Noriaki Kondo

Kitasato University, Edo, Tōkyō, Japan

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Publications (15)57.61 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The hibernation-specific HP-25 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. Our previous studies have revealed two positively acting transcriptional regulatory regions in the 5'-flanking region of the chipmunk HP-25 gene, one from -260 to -80 and another from -80 to -59, and a pivotal role for hepatocyte nuclear factor-4 (HNF-4), which binds to the proximal regulatory region, in HP-25's liver-specific transcription. A database search for transcription factor binding sites in the distal regulatory region indicated the presence of two potential binding sites for upstream stimulatory factor (USF): one between -161 and -156 and the other between -143 and -138. In an electrophoretic mobility shift assay (EMSA), in vitro-translated USF bound only to the sequence from -143 to -138. USF did not bind the corresponding sequence of the tree squirrel HP-25 gene, which has two base substitutions. Transient transfection studies in COS-7 cells showed that USF could activate the transcription of the chipmunk HP-25 gene, and that tree squirrel-type base substitutions in the USF-binding site aborted the transactivation by USF. By chromatin immunoprecipitation (ChIP) analysis, we confirmed that USF bound to the promoter region of the HP-25 gene in the chipmunk liver, and not in the kidney or heart. These results indicate that USF is involved in the transcriptional regulation of the chipmunk HP-25 gene in the liver, and that the base substitutions in the USF-binding site contribute to the lack of HP-25 gene expression in the tree squirrel.
    Gene 08/2007; 396(2):268-72. · 2.20 Impact Factor
  • Noriaki Kondo
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 12/2006; 51(13):1847-53.
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    ABSTRACT: Seasonal hibernation in mammals is under a unique adaptation system that protects organisms from various harmful events, such as lowering of body temperature (Tb), during hibernation. However, the precise factors controlling hibernation remain unknown. We have previously demonstrated a decrease in hibernation-specific protein (HP) complex in the blood of chipmunks during hibernation. Here, HP is identified as a candidate hormone for hibernation. In chipmunks kept in constant cold and darkness, HP is regulated by an individual free-running circannual rhythm that correlates with hibernation. The level of HP complex in the brain increases coincident with the onset of hibernation. Such HP regulation proceeds independently of Tb changes in constant warmth, and Tb decreases only when brain HP is increased in the cold. Blocking brain HP activity using an antibody decreases the duration of hibernation. We suggest that HP, a target of endogenously generated circannual rhythm, carries hormonal signals essential for hibernation to the brain.
    Cell 05/2006; 125(1):161-72. · 31.96 Impact Factor
  • Noriaki Kondo
    Folia Pharmacologica Japonica 03/2006; 127(2):97-102.
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    ABSTRACT: The hibernation-specific HP-27 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. In the promoter region, the chipmunk gene has a potential HNF-1 binding site, and the tree squirrel gene has two base substitutions in the corresponding sequence. In this paper, we investigated the role of HNF-1 in the HP-27 gene promoter activity. Gel retardation assays with in vitro-translated HNF-1 and super-shift assays using HepG2 nuclear extracts and an anti-HNF-1 antibody revealed that HNF-1 bound to the chipmunk gene sequence. HNF-1 also bound to the tree squirrel sequence, but with much lower affinity. In HepG2 cells, HNF-1 activated transcription from the chipmunk HP-27 gene, but not from the tree squirrel gene. In addition, the tree squirrel-type base substitutions in the HNF-1 binding site greatly reduced the promoter activity of the chipmunk HP-27 gene. These results indicate that HNF-1 is required for the promoter activity of the chipmunk HP-27 gene, and that the base substitutions in the HNF-1 binding site are involved in the lack of HP-27 gene expression in the tree squirrel.
    ZOOLOGICAL SCIENCE 05/2004; 21(4):393-6. · 1.08 Impact Factor
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    ABSTRACT: The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.
    Gene 04/2004; 329:71-9. · 2.20 Impact Factor
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    ABSTRACT: The chipmunk hibernation-specific protein HP-27 is a component of the 140-kDa complex that decreases in the blood during hibernation. Although the HP-27 gene is detected in both the chipmunk, a hibernating species of the squirrel family, and the tree squirrel, a nonhibernating species, it is expressed only in the chipmunk, in a liver-specific manner. To understand the difference in HP-27 gene expression between the chipmunk and tree squirrel, we isolated chipmunk and tree squirrel HP-27 genomic clones, and compared their promoter activities. Transient transfection studies in HepG2 cells revealed that the 170 bp 5'-flanking sequence of the chipmunk HP-27 gene was sufficient for liver-specific promoter activity and that deletion of the sequence from -170 to -140 reduced the promoter activity by 90%. Although the corresponding 170 bp 5'-flanking sequence of the tree squirrel HP-27 gene had 89% nucleotide sequence homology to that of the chipmunk, it showed almost no promoter activity in HepG2 cells. In a gel retardation assay using HepG2 or chipmunk liver nuclear extracts, the 5'-flanking sequence of the chipmunk HP-27 gene from -170 to -140 showed a shifted band, but the corresponding tree squirrel sequence did not. Taken together, these data indicate that a transcription factor that binds to this 5'-flanking sequence of the chipmunk HP-27 gene plays an important role in HP-27 gene expression, and the failure of this factor to bind in the case of the tree squirrel HP-27 gene could be responsible for this animal's lack of HP-27 gene expression.
    Gene 02/2003; 302(1-2):193-9. · 2.20 Impact Factor
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    ABSTRACT: The gene for the hibernation-specific protein HP-25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP-25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP-25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP-25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP-25 gene, the 80-bp 5' flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides -67 to -51, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5' flanking sequence had almost no promoter activity in HepG2 cells, and HNF-4 did not bind to the corresponding region of the tree squirrel HP-25 gene. Furthermore, a tree squirrel-type G to A mutation at -57 in the chipmunk HP-25 gene promoter context abolished its binding to and transactivation by HNF-4. Thus, the point mutation in the HNF-4-binding site is likely to be involved in the lack of HP-25 gene expression in the tree squirrel.
    European Journal of Biochemistry 12/2001; 268(22):5997-6002. · 3.58 Impact Factor
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    ABSTRACT: The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.
    Gene 11/2001; 277(1-2):121-7. · 2.20 Impact Factor
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    ABSTRACT: The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5′ flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5′ flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5′ flanking sequence from −74 to −46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5′ flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.
    European Journal of Biochemistry. 07/2000; 267(15):4635 - 4641.
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    ABSTRACT: In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat alpha1-antitrypsin (alpha1-AT); however, the sequence corresponding to the reactive center P1-P1' residues was found to be Met-Leu, whereas it is Met-Ser in the rat alpha1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an alpha1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk alpha1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1-P1' sequences are Met-Leu (HP-55 or CM55-ML), Met-Met (CM55-MM), Met-Ser (CM55-MS), Ser-Ile (CM55-SI) and Ser-Thr (CM55-ST). Each of the alpha1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the alpha1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single alpha1-AT gene.
    Gene 01/1998; 204(1-2):127-32. · 2.20 Impact Factor
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    ABSTRACT: In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat α1-antitrypsin (α1-AT); however, the sequence corresponding to the reactive center P1–P1′ residues was found to be Met–Leu, whereas it is Met–Ser in the rat α1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an α1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk α1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1–P1′ sequences are Met–Leu (HP-55 or CM55-ML), Met–Met (CM55-MM), Met–Ser (CM55-MS), Ser–Ile (CM55-SI) and Ser–Thr (CM55-ST). Each of the α1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the α1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single α1-AT gene.
    Gene. 01/1997;
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    ABSTRACT: In mammals, hibernation is expressed by only a limited number of species, and the molecular mechanisms underlying hibernation are not well understood. Recently, we have found plasma proteins which disappear from blood specifically during hibernation in a mammalian hibernator, the chipmunk. Here, we report the cDNA cloning of these chipmunk hibernation-related proteins, HP-20, -25, and -27, and analyses of their expression. All three proteins contain a collagen-like domain near the N terminus and are highly homologous to each other. Their mRNAs were detected only in liver in nonhibernating chipmunks, and in hibernating chipmunks, the amounts were reduced to less than 1/10 of those in nonhibernating chipmunks, indicating that HP-20, -25, and -27 mRNA expression is regulated similarly in association with hibernation. Southern blot analyses of the squirrel family with each of chipmunk HP-20, -25, and -27 cDNA revealed that a nonhibernating species (tree squirrel) as well as another hibernating species (ground squirrel) retained the corresponding genes. However, their transcripts were detected only with the hibernating species, and in hibernating ground squirrels, their levels were greatly reduced compared with those in nonhibernating animals, as were the cases with the chipmunk. These observations are the first line of evidence for occurrence of hibernation-associated gene regulation. The results would indicate the commitment of HP-20, -25, and -27 to hibernation and support the idea that genetic controls are involved in mammalian hibernation.
    Molecular and Cellular Biology 04/1993; 13(3):1516-21. · 5.37 Impact Factor
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    N Kondo, J Kondo
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    ABSTRACT: Mammalian hibernation is a unique physiological adaptation that allows the sustainment of life under extremely low body temperatures. In the chipmunk, we found four proteins related specifically to hibernation. These proteins started to diminish in concentration in the blood before and disappeared during hibernation. These proteins reappeared in the blood as hibernation ceased and remained during nonhibernation. The complete or partial amino acid sequences of the four proteins showed that three (27-, 25-, and 20-kDa) were previously unknown, whereas another (55-kDa) is highly homologous with alpha 1-antitrypsin. The three novel proteins are homologous, indicating that they are a family. In the NH2-terminal regions of these proteins, a collagen-like amino acid sequence is present, whereas in their COOH-terminal regions, two sequences, Ser-Ala-Phe-Ala-Val-Lys and Val-Trp-Leu-Glu, are conserved. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and gel permeation chromatography under denaturating conditions revealed that the four proteins form a 140-kDa complex in the plasma fraction. The novel proteins were detected in blood of another hibernator, the ground squirrel, but not in rodent nonhibernators, namely tree squirrels and rats. The present finding is the first identification of a hibernation-specific protein. The presence of specific proteins in hibernators suggests the involvement of genetic factors in the control of hibernation. These proteins provide valuable tools for understanding molecular mechanisms of mammalian hibernation.
    Journal of Biological Chemistry 02/1992; 267(1):473-8. · 4.65 Impact Factor
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Publication Stats

163 Citations
57.61 Total Impact Points

Institutions

  • 2003–2007
    • Kitasato University
      • Department of Biosciences
      Edo, Tōkyō, Japan
  • 2006
    • Mitsubishi Chemical Group Science and Technology Research Center, Inc.
      Yokohama, Kanagawa, Japan
  • 1997–2000
    • Kanagawa Academy of Science and Technology
      Kawasaki Si, Kanagawa, Japan