Biomedical subjects

Masahiro Fukuzawa

Publications and source records attributed to Masahiro Fukuzawa.

At least 19 recordsLinked to original sources

PubMed2006

Mutations and polymorphisms of Hirschsprung disease candidate genes in Thai patients.

Mutation and polymorphism data for Hirschsprung disease (HSCR) varies among ethnic groups. Single nucleotide polymorphisms (SNP) of RET proto-oncogene (RET) were recently shown to be associated with the disease, and with disease severity, in different populations. In this study, comprehensive analysis of RET, GDNF, EDNRB, ET-3, and SOX-10 genes among sporadic HSCR in Thailand was conducted by standard PCR-SSCP, RFLP, and sequencing methods. Of 41 patients, 30 cases had rectosigmoid disease (RSD) and 11 cases were assigned to the long-segment disease (LSD) group. Four missense mutations of RET, S100M, R231H, T278N, and G533S, were identified in three patients. One novel missense mutation, V111Q, was detected in EDNRB. For ET-3, two novel missense mutations, D166E and C173R, occurred concomitantly in a patient. The incidence of missense mutation was significantly higher in our female HSCR patient than in the male counterpart. Statistical analysis of the SNPs revealed a significant difference between allele distribution of RET L769L in patients in the LSD and RSD groups. The predominant genotype construct of RET A45A/L769L in our HSCR was GG/GG, which is obviously different from results from all previous studies. The GG/GG genotype construct was associated with RSD and with males. The study also detected a variant allele of RET S836S which has never been reported in Asian cohorts.

Alleles↗

PubMed2006

Features of a newly cloned pig C1 esterase inhibitor.

The pig cDNA encoding C1 esterase inhibitor (C1-INH) was isolated and the homology of the sequence was compared with that from other animals. The structure of pig C1-INH contains a two disulfide bridge pattern identical to the human C1-INH. In the amino acid sequence of the first Cys-91 to the C-terminal end, the pigC1-INH has a 76.2% homology with the human protein, and the sequence of the reactive site is close to the human. A surface-bound form of pig and human C1-INH, pC1-INH-PI and hC1-INH, respectively, were next constructed. Stable Chinese hamster ovarian tumor (CHO) cell lines and pig endothelial cell (PEC) lines expressing these C1-INH-PI were prepared by transfection. The basic function and the species specificity of pCI-INH were then investigated using these transfectants. pC1-INH and hC1-INH have almost the same suppressive effect on pig, human, dog and rabbit sera in complement-dependent cell lysis, indicating little species specificity.

Amino Acid Sequence↗

PubMed2006

Involvement of position-147 for HLA-E expression.

HLA-E functions as an inhibitory signaling molecule of natural killer (NK) cell-mediated cytolysis. However, the cell surface expression of HLA-E molecules is quite restricted because of the limited repertoire of binding peptide sequences, such as signal peptides of other HLA molecules, especially on xenogeneic cells. In this study, we successfully determined that position-147 is an important amino acid position for cell surface expression by producing point substitutions. For further studies concerning transplantation therapy, the point substitution, Ser147Cys, that resulted in a single atom change, oxygen to sulfur, designated as HLA-Ev(147), led to a much higher expression on the human and pig cell surface and a greater inhibitory function against human NK cells than wild type HLA-E in an in vitro model system of pig to human xenotransplantation. Consequently, HLA-Ev(147) might be a promising alternative gene tool for future transplantation therapy such as xenotransplantation.

Amino Acid Sequence↗

PubMed2006

Non-random p53 mutations in pediatric undifferentiated (embryonal) sarcoma of the liver.

Undifferentiated (embryonal) sarcoma of the liver (USL) is a rare hepatic tumor in children. Its pathogenesis is largely unknown, but lines of evidence suggest common links to that of mesenchymal hamartoma of the liver (MH). Previously, we found a p53 mutation in a case of pediatric USL. Recently, there was another published report, demonstrating further evidence of p53 alterations in the adult cases. In this study, we analyzed in three cases of pediatric USL and two cases of MH by using PCR-SSCP and direct sequencing technique. The study identified missense mutations in all three cases of USL, but none of MH. The mutations were found specifically in tumor tissue and not detected in the surrounding normal hepatic tissue. Mutation points were localized in exon 7 (Gly245Ser), exon 6 (Arg196Pro), and exon 8 (Arg273Pro), respectively. Immunohistochemical study of p53 protein expression revealed strong immunoreactivity in cases of USL and negative staining in MH. In summary, this study provided a novel data suggesting that mutations of p53 in pediatric USL are not random genetic events and highly possible to be involved in its tumorigenesis.

Journal Article↗

PubMed2006

Significant inhibition of human CD8(+) cytotoxic T lymphocyte-mediated xenocytotoxicity by overexpression of the human decoy Fas antigen.

BACKGROUND: Human CD8(+) CTL-mediated killing may be important for xenograft rejection. The purpose of this study was to explore the preventing methods for CTL-mediated xenocytotoxicity by overexpression of human decoy Fas, which lacks a death domain in its cytoplasmic region, by binding competition with endogenous pig Fas. Moreover, the cytoprotective effect of this CTL-killing of membrane-bound human FasL, which is resistant to metalloproteolytic cleavage, was also assessed. METHODS: Human CTL were generated by the stimulation of human PBMC with swine endothelial cells (SEC) and human IL-2, subsequently a CD8(+) population were selected by magnetic beads and employed as the effector cells. Stable SEC transfectants expressing either decoy Fas or membrane-bound FasL were established. Double-transfectants were also created. The amelioration of cytotoxicity to these transfectants was examined with Cr release assay. RESULTS.: Human CD8(+) CTL were highly detrimental against parental SEC. This CTL-killing was strongly inhibited by anti-FasL mAb treatment, however partial suppression was observed by Concanamycin A treatment. The overexpression of either decoy Fas or membrane-bound FasL in SEC markedly inhibited CTL-xenocytotoxicity. The double expressions of these molecules also significantly reduced this xenocytotoxicity despite the low levels of expression of either decoy Fas or membrane-bound FasL. CONCLUSION: These findings indicate that the strong xenocytotoxicity of human CD8(+) CTL is mediated mainly by the Fas/FasL pathway. The overexpression of either decoy Fas or membrane-bound FasL were quite effective in preventing CTL-killing. Furthermore, the combined expression of both molecules in pig cells may create a window of opportunity for prolonging xenograft survival.

Animals↗

PubMed2006

Association of 11q loss, trisomy 12, and possible 16q loss with loss of imprinting of insulin-like growth factor-II in Wilms tumor.

We evaluated the WT1 and IGF2 status and performed chromosome and/or comparative genomic hybridization analysis in 43 tumor samples from patients with Wilms tumor. On this basis, we classified them into 4 groups: WT1 abnormality, loss of heterozygosity (LOH) of IGF2, loss of imprinting (LOI) of IGF2, and retention of imprinting (ROI) of IGF2, which were seen in 12%, 30%, 16%, and 42% of the tumors, respectively. Patients in the LOI group were older than those in other groups (P < 0.01), and tumors in the WT1 group had fewer cytogenetic changes than did those in the other groups (P < 0.01). It was found that 11q- and +12 were more frequent in the LOI group than in the WT1+LOH+ROI group (P < 0.01 and P < 0.01). There was no difference in the incidence of 16q- between the LOI group and the other groups; however, when we excluded 16 tumors with LOH on 11p15, 16q- tended to be more frequent in the LOI group than in the WT1+ROI group (P = 0.06). The association of 11q- or +12 with LOI of IGF2 found in the present study suggests that many tumors with no WT1 abnormalities need overexpression of IGF2 together with biallelic inactivation of the tumor-suppressor gene on 11q and/or overexpression of growth-promoting genes on chromosome 12. The 11q gene may code for one of the proteins that constitute a CTCF insulator complex, and its mutation, deletion, or haploinsufficiency may cause insulator abnormalities that might lead to LOI of IGF2.

Child↗

PubMed2006

Histological classification of liver fibrosis and its impact on the postoperative clinical course of patients with congenital dilatation of the bile duct.

PURPOSE: Infants and children with congenital dilatation of the bile duct (CDBD) usually also have some degree of liver fibrosis, which occasionally persists after diversion surgery, leading to liver cirrhosis. We conducted this study to evaluate the histological degree of liver fibrosis using intraoperative biopsy and to determine whether it affects the postoperative clinical course. METHODS: The subjects were 43 infants and children with CDBD. The age at operation ranged from 1 month to 14 years. Histological classification was defined as follows: grade 0, no fibrosis; grade 1, fibrosis confined to the portal area; grade 2, bridge formation with the neighboring portal area; grade 3, widened bridging fibrosis; and grade 4, pseudolobule formation, representative of cirrhosis. RESULTS: There were 21 (48.8%) patients with grade 0, 17 (39.5%) with grade 1, 4 (9.3%) with grade 2, 1 (2.3%) with grade 3, and 0 (0%) with grade 4 fibrosis. There were no differences in preoperative serum total bilirubin, total bile acid, aspartate aminotransferase, or gamma-glutamyl transpeptidase levels among the four groups. On the other hand, the grade 2 and 3 groups tended to include patients with a lower age at operation, and lower amylase levels in the serum and bile. Postoperatively, clinical symptoms resolved and laboratory data normalized in all patients. Two patients underwent liver biopsy for other reasons 3 and 4 years after surgery, which confirmed disappearance of the liver fibrosis. These patients had originally had grade 1 and grade 2 fibrosis, respectively. CONCLUSION: Mild to moderate liver fibrosis is present in almost half of all children with CDBD; however, if it is graded as less than moderate, it will probably not affect the postoperative clinical course.

Adolescent↗

PubMed2006

Long-term outcome in patients with prenatally diagnosed cystic lung disease: special reference to ventilation and perfusion scan in the affected lung.

BACKGROUND/PURPOSE: The natural history of cystic lung disease (CLD) such as congenital cystic adenomatoid malformation (CCAM) and pulmonary sequestration has been altered by the advent of prenatal diagnosis. Although recent advances including fetal therapy have gradually improved outcome, the long-term course and the function of the residual lung have not been well clarified. METHODS: Twenty-two patients with CLD who had been prenatally diagnosed and treated between 1990 and 2004 were reviewed. The clinical outcome and growth measurements were established, and, where possible, all infants underwent ventilation and perfusion lung scan. RESULTS: Mediastinal shift was present in 14 fetuses. Fetal hydrops was present in 5 fetuses. Antenatal intervention was performed for hydrops in 2 fetuses (cyst-amniotic shunt and aspiration). Twenty-one infants underwent appropriate excisional surgery. Final diagnosis included CCAM (n = 12) and pulmonary sequestration (n = 7). No late death was observed. Common complications were failure to thrive (n = 5), frequent respiratory tract infection (n = 4), and asthmatic attack (n = 4). A significant decrease in lung ventilation and perfusion on the affected side was observed in patients with hydrops, lobectomy, and CCAM. CONCLUSION: Long-term follow-up including respiratory care and growth assessment should be performed in prenatally diagnosed patients with CLD, especially those who present with hydrops.

Bronchopulmonary Sequestration↗

PubMed2006

Feasibility of duct-to-duct biliary reconstruction in pediatric living related liver transplantation: report of three cases.

Feasibility of duct-to-duct biliary reconstruction in adult living related liver transplantation (LRLTx) has been recently reported; however, little has been known of its surgical outcome in children. To assess the feasibility and safety of duct-to-duct biliary reconstruction in children, the surgical outcomes of duct-to-duct biliary reconstruction were retrospectively analyzed. The subjects were three children who underwent LRLTx in our hospital each utilizing allografts with a right lobe, a left robe and a lateral segment, respectively. The cause of end-stage liver disease in each of them was fulminant Wilson's disease, fulminant hepatic failure and unresectable hepatoblastoma. Duct-to-duct anastomosis was performed in younger patients and adolescents with interrupted and continuous sutures, respectively. The diameter of bile duct in allografts was from 4 to 6 mm and 12 or 13 stitches were required for anastomosis. Post-operative choledochography from the external tube showed neither stenosis nor leakage and the tube was evacuated within 3 months after LRLTx. No biliary complications were observed with the median follow-up of 28 months. In conclusion, our results show that duct-to-duct biliary reconstructions in pediatric LRLTx seemed to be feasible and safe. Further studies are required to elucidate its real impact on pediatric LRLTx.

Adolescent↗

PubMed2006

Living-related liver transplantation with removal of inferior vena cava for unresectable hepatoblastoma.

We report a case of a two-yr-old boy with hepatoblastoma resectable only by total hepatectomy including the vena cava. Successful LTx was performed with a living donor segment without vena cava reconstruction. The tumor was located in the bilateral lobe, surrounding the IVC. In spite of the high-dose chemotherapy, the tumor did not become resectable. LTx was performed using left lateral segment after removal of the IVC combined with total hepatectomy. Because the collaterals were well developed, the patient tolerated the procedure well. The serum AFP level decreased from 186 699 to 8 ng/mL in 11 months after LTx without local recurrence or distant metastasis.

Child, Preschool↗

PubMed2006

Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.

BACKGROUND: It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement. METHODS: cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis. RESULTS: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration. CONCLUSION: These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.

Animals↗

PubMed2006

In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors.

Mutations of beta-catenin have been identified in the majority of pediatric hepatic malignancies, including hepatoblastoma (HB) and hepatocellular carcinoma (HCC), suggesting its important contribution in hepatic tumorigenesis in this age group. However, the role of beta-catenin/canonical Wnt signaling pathway in the neoplastic growth of cancer cells has not been directly studied. To address beta-catenin's capability in maintaining the malignant phenotype in established pediatric HB and HCC cell lines, HuH-6 and HepG2, harboring mutated and overexpressed beta-catenin, we carried out a series of in vitro analyses through a transfection of short interfering RNAs (siRNAs) to generate a loss-of-function model. HuH-7, another HB cell line derived from a pediatric patient without a stabilizing mutation was used for comparison. RNA interference successfully manipulated the degradation of overexpressed beta-catenin. In all cell lines, beta-catenin mRNA was suppressed by 80-90% after 48 h of transfection, and a reduction of its protein expression was demonstrated. In HuH-6 and HepG2, the pre-existing beta-catenin nuclear accumulation disappeared and reductions of beta-catenin downstream target genes, c-myc and cyclinD1, were also evidenced after the treatment. The in vitro proliferation of both cell lines was transiently inhibited. In contrast, the suppression of beta-catenin in HuH-7 did not lead to a significant change in the expression of target genes or cellular proliferation. Our data indicate that beta-catenin can be considered a specific target for gene therapy in pediatric hepatic tumors with mutations and overexpression of this gene.

Apoptosis↗

PubMed2006

Artificially accumulated beta-catenin inhibits proliferation and induces neurite extension of neuroblastoma cell line NB-1 via up-regulation of trkA.

Recent evidence suggests an association between up-regulation of beta-catenin/Wnt signaling pathway and neuronal differentiation of neuroblastoma. We overexpressed beta-catenin into a human neuroblastoma cell line NB-1 and observed its effect on cellular morphology, growth potential and alteration in a known differentiation related gene, trkA. Expression plasmids containing wild-type and mutated forms of beta-catenin gene were transfected into NB-1 cells, using liposome-based transfection method. The mutated forms were a deletion of three nucleotides of codon 45 and a large deletion involving the whole exon 3. In the transient transfection model, cell viability assay demonstrated significant negative effect of mutated beta-catenin transfection, but not wild-type, on the cell proliferation. To investigate impacts of beta-catenin overexpression in detail, a stable transfection model was established. Clones with comparable expression of beta-catenin at the mRNA level were selected. Only the selected clones with mutated form of beta-catenin exhibited neurite extension pattern and stunned cell proliferation, in association with higher accumulation of total cellular beta-catenin protein as evidenced by Western blot and immunocytochemistry. Cell cycle progression demonstrated significantly higher G0-G1 fraction in each stable cell clone with beta-catenin expression plasmid. In addition, retarded G1/S transition was observed exclusively in the cell clones with mutated form. Concomitantly with overexpressed beta-catenin, up-regulations of trkA and Ha-ras were also identified. Our study suggests a potential availability of beta-catenin/Wnt signaling pathway as a target of molecular manipulation for treatment of high-risk neuroblastoma and a potential association between the pathway and the trkA/neurotrophin cascades.

Blotting, Western↗

PubMed2005

Renal cell carcinoma in a pediatric patient with an inherited mitochondrial mutation.

Renal cell carcinoma (RCC) is a rare pediatric renal cancer. Recent molecular genetic studies discovered a tumor-specific mutation involving translocation of a transcription factor TFE3 in a subset of pediatric RCC with distinct histopathology. We reported a case of a 2-year-old boy with RCC associated with TFE3 translocation resulting in a PRCC-TFE3 fusion gene. Interestingly, the case carried a maternally inherited mitochondrial DNA (mtDNA) alteration at the position which is usually found in MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes) syndrome (A3243G). Although evidence of somatic alterations in mtDNA existed in various cancers, association between inherited mtDNA mutation and pediatric renal cancer has not been reported. Our case provided the first evidence of a co-occurrence between a germ line mutation in mtDNA and the somatic mutation of pediatric RCC. With this information, we speculated a role of mitochondria mutation in the pathogenesis of this cancer.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2005

Novel mutation of Endothelin-B receptor gene in Waardenburg-Hirschsprung disease.

Homozygous mutations of EDNRB in human have been reported to result in Waardenburg-Hirschsprung disease (WS4), while mutated heterozygotes manifested isolated Hirschsprung disease in lower penetrance. We investigated a case of WS4 together with all members of her nuclear family for the alteration of the EDNRB gene by using PCR-SSCP and direct sequencing technique. The index patient, who was born to a family with no history of Hirschsprung disease, presented total colonic aganglionosis with small bowel extension, sensorineural hearing loss and generalized cutaneous pigmentary defects. Interestingly, both irides were normally black. The study detected a homozygous missense mutation at codon 196 in exon 2 (Ser196Asn), which has not been reported. Both parents and four in six siblings harbored heterozygous mutation without any clinical manifestation. Our findings were consistent with previous observations that full spectrum of WS4 occurred to the mutate homozygotes. Moreover, the non-penetrance of heterozygotes in our pedigree, which differs from other reports, demonstrates the high pleiotropic effect of EDNRB mutations in human.

Female↗

PubMed2005

Recurrent acute pancreatitis caused by malrotation of the intestine and effective treatment with laparoscopic Ladd's procedure.

We herein report a rare case of recurrent pancreatitis caused by malrotation of the intestine. Since the age of 10 years, a female patient showed recurrent epigastralgia and was diagnosed at age 16 years as suffering from acute pancreatitis. Upper gastrointestinal series showed that the duodenal bulb was elongated and rotated caudally. Endoscopic retrograde cholangiopancreatography showed that the descending portion of the duodenum was rotated and occluded the papilla of Vater. Under laparoscopy, the duodenal bulb was tracted caudally by the Ladd's ligament. By incision of this ligament, the entire duodenum was mobilized, with subsequent reduction of the rotation and compression of the pancreatic head. The girl's postoperative course was uneventful, and she has been asymptomatic for the last 3 years. In conclusion, malrotation of the intestine may be a rare cause of recurrent pancreatitis, and laparoscopic Ladd's procedure may be a less invasive and useful treatment.

Acute Disease↗

PubMed2005

LDOC1, a novel MZF-1-interacting protein, induces apoptosis.

LDOC1 was isolated as a gene encoding a leucine-zipper protein whose expression was decreased in pancreatic and gastric cancer cell lines in 1999. Here, we found that overexpression of LDOC1 caused externalization of the cell membrane phosphatidylserine, which was characteristic for early-phase apoptotic events, and reduced cell viability in some human cell lines. The apoptotic process was triggered by a loss of the mitochondrial membrane potential, leading to both caspase-3-dependent and -independent pathways. Furthermore, a transcription factor, MZF-1, was revealed to interact with LDOC1 and enhance the activity of LDOC1 for inducing apoptosis.

Apoptosis↗

PubMed2005

Cytogenetic abnormalities in hepatoblastoma: report of two new cases and review of the literature suggesting imbalance of chromosomal regions on chromosomes 1, 4, and 12.

Two cases of hepatoblastoma with unique karyotypic changes are described. One case was that of a 2-year-old boy with an unbalanced chromosomal translocation involving 4q35 as the sole chromosomal abnormality. The clonal karyotype of this tumor was 46,XY,add(4)(q35)[3]/46,XY[9]. In the other case, that of a 2-year-old boy, karyotypic analyses revealed the clonal karyotype as 57,XY,+del(1)(p22),+2,+5,+6,+7,+8,+del(12)(p12),+18,+19,+20,+22[4]/46,XY[12]. Review of these two cases, together with previous reports, underscored the significance of numerical and/or structural chromosomal abnormalities of 1q, 4q, 2, 8, and 20 in the development of hepatoblastoma. The present results show that imbalance of the terminal region of 4q could be the sole chromosomal abnormality in a hepatoblastoma. We also found that imbalance of chromosomal regions on chromosomes 1 and 12 may contribute to the development of hepatoblastoma.

Aneuploidy↗