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Regulation of cell growth and cell differentiation during muscle development.

Research Project

Project/Area Number 08458242
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Developmental biology
Research InstitutionNational Institute of Neuroscience, NCNP

Principal Investigator

NABESHIMA Yo-ichi  National Institute of neuroscience, Director, 神経研究所遺伝子工学, 部長 (60108024)

Co-Investigator(Kenkyū-buntansha) YOSHIDA Shosei  Osaka Univetsity, IMCB Investigator, 細胞生体工学センター, 助手 (60294138)
TAKEDA Sin-ichi  National Institute of neuroscience, Section Chief, 神経センター・神経研究所遺伝子工学, 室長 (90171644)
FUJISAWA Atsuko  National Institute of neuroscience, Section Chief, 神経センター・神経研究所遺伝子工学, 室長 (60209038)
NABESHIMA Yo-ichi  National Institute of neuroscience, Director (60108024)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥5,500,000 (Direct Cost: ¥5,500,000)
KeywordsMuscle development / MyoD family / LPA / bFGF / Cell cycle / Myogenesis / 細胞培養 / bHLH
Research Abstract

1) LPA and bFGF control different modes in proliferating myoblasts We discoverd that LPA stimulates the growth and inhibits the differentiation of mouse C2C12 myoblasts cells, in a distinct manner from bFGF whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases. LPA and bFGF act differently in regulating the myogenic bHLH proteins. LPA stimulates the proliferation of undifferentiated myoblasts allowing the continued expression of MyoD,but in contrast, bFGF does so with the MyoD expression suppressed at the mRNA level. Both compounds maintain the myf-5 expression and suppress the myogenin expression. In addition, while LPA did not inhibit cell-cell contact-induced differentiation. bFGF strongly inhibited process.
2) Variation of MyoD and Myf-5 specifies the differentiating and Reserve cells upon myogenesis. We investigated that homogeneously growing C2 … More C12 mouse myoblasts generate two distinct types of subpopulatioin upon serum deprivation ; differentiating cells and reserve cells. A number of events known to be related to terminal differentiation including myogenin expression, biochemical differentiation, permanent withdrawal from cell cycle which is parallel to P21^<WAF1> expression, and cell fusion, occur only in the former type of cells. In contrast, the latter do not cause these events and remain undifferentiated. However, they retain their myogenic potentials and cause differentiation identically to the original myoblasts after serum re-stimulation.
These two cell types are distinguishable in very early step of differentiation by MyoD and Myf-5 expression. Whereas the differentiating cells continue to express MyoD and Myf-5, the reserve cell rapidly lose or decrease their expression. This variation proceeds the myogenin expression. Moreover, ectopic expression of MyoD pretein converts the reserve cells to differentiating cells. indicating that MyoD specifies the cells to differentiate. Less

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (57 results)

All Other

All Publications (57 results)

  • [Publications] Yoshida S., Fujisawa-Sehara A., Arai K.and Nabeshima Y.: "Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts." J.Cell Biol.132. 181-193 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hoshino M., Suzuki E., Nabeshima Y.and Hama C.: "hikaru genki protein secreted into synaptic cleft from an early stage of synapse formation in Drosophila." Development. 122. 589-597 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Ohbayashi T., Schmidt E.E., Makino Y., Kishimoto T., Nabeshima Y., Muramatsu M., and Tamura T.: "Promoter structure of the mouse TATA-binding protein(TBP)gene." Biochem.Biophys.Res.Comm.225. 275-280 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sone M., Hoshino M., Suzuki E, .Saigo K., Nabeshima Y., and Hama C.: "Still life,a protein in synaptic tereminals of Drosophila homologous to GDP-GTP exchanger." Science. 275. 543-547 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takahashi S., Onodera K., Motohashi H., Suwabe N., Hayashi N., Yanai N., Nabeshima Y., Yamamoto M.: "Arrest in primitive erythroid cell development caused by promotea-specific distruption of the GATA-1 gene." J.Biol.Chem.272. 12611-12615 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sakamoto K., Yan L., Imai H., Takagi M., Nabeshima Y., Takeda S., Katsube K.: "Idenification of a chick homologue,C-Fringe 1.Involvement in the neurogenesis and the somitogenesis." Biochem.Biophys.Comm.234. 754-759 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Itoh K., Chiba T., Takahashi S., Ishii T., Igarshi K., Katoh Y., Oyake T., Hayashi N., Satoh K., Yamamoto M., Nabeshima Y.: "An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying genes through antixidant response elements" Biochem.Biophys.Res.Comm.236.313-322 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Miyagoe Y., Terashima T., Nonaka I., Hanaoka K., Hayasaka M., Nabeshima Y., Arahata K., Nabeshima Y., and Takeda S.: "Laminin α2chain-null mutant mice by targeted disruption of the Lama 2 gene: A new model of merosin(Iaminin 2)-deficient congenital muscular dystophy." FEBS Letters. 415. 33-39 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kuro-o M., Matsumura Y., Aizawa H., Kawaguchi H., Suga T.Vtugi T., Ohyama Y., Kaname T., Kume E., Iwasaki H., Iida A., Shiraki-Iida T., Nishikawa S., Ryozo N.and Nabeshima Y.: "Mutation of the mouse Klotho gene leads to a syndrome resembling ageing." Nature. 390,. 45-51 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kameya S., Araki E., Katsuki M., Mizota A., Adachi E., Nakahara K., Nonaka I., Sakuragi S., Takeda S., an Nabeshima Y.: "Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of β-dystoglycan in the outer pleviform layea of retina." Human Molecular Genetics. 6. 2195-2203 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Ikeshima-Kataoka H., Skeath J.B., Nabeshima Y., Doe C.Q., and Matsuzaki F.: "Miranda directs Prospei to a daughter cell during Drosophila asymmeteric divisions." Nature. 390. 625-629 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshida N.Yoshida S., Fujisawa-Sehara A., and Nabeshima Y.: "Variation of MyoD and myf-5,which precedes myogenin expression and irreversible cwmmiment,specifies the differentiating and"Reserve Cells"upon myogenesis." J.Cell Sci.in press.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Matsumura Y., Aizawa H., Shiraki-Iida T., Nagai R., Koro-o M., Nabeshima Y.: "Identification of the human klotho gene and its two transcripts encoding membrane and secreted Klotho protein." B.B.R.C.in press.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shiraki-Ijda T., Aizawa H., Matsumura Y., Sekine S., Iida A., Anazawa H., Nagai R., Kuro-o M., Nabeshim Y.: "Structure of the mouse Klotho gene and its two transcripts encoding membrane and secreted protein." FEBS Letters. in press.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yamashita T., Nifuji A., Furya K., Nabeshima Y., Noda M.: "Elongation of the epiphyseal trabecular bon associated with precocious aging in transgenic mice carrying an inserational mutation in Klotho gene locus." J.Bone Mineral Res.in press.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Okuda A., Fukushima A., Nishimoto M., Nabeshima Y., Kuro-oM., Nabeshima Y., Boon K., Keaveney M.Stunneneberg H.G., Muramatsu M.: "UTF1,a transcriptional coacruvator representing part of the E1A-like activity in pluripotent embryonic stem cells." EMBO J.in press.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshida S., Fujisawa-Sehara A., Arai K.and Nabeshima Y.: "Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts." J.Cell Biol. 132. 181-193 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Hoshino M., Suzuki E., Nabeshima Y.and Hama C.: "hikaru genki protein secreted into synaptic clefts from an early stage of synapse formation in Drosophila." Development. 122. 589-597 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Ohbayashi T., Schimidt E.E., Makino Y., Kishimoto T., Nabeshima Y., Muramatsu M., and Tamura T.: "Promoter structure of the mouse TATA-binding pretein (TBP) gene." Biochem.Biophys.Res.Comm.225. 275-280 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sone M., Hoshino M., Suzuki E., Saigo K., Nabeshima Y., and Hama C.: "Still life, a protein in synaptic tereminals of Drosophila homologous to GDP-GTP exchanger." Science. 275. 543-547 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takahashi S., Onodera K., Motohashi H., Suwabe N., Hayashi N., Yanai N., Nabeshima Y., Yamamoto M.: "Arrest in primitive erythroid cell development caused by promoter-specific distruption of the GATA-1 gene." J.Biol.Chem. 272. 12611-12615 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sakamoto K., Yan L., Imai H., Takagi M., Nabeshima Y., Takeda S., Katsube K.: "Identification of a chick homologue of Fringe, C-Fringe 1. Involvement in the neurogenesis and the somitogeneses." Biochem.Biphys.Res.Comm.234. 754-759 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Itoh K., Chiba T., Takahashi S., Ishii T., Igarashi K,Katoh Y., Oyake T., Hayashi N., Satoh K., Yamamoto M., Nabeshima Y.: "An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying genes through antioxidant response elements" Biochem.Biophys.Res.Comm.236. 313-322 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Miyagoe Y., Terashima T., Nonaka I., Hanaoka K., Hayasaka M., Nabeshima Y., Arahata K., Nabeshima Y., and Takeda S.: "Laminin alpha2 chain-null mutant mice by targeted disruption of the Lama 2 gene : A new model of merosin (laminin 2) -deficient congenital muscular dystrophy" FEBS Letters. 415. 33-39 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kuro-o M., Matsumura Y., Aizawa H., Kawaguchi H., Suga T.Ursugi T., Ohyama Y., Kaname T., Kume E., Iwasaki H., Iida A., Shiraki-Iida T., Nishikawa S., Ryozo N.and Nabeshima Y.: "Mutation of the mouse Klotho gene leads to a syndrome resembling ageing" Nature. 390. 45-51 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kameya S., Araki E., Katsuki M., Mizota A., Adachi E., Nakahara K., Nonaka I., Sakuragi S., Takeda S., and Nabeshima Y.: "Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layr of retina." Human Molecular Genetics. 6. 2195-2203 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Ikeshima-Kataoka H., Skeath J.B., Nabeshima Y., Doe C.Q., and Matsuzaki F.: "Miranda directs Prospero to a daughter cell during Drosophila asymmeteric divisions." Nature. 390. 625-629 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshida N.Yoshida S., Fujisawa-Sehara A., and Nabeshima Y.: "Variation of MyoD and myf-5 which precedes myogenin expression and irreversible commitment, specifies the differentiating and "Reserve Cells" upon myogenesis." J.Cell Sci.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Matsumura Y., Aizawa H., Shiraki-Iida T., Nagai R., Koro-o M., Nabeshima Y.: "Identification of the human klotho gene and its two transcripts encoding membrane and secreted Klotho pretein." B.B.R.C.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Shiraki-Iida T., Aizawa H., Matsumura Y., Sekine S., Iida A., Anazawa H., Nagai R., Kuro-o M., Nabeshima Y.: "Structure of the mouse Klotho gene and its two transcripts encoding membrane and secreted protein." FEBS Letters. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yamashita T., Nifuji A., Furuya K., Nabeshima Y., Noda M.: "Elongation of the epiphyseal trabecular bone associated with precocious aging in transgenic mice carrying an insertional mutatiion in Klotho gene locus." J.Bone Mineral Res.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Okuda A., Fukushima A., Nishimoto M., Nabeshima Y., Kuro-o M., Nabeshima Y., Boon K., Keaveney M., Stunnenberg H.G., Muramatsu M.: "UTF1, a transcriptional coacruvator representing part of the E1A-like activity in pluripotent embryonic stem cells." EMBO J.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshida S., FujisawaーSehara., Arai K. and Nabeshima Y.: "Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts." J.Cell.Biol.132. 181-193 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Hoshino M., Suzuki E., Nabeshima Y. and Hama C.: "hikaru genki protein secreted into synaptic cleft from an early stage of synapse formation in Drosopiola." Development. 122. 589-597 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Ohbayashi T., Schmidt E.E., Makino Y., Kishimoto T., Nabeshima Y., Muramatsu M. and Tamura T.: "Promotor structure of the mouse TATA-binding protein (TBP)gene." Biochem.Biophys.Res.Comm.225. 257-280 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sone M., Hoshino M., Suzuki E., Saigo K., Nabeshima Y. and Hama C.: "Still life,a protein in synaptic tereminals of Drosophila homologous to GDP-GTP exchanger." Science. 275. 543-547 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Takahashi S., Onodera K., Motohashi H., Suwabe N., Hayashi N., Yanai N., Nabeshima Y., Yamamoto M.: "Arrest in primitive erythroid cell development caused by promoter-specific distruption of the GATA-1 gene." gene.J.Biol.Chem.272. 12611-12615 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sakamoto K., Yan L., Imai H., Takagi M., Nabeshima Y., Takeda S., Katsube K.: "Identifiation of a chick homologue of Fringe,C-Fringe1.Involvement in the neurogenesis and the somitogenesis." Biochem.Biophys.Res.Comm.234. 754-759 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Itoh K., Chiba T., Takahashi S., Ishii T., Igarashi K., Katoh Y., Oyake T., Hayashi N., Satoh K., Yamamoto M., Nabeshima Y.: "An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying genes through antioxidant response elements." Biochem.Biophys.Res.Comm.236. 313-322 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Miyagoe Y., Terashima T., Nonaka I., Hanaoka K., Hayasaka M., Nabeshima Y., ArahataK., Nabeshima Y.,and Takeda S.: "Liminin α2 chain-null mutant mice by targeted disuruption of the Lama2 gene: A new model of merosin (laminin 2)-deficient congenital muscular dystrophy." FEBS Letters. 415. 33-39 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kuro-o M., Matusumura Y., Aizawa H., Kawaguchi H., Suga T., Utsugi T., Ohyama Y., Kaname T., Kume E., Iwasaki H., Iida A., Shiraki-Iida T., Nishikawa S., Nagai R.and Nabeshima Y.: "Mutation of the mouse Klotho gene leads to a syndrome resembling ageing." Nature. 390. 45-51 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kameya S., Araki E., Katsuki M., Mizota A., Adachi E., Nakahara K., Nonaka I.,Sakuragi S., Takeda S.,and Nabeshima Y.: "Dp260 dusrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of β-dystroglycan in the outer plexiform layer of retina." Human Molocular Genetics. 6. 2195-2203 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Ikeshima-Kataoka H., Skeath J.B., Nabeshima Y., Doe C.Q.,and Mtsuzaki F.: "Miranda directs Prospero to a daughter cell during Drosophila asymmeteric divisions." Nature. 390. 625-629 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Yoshida N., Yoshida S., Fujisawa-Sehara A.,and Nabeshima Y.: "Variation of MyoD and myf-5,which presedes myogenin expression and irreversible commitment,specifies the differentiating and“Reserve Cells " upon myogenesis." J.Cell Sci.(in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] MatsumuraY., Aizawa H., Shiraki-Iida T., Nagai R., Kuro-o M., Nabeshima Y.: "Identification of the human klotho gene and its two transcripts encoding membrane and secreted Koltho protein." B.B.R.C. (in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Shiraki-Iida T., Aizawa H., Matsumura Y., Sekine S., Iida A., Anazawa H., Hagai R., Kuro-o M., Nabeshima Y.: "Structure of the mouse Klotho gene and its two transcripts encoding membrane and secreted protein." FEBS Letters. (in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Yamashita T., Nifuzi A., Furuya K.,Nabeshima Y., Noda M.: "Elongation of the epiphyseal trabecular bon associated with precocious aging in transgenic mice carrying an insertional mutation in Klotho gene locus." J.Bone Mineral Res.(in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Okuda A., Fukushima A., Nishimoto M., Nabeshima Y., Kuro-o M., Nabeshima Y., Boon K.,Keaveney M., Stunnenberg H.G., Muramatsu M.: "UTF1,a transcriptional coacruvator representing part of the E1A-like activity in plutripotent embryonic stem cells." EMBOJ.(in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Yoshida S.,Fujisawa-Sehara A.,Arai K.and Nabeshima Y.: "Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts." J.Cell Biol.132. 181-193 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Hoshino M.,Suzuki E.,Nabeshima Y.and Hama C.: "hikaru genki protein secreted into synaptic clefts from an early stage of synapse formation in Drosophila." Development. 122. 589-597 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Ohbayashi T.,Schmidt E.E.,Makino Y.,Kishimoto T.,Nabeshima Y.,Muramatsu M.,and Tamura T.: "Promoter structure of the mouse TATA-binding protein (TBP) gene." Biochem Biophys.Res.Comm.225. 275-280 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Sone M.,Hoshino M.,Suzuki E.,Saigo K.,Nabeshima Y.,and Hama C.: "Still life,a protein in synaptic tereminals of Drosophila homologous to GDP-GTP exchanger." Science. 275. 543-547 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] 石井亜紀子、武田伸一、鍋島陽一: "筋細胞" 実験医学別冊「遺伝子治療の基礎技術」. 172-179 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 鍋島陽一: "筋細胞分化の遺伝子制御ネットワーク" The Bone 「硬組織細胞と分子発生生物学」. 10. 97-106 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 鍋島陽一: "筋細胞分化の遺伝子制御ネットワーク" 蛋白質、核酸、酵素 増刊号「遺伝子機能と転写因子」. 41. 1008-1017 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 鍋島陽一: "筋細胞分化における遺伝子の段階的活性化" 生体の科学 特集「細胞分化」. 47. 184-189 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 鍋島陽一: "筋細胞の増殖因子" 脳と神経科学シリーズ 1 「遺伝子異常からみた神経疾患」. 36-44 (1996)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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