- Email: [email protected]
Molecular cloning and expression of human neurochondrin-1 and -21
Biochimica et Biophysica Acta 1446 (1999) 397^402 www.elsevier.com/locate/bba
Short sequence-paper
Molecular cloning and expression of human neurochondrin-1 and -21 Reiko Mochizuki
a;b
, Yasuyuki Ishizuka a , Kazuyuki Yanai b , Yoshihiko Koga a , Akiyoshi Fukamizu b;c;d; *
a
d
Sumitomo Pharmaceuticals Research Center, Sumitomo Pharmaceuticals, Osaka 554-0022, Japan b Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan c The National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba, Ibaraki 305-8572, Japan Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Received 7 April 1999; accepted 2 July 1999
Abstract Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucinerich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurochondrin; cDNA; Alternative splicing; Expression; Brain; Human
Neurochondrin has been cloned as a bone and brain speci¢c leucine-rich protein from murine, which associated with hydroxyapatite-resorptive activity in vitro [1]. As the cDNA product was able to create lacunae on the hydroxyapatite plate in the bone marrow culture system, neurochondrin is thought to be related to bone metabolism in vivo [2^4]. Two cDNAs encoding mouse neurochondrin, neurochondrin-1 (729 amino acids) and neurochondrin-2 (712 amino acids), have been identi¢ed. From the comparison of the nucleotide sequences, these isoforms are thought to be the resultant from an * Corresponding author. Tel.: +81 (298) 53 4605; Fax: +81 (298) 53 4605; E-mail: [email protected] 1 The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB018739 (neurochodrin-1) and AB018740 (neurochondrin-2).
alternative splicing [1]. On the other hand, the rat homologue of neurochondrin-1, norbin, was independently identi¢ed from a study of long-term potentiation by subtraction screening between the mRNA of tetraethylammonium-treated slices and that of untreated whole brain [5]. Overexpression of norbin in cultured neuro 2a cells induced neurite outgrowth, so it has been thought that this transcript may play an important role in neural plasticity [5^ 8]. The amino acid sequence of mouse neurochondrin-1 shows 99.7% identity (727 out of 729) to rat norbin, but there is no description about the existence of a neurochondrin-2-type transcript in rat. In this report, we present the molecular cloning and the expression of human neurochondrin-1 and -2. We also describe the homology of this protein with mouse neurochondrin-1 (AB017608) and the rat norbin (AB006461). The nucleotide sequence data reported in this paper will appear in the
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 2 0 - 7
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Fig. 1. Molecular cloning and primary sequences of human neurochondrin cDNAs. (A) Schematic representation of the human neurochondrin clones. Clones were obtained from a human fetal brain cDNA library by PCR using the primers indicated by arrowheads. (B) Nucleotide and deduced amino acid sequences of human neurochondrin-1 and -2. The positions of the nucleotides are indicated in the left. Amino acids are written below the nucleotide sequence with a one-letter code and the positions are indicated in the right margin. Open circles indicate the putative initiating codons. The stop codon is indicated by an asterisk. The RGD motif is underlined. The segment of 68 nucleotides deleted in neurochondrin-2 is boxed. (GenBank accession number AB018739 for neurochondrin-1, AB018740 for neurochondrin-2).
6
DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers AB018739 (neurochondrin-1) and AB018740 (neurochondrin-2). To obtain the human neurochondrin cDNA, PCR ampli¢cation was performed using a human fetal brain Marathon-Ready cDNA kit (Clontech). As shown in Fig. 1A, PCR was subjected using primer AP1 (5P-CCATCCTAATACGACTCACTATCGGGC-3P: adaptor primer) and primer 1 (5P-CTGGTAGGTGTCATCGATCATGGA-3P: corresponding to nucleotides 846^869 of the mouse neurochondrin-1). The nested PCR was done with the ¢rst PCR products as templates using primer AP2 (5P-ACTCACTATAGGGCTCGAGCGGC-3P: adaptor primer) and primer 2 (5P-AATGTGCTAAGGATGGGGATCTTGTT-3P: corresponding to nucleotides 780^805 of the mouse neurochondrin-1). The PCR products obtained by the nested PCR were subcloned into the pCRII vector (TA Cloning kit, Invitrogen) and sequenced. They were divided into two groups, according to the presence or absence of a 68 nucleotide segment (clone 1 and 2 in Fig. 1A). To obtain a fragment containing the complete open reading frame, the next PCR was performed using primer 3 (5P-CCTCCGTGAGGCCAAGAATGACAG-3P: corresponding to nucleotides 237^260 of the human neurochondrin-1) and primer 4 (5P-AAAGCCAGAAAGCTGGGGTG-3P: corresponding to nucleotides 2693^2712 of the mouse neurochondrin-1). As shown in Fig. 1A, the 2178 bp fragment (clone 3) was obtained. Double-stranded sequencing was performed by primer walking using the AutoRead Sequencing kit on an ALFred DNA sequencer (Pharmacia Biotech). The full-length cDNA sequences were obtained by connecting the PCR fragments. As shown in Fig. 1B, the neurochondrin-1 cDNA (connected with clone 1 and 3) consisted of 2413 nucleotides, which contains 2187 nucleotides coding a protein of 729 amino acids with an estimated molecular weight of 78 863 and a pI of 5.22. The neuro-
chondrin-2 cDNA (connected with clone 2 and 3) encoded a protein of 712 amino acids with an estimated molecular weight of 77 242 and a pI of 5.22, which had a deletion of 68 nucleotides corresponding to nucleotides 77^144 of the human neurochondrin1. It suggests alternative splicing as observed in the mouse, except that the size of the deleted segment is di¡erent from that of mouse [1]. While the functional signi¢cance of the heterogeneous neurochondrin 5Psequence is unclear, it is possible that these alternative 5P-sequences may have an important role, because these isoforms are conserved between mouse and human species. Judging from the above, there may be a homologue of neurochondrin-2 in rat. The sequence surrounding the initiation codon of neurochondrin-2 (ATCATGG) is in good agreement with the consensus sequence (A(G)XXATGG) for the start of translation in eukaryotes proposed by Kozak [9]. As shown in Fig. 1B, a RGD motif, implicated in cell attachment to integrin [10] and apoptosis [11], is conserved in the neurochondrin-1 and -2. The predicted amino acid sequence from the neurochondrin-1 and -2 is rich in leucine (15.8 and 15.9%, respectively). It contains a putative leucine zipper motif between residues 565^586 of neurochondrin1, but proline and glycine residues exist in this region. Therefore, it is thought not to be a functional leucine zipper motif as observed in the mouse [1,12]. Other consensus sequences of leucine-rich repeats [13], associated to cellular adhesion, protein interactions and other di¡erent functions, were not observed in the neurochondrin-1 and -2. The expression of neurochondrin mRNA was studied by Northern blotting with a Human Multiple Tissue Northern blot (Clontech) according to the manufacturer's protocol. The hybridization was carried out at 50³C using clone 3 as a 32 P-labelled probe. These blots were subsequently probed with a human L-actin cDNA. As shown in Fig. 2A, the neurochondrin mRNA is highly expressed in the
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adult brain as observed in mouse [1] and has a size of about 4 kb. A very faint band can be observed in the lanes corresponding to the ovary and testis. In fetal human, neurochondrin mRNA was detected only in the brain. In the adult brain, neurochondrin tran-
scripts are widely expressed in the 16 di¡erent brain regions, including the spinal cord (Fig. 2B). Fig. 3 shows the deduced amino acid sequence of the human neurochondrin-1 and its comparison with those of mouse and rat norbin. Amino acid identity
Fig. 2. Northern blot analysis of human neurochondrin mRNA. A Human Multiple Tissue Northern blot (Clontech) of 16 adult tissues and four fetal tissues (A) and 16 di¡erent brain regions (B) was probed with a human neurochondrin cDNA probe or with a human L-actin probe. Positions of the RNA markers are indicated in the left.
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Fig. 3. Comparison of the amino acid sequence of human neurochondrin-1 with the sequence of mouse and rat forms of the protein. The amino acid sequence of human neurochondrin-1 is aligned against sequences from mouse neurochondrin-1 (AB017608) and rat norbin (AB006461). The conserved residues between human and other mammals are indicated by asterisks. The putative initiating codon of neurochondrin-2 is indicated by an arrow. The RGD motif is boxed.
between the human and the mouse neurochondrin-1 was 98.2% with a nucleotide sequence homology of 90.7% and that between the human and the rat homologue was 98.1% with a nucleotide sequence homology of 90.7%. The putative RGD motif and the surrounding amino acids are also conserved among these species. In conclusion, we have cloned and characterized
the human neurochondrin-1 and -2. The expression of human neurochondrin mRNA was predominant in the fetal brain and the adult multi-brain regions. Though expression of neurochondrin in the early mouse embryo was previously reported [1], this is the ¢rst report about the expression of neurochondrin in fetal brain. The amino acid sequence of neurochondrin is highly conserved among human,
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mouse and rat. This strong conservation implies that neurochondrin has an important physiological function in these organisms. The presence of the conserved RGD motif implies the possibility that neurochondrin may have an important function in proliferation, di¡erentiation and apoptosis in these organisms. This is the ¢rst step directed towards the isolation of their respective genes and the study of their regulatory mechanisms.
[5]
[6] [7]
Acknowledgements
[8]
We thank Miki Takatsuka for her excellent technical assistance.
[9]
[10]
References [1] Y. Ishizuka, R. Mochizuki, K. Yanai, M. Takatsuka, T. Nonomura, S. Niida, H. Horiguchi, N. Maeda and A. Fukamizu, Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to ba¢lomycin A1 by a factor with restricted expression to bone and brain, neurochondrin. Biochim. Biophys. Acta, 1999, (in press). [2] J.E. Davies, G. Shapiro, B.F. Lowenberg, Osteoclastic resorption of calcium phosphate ceramic thin ¢lms, Cells Mater. 3 (1993) 245^246. [3] H.C. Blair, S.L. Teitelbaum, R. Ghiselli, S. Gluck, Osteoclastic bone resorption by a polarized vacuolar protonpump, Science 245 (1989) 855^857. [4] H.K. Vaananen, E.K. Karhukorpi, K. Sundquist, B. Wall-
[11]
[12]
[13]
mark, I. Roininen, T. Hentunen, J. Tuukkanen, P. Lakkakorpi, Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ru¥ed borders of osteoclasts, J. Cell Biol. 111 (1990) 1305^1311. K. Shinozaki, K. Maruyama, H. Kume, H. Kuzume, K. Obata, A novel brain gene, norbin, induced by treatment of tetraethylammonium in rat hippocampal slice and accompanied with neurite-outgrowth in neuro 2a cells, Biochem. Biophys. Res. Commun. 240 (1997) 766^771. T.J. Teyler, P. DiScenna, Long-term potentiation, Annu. Rev. Neurosci. 10 (1987) 131^161. G.L. Collingridge, T.V.P. Bliss, Memories of NMDA receptors and LTP, Trends Neurosci. 18 (1995) 54^56. Y.Y. Huang, R.C. Malenka, Examination of TEA-induced synaptic enhancement in area CA1 of the hippocampus: the role of voltage-dependent Ca2 channels in the induction of LTP, J. Neurosci. 13 (1993) 568^576. M. Kozak, An analysis of 5P-noncoding sequences from 699 vertebrate messenger RNAs, Nucleic Acids Res. 15 (1987) 8125^8148. E. Ruoslahti, M.D. Pierschbacher, New perspectives in cell adhesion: RGD and integrins, Science 238 (1987) 491^ 497. C.D. Buckley, D. Pilling, N.V. Henriquez, G. Parsonage, K. Threlfall, D. Scheel-Toellner, D.L. Simmons, A.N. Akbar, J.M. Lord, M. Salmon, RGD peptides induce apoptosis by direct caspase-3 activation, Nature 397 (1999) 534^539. W.H. Landschulz, P.F. Johnson, S.L. McKnight, The leucine zipper : a hypothetical structure common to a new class of DNA binding proteins, Science 240 (1988) 1759^1764. F. Tan, D.K. Weerasinghe, R.A. Skidgel, H. Tamei, R.K. Kaul, I.B. Roninson, J.W. Schilling, E.G. Erdos, The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats, J. Biol. Chem. 265 (1990) 13^19.
BBAEXP 91295 19-8-99
Short sequence-paper
Molecular cloning and expression of human neurochondrin-1 and -21 Reiko Mochizuki
a;b
, Yasuyuki Ishizuka a , Kazuyuki Yanai b , Yoshihiko Koga a , Akiyoshi Fukamizu b;c;d; *
a
d
Sumitomo Pharmaceuticals Research Center, Sumitomo Pharmaceuticals, Osaka 554-0022, Japan b Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan c The National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba, Ibaraki 305-8572, Japan Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Received 7 April 1999; accepted 2 July 1999
Abstract Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucinerich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurochondrin; cDNA; Alternative splicing; Expression; Brain; Human
Neurochondrin has been cloned as a bone and brain speci¢c leucine-rich protein from murine, which associated with hydroxyapatite-resorptive activity in vitro [1]. As the cDNA product was able to create lacunae on the hydroxyapatite plate in the bone marrow culture system, neurochondrin is thought to be related to bone metabolism in vivo [2^4]. Two cDNAs encoding mouse neurochondrin, neurochondrin-1 (729 amino acids) and neurochondrin-2 (712 amino acids), have been identi¢ed. From the comparison of the nucleotide sequences, these isoforms are thought to be the resultant from an * Corresponding author. Tel.: +81 (298) 53 4605; Fax: +81 (298) 53 4605; E-mail: [email protected] 1 The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB018739 (neurochodrin-1) and AB018740 (neurochondrin-2).
alternative splicing [1]. On the other hand, the rat homologue of neurochondrin-1, norbin, was independently identi¢ed from a study of long-term potentiation by subtraction screening between the mRNA of tetraethylammonium-treated slices and that of untreated whole brain [5]. Overexpression of norbin in cultured neuro 2a cells induced neurite outgrowth, so it has been thought that this transcript may play an important role in neural plasticity [5^ 8]. The amino acid sequence of mouse neurochondrin-1 shows 99.7% identity (727 out of 729) to rat norbin, but there is no description about the existence of a neurochondrin-2-type transcript in rat. In this report, we present the molecular cloning and the expression of human neurochondrin-1 and -2. We also describe the homology of this protein with mouse neurochondrin-1 (AB017608) and the rat norbin (AB006461). The nucleotide sequence data reported in this paper will appear in the
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 2 0 - 7
BBAEXP 91295 19-8-99
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R. Mochizuki et al. / Biochimica et Biophysica Acta 1446 (1999) 397^402
399
Fig. 1. Molecular cloning and primary sequences of human neurochondrin cDNAs. (A) Schematic representation of the human neurochondrin clones. Clones were obtained from a human fetal brain cDNA library by PCR using the primers indicated by arrowheads. (B) Nucleotide and deduced amino acid sequences of human neurochondrin-1 and -2. The positions of the nucleotides are indicated in the left. Amino acids are written below the nucleotide sequence with a one-letter code and the positions are indicated in the right margin. Open circles indicate the putative initiating codons. The stop codon is indicated by an asterisk. The RGD motif is underlined. The segment of 68 nucleotides deleted in neurochondrin-2 is boxed. (GenBank accession number AB018739 for neurochondrin-1, AB018740 for neurochondrin-2).
6
DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers AB018739 (neurochondrin-1) and AB018740 (neurochondrin-2). To obtain the human neurochondrin cDNA, PCR ampli¢cation was performed using a human fetal brain Marathon-Ready cDNA kit (Clontech). As shown in Fig. 1A, PCR was subjected using primer AP1 (5P-CCATCCTAATACGACTCACTATCGGGC-3P: adaptor primer) and primer 1 (5P-CTGGTAGGTGTCATCGATCATGGA-3P: corresponding to nucleotides 846^869 of the mouse neurochondrin-1). The nested PCR was done with the ¢rst PCR products as templates using primer AP2 (5P-ACTCACTATAGGGCTCGAGCGGC-3P: adaptor primer) and primer 2 (5P-AATGTGCTAAGGATGGGGATCTTGTT-3P: corresponding to nucleotides 780^805 of the mouse neurochondrin-1). The PCR products obtained by the nested PCR were subcloned into the pCRII vector (TA Cloning kit, Invitrogen) and sequenced. They were divided into two groups, according to the presence or absence of a 68 nucleotide segment (clone 1 and 2 in Fig. 1A). To obtain a fragment containing the complete open reading frame, the next PCR was performed using primer 3 (5P-CCTCCGTGAGGCCAAGAATGACAG-3P: corresponding to nucleotides 237^260 of the human neurochondrin-1) and primer 4 (5P-AAAGCCAGAAAGCTGGGGTG-3P: corresponding to nucleotides 2693^2712 of the mouse neurochondrin-1). As shown in Fig. 1A, the 2178 bp fragment (clone 3) was obtained. Double-stranded sequencing was performed by primer walking using the AutoRead Sequencing kit on an ALFred DNA sequencer (Pharmacia Biotech). The full-length cDNA sequences were obtained by connecting the PCR fragments. As shown in Fig. 1B, the neurochondrin-1 cDNA (connected with clone 1 and 3) consisted of 2413 nucleotides, which contains 2187 nucleotides coding a protein of 729 amino acids with an estimated molecular weight of 78 863 and a pI of 5.22. The neuro-
chondrin-2 cDNA (connected with clone 2 and 3) encoded a protein of 712 amino acids with an estimated molecular weight of 77 242 and a pI of 5.22, which had a deletion of 68 nucleotides corresponding to nucleotides 77^144 of the human neurochondrin1. It suggests alternative splicing as observed in the mouse, except that the size of the deleted segment is di¡erent from that of mouse [1]. While the functional signi¢cance of the heterogeneous neurochondrin 5Psequence is unclear, it is possible that these alternative 5P-sequences may have an important role, because these isoforms are conserved between mouse and human species. Judging from the above, there may be a homologue of neurochondrin-2 in rat. The sequence surrounding the initiation codon of neurochondrin-2 (ATCATGG) is in good agreement with the consensus sequence (A(G)XXATGG) for the start of translation in eukaryotes proposed by Kozak [9]. As shown in Fig. 1B, a RGD motif, implicated in cell attachment to integrin [10] and apoptosis [11], is conserved in the neurochondrin-1 and -2. The predicted amino acid sequence from the neurochondrin-1 and -2 is rich in leucine (15.8 and 15.9%, respectively). It contains a putative leucine zipper motif between residues 565^586 of neurochondrin1, but proline and glycine residues exist in this region. Therefore, it is thought not to be a functional leucine zipper motif as observed in the mouse [1,12]. Other consensus sequences of leucine-rich repeats [13], associated to cellular adhesion, protein interactions and other di¡erent functions, were not observed in the neurochondrin-1 and -2. The expression of neurochondrin mRNA was studied by Northern blotting with a Human Multiple Tissue Northern blot (Clontech) according to the manufacturer's protocol. The hybridization was carried out at 50³C using clone 3 as a 32 P-labelled probe. These blots were subsequently probed with a human L-actin cDNA. As shown in Fig. 2A, the neurochondrin mRNA is highly expressed in the
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R. Mochizuki et al. / Biochimica et Biophysica Acta 1446 (1999) 397^402
adult brain as observed in mouse [1] and has a size of about 4 kb. A very faint band can be observed in the lanes corresponding to the ovary and testis. In fetal human, neurochondrin mRNA was detected only in the brain. In the adult brain, neurochondrin tran-
scripts are widely expressed in the 16 di¡erent brain regions, including the spinal cord (Fig. 2B). Fig. 3 shows the deduced amino acid sequence of the human neurochondrin-1 and its comparison with those of mouse and rat norbin. Amino acid identity
Fig. 2. Northern blot analysis of human neurochondrin mRNA. A Human Multiple Tissue Northern blot (Clontech) of 16 adult tissues and four fetal tissues (A) and 16 di¡erent brain regions (B) was probed with a human neurochondrin cDNA probe or with a human L-actin probe. Positions of the RNA markers are indicated in the left.
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Fig. 3. Comparison of the amino acid sequence of human neurochondrin-1 with the sequence of mouse and rat forms of the protein. The amino acid sequence of human neurochondrin-1 is aligned against sequences from mouse neurochondrin-1 (AB017608) and rat norbin (AB006461). The conserved residues between human and other mammals are indicated by asterisks. The putative initiating codon of neurochondrin-2 is indicated by an arrow. The RGD motif is boxed.
between the human and the mouse neurochondrin-1 was 98.2% with a nucleotide sequence homology of 90.7% and that between the human and the rat homologue was 98.1% with a nucleotide sequence homology of 90.7%. The putative RGD motif and the surrounding amino acids are also conserved among these species. In conclusion, we have cloned and characterized
the human neurochondrin-1 and -2. The expression of human neurochondrin mRNA was predominant in the fetal brain and the adult multi-brain regions. Though expression of neurochondrin in the early mouse embryo was previously reported [1], this is the ¢rst report about the expression of neurochondrin in fetal brain. The amino acid sequence of neurochondrin is highly conserved among human,
BBAEXP 91295 19-8-99
402
R. Mochizuki et al. / Biochimica et Biophysica Acta 1446 (1999) 397^402
mouse and rat. This strong conservation implies that neurochondrin has an important physiological function in these organisms. The presence of the conserved RGD motif implies the possibility that neurochondrin may have an important function in proliferation, di¡erentiation and apoptosis in these organisms. This is the ¢rst step directed towards the isolation of their respective genes and the study of their regulatory mechanisms.
[5]
[6] [7]
Acknowledgements
[8]
We thank Miki Takatsuka for her excellent technical assistance.
[9]
[10]
References [1] Y. Ishizuka, R. Mochizuki, K. Yanai, M. Takatsuka, T. Nonomura, S. Niida, H. Horiguchi, N. Maeda and A. Fukamizu, Induction of hydroxyapatite resorptive activity in bone marrow cell populations resistant to ba¢lomycin A1 by a factor with restricted expression to bone and brain, neurochondrin. Biochim. Biophys. Acta, 1999, (in press). [2] J.E. Davies, G. Shapiro, B.F. Lowenberg, Osteoclastic resorption of calcium phosphate ceramic thin ¢lms, Cells Mater. 3 (1993) 245^246. [3] H.C. Blair, S.L. Teitelbaum, R. Ghiselli, S. Gluck, Osteoclastic bone resorption by a polarized vacuolar protonpump, Science 245 (1989) 855^857. [4] H.K. Vaananen, E.K. Karhukorpi, K. Sundquist, B. Wall-
[11]
[12]
[13]
mark, I. Roininen, T. Hentunen, J. Tuukkanen, P. Lakkakorpi, Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ru¥ed borders of osteoclasts, J. Cell Biol. 111 (1990) 1305^1311. K. Shinozaki, K. Maruyama, H. Kume, H. Kuzume, K. Obata, A novel brain gene, norbin, induced by treatment of tetraethylammonium in rat hippocampal slice and accompanied with neurite-outgrowth in neuro 2a cells, Biochem. Biophys. Res. Commun. 240 (1997) 766^771. T.J. Teyler, P. DiScenna, Long-term potentiation, Annu. Rev. Neurosci. 10 (1987) 131^161. G.L. Collingridge, T.V.P. Bliss, Memories of NMDA receptors and LTP, Trends Neurosci. 18 (1995) 54^56. Y.Y. Huang, R.C. Malenka, Examination of TEA-induced synaptic enhancement in area CA1 of the hippocampus: the role of voltage-dependent Ca2 channels in the induction of LTP, J. Neurosci. 13 (1993) 568^576. M. Kozak, An analysis of 5P-noncoding sequences from 699 vertebrate messenger RNAs, Nucleic Acids Res. 15 (1987) 8125^8148. E. Ruoslahti, M.D. Pierschbacher, New perspectives in cell adhesion: RGD and integrins, Science 238 (1987) 491^ 497. C.D. Buckley, D. Pilling, N.V. Henriquez, G. Parsonage, K. Threlfall, D. Scheel-Toellner, D.L. Simmons, A.N. Akbar, J.M. Lord, M. Salmon, RGD peptides induce apoptosis by direct caspase-3 activation, Nature 397 (1999) 534^539. W.H. Landschulz, P.F. Johnson, S.L. McKnight, The leucine zipper : a hypothetical structure common to a new class of DNA binding proteins, Science 240 (1988) 1759^1764. F. Tan, D.K. Weerasinghe, R.A. Skidgel, H. Tamei, R.K. Kaul, I.B. Roninson, J.W. Schilling, E.G. Erdos, The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats, J. Biol. Chem. 265 (1990) 13^19.
BBAEXP 91295 19-8-99