Research Themes

• Basic research to develop a method for the prevention of acute appendicitis
  Grant-in-Aid for Scientific Research (C)
  Saitama Medical University
  01 Apr. 2020 - 31 Mar. 2023
  In order to elucidate abnormalities in the oral and intestinal microbiota associated with acute appendicitis, the microbiota (oral, intestinal, appendix) of children with acute appendicitis, the microbiota (oral, intestinal) of healthy children were examined. In addition, lifestyle habits and medical history related to acute appendicitis (e.g., presence or absence of caries) were examined.
  Oral bacteria such as Fuzobacteriaceae bacteria predominated in the appendicitis group, accounting for 52% of the bacterial composition. This was due to the very high diversity of microbiota, but it was difficult to identify abnormalities in the microbiota (oral, intestinal) associated with acute appendicitis.
• 難治性小児消化器疾患の医療水準向上および移行期・成人期の QOL 向上に関する研究　　　　　　　　　　　　　　　
  01 Apr. 2020
• Comprehensive research about medical care and nursing for transitional care of the excretion function child with a disability.
  Grant-in-Aid for Scientific Research (C)
  University of Yamanashi
  Apr. 2014 - Mar. 2018
  We reviewed it and based on the report of transition programs in North America, referred to as the "transition tool kit" issued by DSCC (Special Division of Children with Chronic Disease) in the state of Illinois. Three adolescents were surveyed with a questionnaire whom had excretory function disabilities, and the results were, (1) health management skills and abilities, (2) drug treatment skills and capabilities, (3) insurance skills and ability were almost acquired. Also, we hosted the Stoma ·Continence Rehabilitation Conference (33rd) which is the field of adult surgery, and in the panel discussion, we were able to discuss the transition toward adult medical treatment of a child with excretory function disabilities. Additionally, the research plan did not proceed as planned due to staff transitioning and movement, including due to retirement. However, it is believed that by providing lectures to surgical nurses at Stoma Rehabilitation seminars leads to future support.
• Purification of the antibody against synthetic peptide from N-myc cncoprotein
  Grant-in-Aid for Scientific Research (C)
  University of Tokyo
  1996 - 1997
  The importance of determining N-myc oncoprotein rather than genomic N-myc amplification has been emphasized in neuroblastoma. N-myc-specific peptides, HGRGPPTAGSTAQSPG (codon. 136-151) and GVAPPRPGGRQTSGGDH (codon. 223-239), were synthesized and injected into rabbits in conjugation with either hemocyanin or lysine core (multiple antigen peptide method). Synthesized peptides conjugated to the lysine core raised more potent antibodies than those conjugated to hemocyanin. IgG against GVAPPRPGGRQTSGGDH purified by affinity column showed a precipitation line identical to that of N-myc oncoprotein present in N-myc amplified neuroblastomas, and strongly stained the nuclei of neuroblastoma cells with N-myc amplification, while it. did not react with N-myc unamplified tumors and with c-myc/L-myc amplified tumors either with immunoblot analysis or with immunostaining. A polyclonal antibody specific for a synthetic peptide from the N-Myc oncoprotein was thus obtained.
• Quantification of myc gene products and its clinical implication
  Grant-in-Aid for Scientific Research (B)
  UNIVERSITY OF TOKYO
  1995 - 1996
  The importance of determining N-Myc oncoprotein rather than genomic N-myc amplification has been emphasized in neuroblastoma. In order to develop an ELISA method for N-Myc oncoprotein quantification, an effort was made to raise antibodies specific for N-Myc, and also to produce a relatively smaller-sized N-Myc oncoprotein, which should be water soluble. N-Myc-specific peptide (codon.223-239) were synthesized and injected into rabbits in conjugation with lysine core (MAP method) plus adjuvant. Synthesized peptide conjugated to the lysine core raised a potent antibody. Both crude antibody and IgC purified on an affinity column on such peptide coupled to EAH Sepharose showed a precipitation line identical to that of N-Myc oncoprotein by immunoblot analysis. The purified IgG against N-Myc-specific peptide (codon.223-239) strongly stained the nuclei of neuroblastoma cells with N-myc amplification ; thus a polyclonal anti-body specific for a synthetic peptide from the N-Myc oncoprotein was obtained.
  For preparing standard protein, partial exon 2 and exon 3 of the N-myc gene was cloned and inserted into an expression vector, pET16b. A water-soluble recombinant N-Myc oncoprotein (rN-Myc) with a molecular weight of 38 kDa was expressed by the Escherichia coli, and was purified with Ni^<2+> affinity column chromatography. In immunoblot analysis, the purified anti-N-Myc-specific peptide (codon.223-239) IgG reacted with the rN-Myc oncoprotein. Thus, a water-soluble rN-Myc was successfully obtained, which will enable the establishment of a quantitative method for measuring N-Myc oncoprotein expression.
• MOLECULAR BIOLOGICAL STUDIES OF APOPTOSIS ENDONUCLEASE IN HUMAN NEUROBLASTOMAS
  Grant-in-Aid for Scientific Research (B)
  UNIVERSITY OF TOKYO
  1995 - 1996
  During the research period in 1995, apoptosis induction was examined for each of 16 human neuroblastoma cell lines, using the following three apoptosis inducers, namely, actinomycin D,adriamycin, and camptothecin, . We have already found that the cleavage of DNA in apoptotic cells occurs in at least two steps, i.e., large fragmentation of DNA of 2-50 kilobase pairs and internucleosomal fragments of 180 basepairs. Depending upon the size of DNA fragmentation oberved in each tumor cell line, we found that 16 human neuroblastoma cell lines investigated were classified into the followign three categories ; (1) cell line in which even large fragmentation was not induced by any of the aforementioned three apoptotic inducers, (2) cell line in which only large fragmentation was induced followed by no further small fragmentaton, and (3) cell line in which small fragmentation into internucleosomal fragments with typical apoptotic DNA ladder formation was induced following large fragmentation.
  During the year 1996, our study was directed mainly toward identification of DNase lambda, a newly recognized key-endonuclease responsible for large fragmentation, by comparing the enzymatic activity between a pair of neruoblastoma cell lines ; one cell line (TGW) with positive DNase lambda activity and the oher one (GOTO) lacking it. We exmined whether large DNA fragmentation can be induced in vitro by autodigested nuclei of DNase lambda positive TGW cells, and confirmed the induction of large fragmentation with about 20 kb fragments.
  Using this autodigestion system, fragmentation induction in vitro was further exmined in GOTO cells without DNase lambda, and in SK-N-SH cells with both DNase lambda and DNase gamma (a key-endonuclease for small, internucleosomal fragmentation). As has been expected, no induction was seen in GOTO cells, while in SK-N-SH cells small fragmentation in vitro was shown.
  For further genetic analysis studies on DNase gamma in small DNA fragmentation b, three neuroblastoma cells with distinct biological characteristics (GOTO,TWGW,and SK-N-SH) were propagated and kept frozen.
• Clinical and Histopathological Study on Diagnostic Value of Total Renin
  Grant-in-Aid for General Scientific Research (B)
  University of Tokyo
  1992 - 1993
  No tumor maker specific for Wilms'tumor has been known. Total renin consists of active and inactive forms, and it is known that inactive form of renin constitutes 70% to 90% of total renin in plasma. Since it was reported in 1985 that inactive renin can be a good marker for Wilms' tumor, we started to investigte whether or not total renin in plasma as determined by an RIA with monoclonal antibodies can become a much better marker for this tumor. As a preliminary study, we could confirm that an RIA system developed by Nishiura and his associates truly determines total renin concentrations, by a good correlation with data obtained withthe conventional enzymatic assay, and also by an immunohistochemical study ofmonoclonal antibodies which stained exclusively juxtaglomurular apparatuses of the kidney. We also found the upper border of the normal ranges of plasma total renin is 301 pg/ml in children. Total renin in plasma of 13 patients with Wilms' tumor ranged from 231 pg/ml to 1,289 pg/ml, and 9 out of the 13 patients showed values beyond the upper border of the normal ranges. Postoperatively, plasma total renin decreased to the normal ranges in all 5 patients studied. However, there was no correlation between the plasma values and stages. Two patients each with congenital mesoblastic nephroma and multicystic dysplastic kidney showed exessively high values of total renin, which was also confirmed immunohistochemically. Total renin was normal in all 16 patients with neuroblastoma. Three of 5 cases of hepatoblastoma and one of 10 cases of teratoma showed high values of plasma total renin, and they were considered as examples of extrarenal renin production.