adolescent↗
PubMed2001

Spontaneous mutation in Mitf gene causes osteopetrosis in silver homozygote quail.

Silver homozygous quail was recently reported to have mutations in Mitf gene. Although numerous mutations in Mitf gene have been reported in mice, no mutations corresponding to the mutation in the homozygous silver (B/B) quail in Mitf gene have been reported to cause defects in pigmentation and bone. Therefore, we investigated the bones of the B/B homozygotes. Comparison of the bones of the B/B homozygotes with those of wild-type by X-ray examination revealed osteopetrosis in the long bones of B/B homozygotes. However, osteopetrosis in B/B homozygotes was less severe than that observed in mi/mi mice. Histological examination showed that there were less TRAP-positive multinucleated cells in the trabecular bones in B/B homozygote tibia than in the wild type. In vitro osteoclastogenesis study also suggested that formation of TRAP-positive multinucleated cell was suppressed in the marrow cells of the long bones of the B/B homozygotes. Furthermore, overexpression of chicken Mitf via retroviral transfection into B/B homozygote bone marrow cells in cultures increased the number of TRAP-positive cells 2-3 fold more than that in control. These results indicated that in addition to the previously reported defect in melanogenesis, osteoclastogenesis was inhibited in B/B homozygotes. These results indicate that the novel mutations in Mitf gene observed in the B/B homozygote quail impair osteoclastic bone resorption.

Aging↗
PubMed2001

Differences in anti-LKM-1 autoantibody immunoreactivity to CYP2D6 antigenic sites between hepatitis C virus-negative and -positive patients.

Anti-liver kidney microsome type 1 autoantibodies (anti-LKM-1) are known to be present in sera of autoimmune hepatitis type II and a subset of chronic hepatitis C patients. The autoantigen to anti-LKM-1 has been identified to be cytochrome P450 IID6 (CYP2D6) and the most frequently cited CYP2D6 antigenic sites of anti-LKM-1 in sera from autoimmune hepatitis type II patients spans the region aa 256-269. Other antigenic sites on CYP2D6 exist and have been identified in the two patient groups. However, most of these sites are concentrated on the carboxyl-terminal side of the protein, and the amino-terminal region has not been thoroughly investigated. Here, we have studied the antigenicity of the CYP2D6 amino region and compared reactivities between hepatitis C virus (HCV)-negative and -positive Japanese patient groups. A total of 34 anti-LKM-1-positive sera (eight with autoimmune hepatitis type II and 26 with chronic hepatitis C) were included. The immunoreactivity of patients' sera was examined against four conformational and one linear CYP2D6 peptide fragments. A defined antigenic site spanning aa 181-245 was found to react with 88% (7/8) of autoimmune hepatitis type II patients, as opposed to only 38% (10/26) of chronic hepatitis C patients. This was a significant difference (P< 0.043). Among these positively reacting samples, five of the seven autoimmune hepatitis type II sera and four of the ten chronic hepatitis C sera also reacted with a synthetic peptide spanning aa 256-269. Anti-LKM-1 thus may be able to recognize simultaneously at least two antigenic sites on the CYP2D6 protein, and reactivities against individual epitopes may differ according to HCV infectivity status.

Adult↗
PubMed2001

Effects of gene transfection of human bcl-2 on concordant cardiac xenografts in hamster to rat model.

OBJECTIVES: Concordant cardiac xenografts are known for delayed vascular rejection. Therapy combining with FK506 and cobra venom factor prolongs graft survival. The proposed underlying mechanism holds that cytoprotective proteins such as Bcl-2 play a role here. We studied the effects of gene transfection of human-bcl-2 on graft survival and coronary artery lesions in concordant cardiac xenografts, and discuss the role of cytoprotective genes in vascular xenograft rejection. METHODS: Golden-Syrian-hamster hearts were heterotopically transplanted into Lewis rats given FK506 (1 mg/kg daily) and cobra venom factor (0.2 mg/kg; day 0 and 1) intramuscularly. They were divided into 2 groups--grafts transfected vector with the human-bcl-2 gene (Group-B(+)) and vector without the gene (Group-B(-)) using the HVJ liposome method; 4 or 5 grafts from each group were explanted 1, 2, 3, or 4 weeks and more than 1 month after transplantation and evaluated by H-E, Elastic-Van-Gieson and immunohistochemical staining of Bcl-2. Coronary arterial lesions were examined using a scoring method. RESULTS: Bcl-2 expression in endothelial cells in Group-B(+) was confirmed within 2 weeks after transplantation but not thereafter. The coronary score in Group-B(+) was significantly lower than that in Group-B(-) within 2 weeks after transplantation but not thereafter. CONCLUSIONS: In this hamster-to-rat cardiac xenograft model, the bcl-2 gene was successfully transfected to the coronary endothelium and lasted 2 weeks. During Bcl-2 expression, coronary vascular lesions were suppressed more than in the untransfected group.

Animals↗
PubMed2001

High sensitivity of a novel ELISA for anti-M2 in primary biliary cirrhosis.

The use of an ELISA for the detection of anti-M2, a specific autoantibody in primary biliary cirrhosis (PBC), has been common in Japan. However, there are some problems in the sensitivity of this ELISA, especially in PBC patients showing antimitochondrial antibody (AMA)-negative sera or low AMA titers by immunofluorescence. Recently, a new ELISA for anti-M2 was developed, using porcine heart mitochondrial protein as the antigen. We report here comparative studies of the new and the former anti-M2 ELISAs. Porcine heart mitochondrial protein was prepared and used as the antigen for the new ELISA for anti-M2. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this protein showed three major M2 antigen proteins. As the second antibody, peroxidase-conjugated anti-human mouse monoclonal IgM, in addition to monoclonal IgG, was included. The sera of 171 PBC patients were examined. As controls, we examined the sera of 167 non-PBC patients and the sera of 115 normal controls. The cut-off index was set at 10 U/ml, based on the results for the normal controls. No sera from the non-PBC patients or the normal controls were positive for anti-M2 by either the new or the former ELISA. However, the positivity rate for anti-M2 in PBC patients with the new ELISA was 78%; in contrast, that with the former ELISA was only 54%; this difference was significant (P = 0.00001). In particular, in 65 patients showing AMA titers of 1:20 or less, the positivity rate with the new ELISA was 51%; in contrast, that with the former ELISA was only 17%. As the sensitivity of the new ELISA is significantly higher than that of the former ELISA, especially for sera from patients showing AMA-negativity or low titers of AMA, the new ELISA is considered to be more effective than the former ELISA for use in anti-M2 screening assays in patients with PBC.

Animals↗
PubMed2001

Change of c-Myc expression and cardiac hypertrophy in patients with aortic valve replacement.

BACKGROUND: Long-term volume overload to the left ventricle (LV) due to aortic regurgitation (AR) tends to cause severe impairment in LV function that cannot be reversed even with aortic valve replacement (AVR). Recently, we reported that the protooncogene c-myc is related to the onset of the cardiac hypertrophy and LV dysfunction in patients with chronic AR. However, it is still unclear whether c-myc is related to reversibility of the cardiac hypertrophy or LV dysfunction after AVR. METHODS AND RESULTS: Twenty patients with isolated chronic AR who underwent AVR were included in this study. LV function was calculated before and after AVR. After AVR, end-systolic volume index (ESVI) and enddiastolic volume index (EDVI) were improved, but not mass index (LVMI). However, normalization of ESVI and EDVI was observed only in 12 and 9 patients, respectively. Preoperatively, c-Myc protein was expressed in the myocardium of 16 out of 20 patients with an average point count of 35+/-30%. After AVR, c-Myc protein was observed only in 2 patients. Preoperative ejection fraction (EF), ESVI, and postoperative end-systolic stress (ESS)/ESVI had significant correlation to postoperative cell diameter (CD). Percent c-Myc protein expression before the operation was significantly correlated to postoperative CD, ESVI, and ESS/ESVI. Average c-Myc expression was higher in patients who showed normalization of CD and ESS/ESVI after AVR than the patients who did not. CONCLUSIONS: These data suggest that preoperative expression of c-Myc can be indicative of the reversibility of myocardial cellular hypertrophy and LV dysfunction.

Adult↗
PubMed2001

False positive reaction in ELISA for IgM class anti-M2 antibody and its prevention.

Anti-M2 of anti-mitochondrial antibodies is recognized as the specific autoantibody detected in sera from patients with primary biliary cirrhosis (PBC). The IgG class and IgM class of this antibody can be separately measured using each ELISA. In the present study, false positive reactions were found in some sera from non-PBC patients such as acute hepatitis A, syphilis and rheumatoid arthritis using the IgM anti-M2 ELISA. They showed an increase of polyclonal IgM, and positivity for IgM anti-cardiolipin or rheumatoid factors, respectively. So, we developed a means to prevent these false positive reactions. First, dilutions of test sera at 1:1000-fold were carried out in addition to the original method at 1:100-fold. Secondly, some blocking reagents were added into the buffer system. By serum dilution, non-specific bindings disappeared in most samples other than showing an increase in polyclonal IgM. Moreover, the addition of suitable blocking reagents such as fetal bovine serum (FBS) and skimmed milk into the buffer system could prevent these non-specific bindings. From these findings, the procedure of optical serum dilution and the addition of suitable blocking reagents successfully prevented false positive reactions in this IgM anti-M2 ELISA.

Journal Article↗
PubMed2001

Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis.

The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.

Journal Article↗
PubMed2001

Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens.

Antimitochondrial antibodies (AMA) are the serologic hallmark of primary biliary cirrhosis (PBC). However, depending on the clinical laboratory, from 5% to 17% of PBC patients are consistently AMA-negative, using native mitochondrial antigens and a variety of conventional assays including immunofluorescence (IMF) and enzyme-linked immunosorbent assay (ELISA). The major immunoreactive mitochondrial autoantigens are the E2 members of the 2-oxo-acid dehydrogenase complex family, including pyruvate dehydrogenase complex-E2 (PDC-E2), branched chain 2-oxo acid dehydrogenase complex-E2 (BCOADC-E2), and oxo-glutarate dehydrogenase complex-E2 (OGDC-E2); cDNAs of these proteins have now been cloned, sequenced, and their B-cell epitopes defined. In the present study, we cloned cDNAs encoding these proteins from human, not bovine, sources, and expressed the recombinant proteins in a newly developed ELISA that employs a unique Escherichia coli buffer, and compared the data with previous assays using both AMA-positive and -negative patients. Using this new assay and our criteria for positive as an optical density (OD) greater than 10 SD above the mean of control sera, the AMA-positive rate of 191 PBC sera was 94% (179 of 191) compared with 84% (161 of 191) by IMF. None of the 316 control sera were reactive. Using our recombinant assays, we focused attention on the 30 IMF-AMA-negative patients. Twenty-two of 30 (73%) of these patients were positive using this new ELISA. The group of 30 IMF-AMA-negative/ELISA-positive patients did not differ significantly from a comparable population of IMF-AMA-positive patients with respect to age, sex distribution, liver function tests, elevation of serum IgM, or pathologic stage.

Autoantibodies↗
PubMed2001

Pasteurized autogenous bone graft for reconstruction after resection of malignant bone and soft tissue tumors: imaging features.

Our experimental study revealed that pasteurization was sufficient for killing all tumor cells in any massive bone and that the osteoinductivity and mechanical strength of pasteurized bone were about same as that of a fresh bone graft and better than boiled or autoclaved bone. We have performed reconstruction surgery using pasteurized autogenous bone graft since 1990. Resected bone was warmed for 30 minutes in a homeothermal heater set at 60 degrees C and then placed back to the original anatomic site. The graft was fixed with a plate or intramedullary nail with or without bone cement. Based on our experience in 31 patients, no local recurrence was seen in the grafts. Among 24 patients who can be adequately evaluated, incorporation of graft was complete in 17 cases and partial in 4 cases. In 18 of 21 patients, uptake of radionuclide in the grafted pasteurized bone was detected on bone scintigraphy approximately 6 months after surgery, and the uptake increased gradually, which suggested revascularization and remodeling of grafted bone.

Adolescent↗
PubMed2001

Klotho-deficient mice are resistant to bone loss induced by unloading due to sciatic neurectomy.

Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.

Animals↗
PubMed2000

Mitf is expressed in osteoclast progenitors in vitro.

Microphthalmia mutant (mi/mi) mice reveal defects in osteoclastogenesis and exhibit osteopetrosis. However, there have been no studies to test the importance of Mitf in in vitro osteoclastogenesis using the cells derived from mi/mi mice. Therefore, we investigated in vitro osteoclastogenesis using the cells derived from mi/mi mice. We cocultured spleen cells prepared from either wild-type or mi/mi mice with ST2 or TM8 stromal cells and found that formation of TRAP-positive cells was significantly reduced in the cocultures of mi/mi spleen cells compared to wild-type spleen cells in the presence of 1,25(OH)(2) vitamin D(3) (vitamin D). We further investigated Mitf expression by Northern blot analysis in relation to the differentiation of osteoclasts using the cocultures of bone marrow cells with stromal/osteoblastic cells and found positive correlation in expression levels of c-fms and Mitf. Moreover, osteoclast-progenitor-like C7 cells expressed c-fms as well as Mitf mRNAs when cultured alone. C7 cells also expressed Mitf protein in their nuclei. Similar results were obtained when we used primary spleen cells, which differentiate into osteoclasts cultured in the presence of M-CSF and RANKL/ODF. Mitf expression levels in the cocultures of C7 cells and ST2 cells were not changed by treatment with vitamin D in the presence or absence of dexthamethasone. These results suggest that Mitf is expressed in osteoclast progenitors and its presence facilitates osteoclastogenesis.

Animals↗
PubMed2000

Analysis of two major anti-M2 antibodies (anti-PDC-E2/anti-BCOADC-E2) in primary biliary cirrhosis: relationship to titers of immunofluorescent anti-mitochondrial antibody.

To analyze anti-M2 components in primary biliary cirrhosis (PBC) we measured two major anti-M2 antibodies (anti-PDC-E2 and anti-BCOADC-E2) by immunoblotting and ELISA, and compared the results between 38 immunofluorescent anti-mitochondrial antibody (AMA)-negative PBC patients (group A) and 39 strongly AMA-positive PBC patients (group B) with titers of 1:640. Using bovine heart mitochondrial fraction as antigen, the immunoblot positivity rate of anti-PDC-E2 in group B was significantly higher than that in group A, whereas the positivity rate of anti-BCOADC-E2 was not significantly different between the two groups. This result was similar to that obtained by ELISA using recombinant fusion proteins. In group A there was a significant inverse correlation between ELISA optical density values of anti-PDC-E2 and of anti-BCOADC-E2, but in group B there was no correlation between the two values. Only three patients from group A and 21 from group B were positive for both antibodies. Taken together these results appear to indicate that the detection of anti-BCOADC-E2 is critical for the accurate serological diagnosis of AMA-negative PBC patients. The detection of anti-BCOADC-E2 may also help to distinguish between AMA-negative PBC and autoimmune cholangitis patients.

Journal Article↗
PubMed2000

Retardation in bone resorption after bone marrow ablation in klotho mutant mice.

The klotho gene mutant mice exhibit both osteopetrotic phenotype, including elongation of trabeculae in the epiphyses of long bones and vertebral bodies, and osteopenic phenotype, such as thin cortical bones in the diaphyses of these bones. These diverse features raise the question of whether the klotho gene defect results in alteration in bone resorption in vivo. Therefore, we examined the effect of the klotho gene defect on bone resorption by using bone marrow ablation model. At 1 week after bone marrow ablation, trabecular bones were formed in the ablated marrow cavity to levels higher than those in unablated bones in both klotho mutant and wild-type mice. At 2 weeks postsurgery, newly formed trabecular bones were resorbed in wild-type mice to resume normal bone marrow and trabecular bone volume fraction as reported previously. In contrast, the newly formed trabecular bones in the ablated marrow in klotho mutant mice remained at levels similar to those at 1 week. The defect in the bone resorption phase in klotho mutant mice is associated with site-specific reduction of the number and size of osteoclasts in klotho mutant mice. Moreover, the expression levels of osteoprotegerin messenger RNA in the ablated femora of klotho mutant mice were higher than those in wild-type mice. These results indicate that lack of klotho gene expression suppressed bone resorption that should normally take place 2 weeks after bone marrow ablation.

Animals↗
PubMed2000

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases.

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.

Amino Acid Sequence↗