Seikou Shintani

Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts.

Although osteocytes are the most abundant cells in bone, little is known about their function, and no specific marker protein for osteocytes has been described. Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein expressed in tooth organ and bone. Our previous work showed that in the chicken, which is not capable of forming tooth, DMP1 messenger RNA (mRNA) is highly expressed in bone by Northern blot analysis. To clarify the significance of DMP1 expression in bone, the expression of DMP1 mRNA and its protein was examined in the chicken and rat. In the chicken, DMP1 mRNA was detected only in bone tissues and was localized in osteocytes and preosteocytes but not in osteoblasts. Similarly, in the rat, DMP1 mRNA was predominantly expressed in osteocytes and preosteocytes in bone matrix but not in osteoblasts located at the bone surface. Antiserum was raised against the peptide from rat DMP1, and the localization of DMP1 was examined by immunohistochemistry. In the development of bone, DMP1 was first detected in newly formed bone matrix after osteoblastic cells had been embedded within it. After the appearance of typical osteocytes, DMP1 was localized in the pericellular bone matrix of osteocytes, including their processes. These data show that DMP1 is a bone matrix protein specifically expressed in osteocytes and preosteocytes and suggest that DMP1 plays a role in bone homeostasis because of its high calcium ion‐binding capacity.

STIMULATION OF PROLIFERATION AND DIFFERENTIATION OF DOG DENTAL PULP CELLS IN SERUM-FREE CULTURE MEDIUM BY INSULIN-LIKE GROWTH FACTOR

Insulin, insulin-like growth factors (IGF) I and II are considered to play an important part in the growth and differentiation of dental pulp cells. The present study examined the effects of these factors on pulp cells in serum-free culture conditions. The DNA content and alkaline phosphatase (ALPase) activity of dog pulp cells increased when they were cultured in a serum-free medium supplemented with transferrin, yolk lipoprotein and basic fibrobrast growth factor (TYF medium). The pulp cells produced type I collagen but not type III, suggesting that they might proliferate and differentiate into odontoblast-like cells in a serum-free culture. Both IGF-I and IGF-II enhanced the ALPase activity of pulp cells cultured in TYF medium to an equivalent level, but a higher concentration of IGF-II was necessary to produce a similar effect on DNA synthesis to that of IGF-I. Insulin dose-dependently enhanced DNA synthesis and increased ALPase activity, but its effects were weaker than those of the IGFs. These findings suggest that IGF-I might have a primary role in the growth and differentiation of pulp cells.

Cariogenicity of the Probiotic Bacterium Lactobacillus salivarius in Rats

Probiotic bacteria such as lactobacilli and bifidobacteria are considered to be non-pathogenic and non-toxigenic on the basis of long years of safe usage. However, some species of lactobacilli are thought to be associated with the development of dental caries. The purpose of the present study was to examine the cariogenicity of the probiotic bacterium Lactobacillus salivarius in rats. Rats were divided into six groups, and infected with L. salivarius LS1952R and/or Streptococcus mutans MT8148R. L. salivarius LS1952R became established in the oral cavity of rats and induced significant level of dental caries even when infected for only 5 days from 18 to 22 days of age. In addition, the caries scores of rats superinfected with both Streptococcus mutans MT8148R and L. salivarius LS1952R from 18 days of age were significantly higher than those infected with either L. salivarius LS1952R or S. mutans MT8148R alone. Since strain LS1952R can adhere to saliva-coated hydroxyapatites, it is concluded that L. salivarius strain LS1952R possesses an inherent cariogenic activity following adherence to the tooth surface.

Cloning and characterization of the human ameloblastin gene.

We isolated the full-length human ameloblastin (AMBN) cDNA clone using reverse transcription-polymerase chain reaction (RT-PCR) methods. Sequence analysis of the AMBN cDNA revealed an open reading frame of 1341bp encoding a 447-amino-acid protein. Comparison with pig, cattle, rat, and mouse AMBN sequences showed a high amino acid sequence similarity and led to the identification of a novel 78bp (26 amino acids) insert resulting from internal sequence duplication. By DNA analysis of a human genomic clones, the AMBN gene was shown to consist of 13 exons and a novel 78bp segment, which proved to comprise two small exons. Human ameloblastomas express AMBN transcripts that contain some mutations.

Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation.

Abstract Two cichlid species, the haplochromine Aulonocara hansbaenschi and the tilapiine Oreochromis niloticus, were used to study the major histocompatibility complex (Mhc) class II A variation within this group. Multiple class II A sequences were recovered from A. hansbaenschi and O. niloticus cDNA libraries and three sequence families, DAA, DBA, and DCA, were identified. Sets of O. niloticus haploid embryo families were used to determine the linkage relationships of these genes. Two independently assorting linkage groups were detected, DAA and DBA/DCA, neither of which is linked to the previously described Mhc class I gene cluster. Three DCA genes and up to four DBA genes were found to segregate in different haplotypes, whereas DAA occurred as a single locus. Four DBA haplotypes, DBA*H1-H4, were identified and shown to co-segregate with the previously described class II B haplotypes. Four DCA haplotypes, DCA*H1-H4, were found at a distance of 37 cM from the DBA/class II B cluster; in one DCA haplotype, DCA*H5, the genes were tightly linked to the DBA/class II B clusters. Transcripts of DAA and DBA genes were found in O. niloticus hepatopancreas and spleen; transcripts of DCA genes were detected in the A. hansbaenschi cDNA library, but not in O. niloticus. These findings provide a basis for using class II haplotypes as markers in the study of adaptive radiation in the cichlid species flocks of the East African Great Lakes.

Linkage Relationships of Genes Coding for α2‐Macroglobulin, C3 and C4 in the Zebrafish: Implications for the Evolution of the Complement and Mhc Systems

The α2‐macroglobulin (A2M) and the complement components C3 and C4 are related proteins derived from a common ancestor. Theoretically, this derivation could have occurred either by tandem duplications of their encoding genes or by polyploidization involving chromosomal segments, a chromosome or the whole genome. In tetrapods the A2M‐, C3‐ and C4‐encoding genes are generally each located on a different chromosome. This observation has been interpreted as supporting their origin by polyploidization. We identified and mapped (with the help of a radiation hybrid panel of cell lines) the A2M, C3 and C4 loci in the zebrafish, Danio rerio. Each of the three types of loci is present in the zebrafish in multiple copies, but all of the identified copies of a given type map to the same region in linkage groups 1 (C3) and 15 (A2M, C4). The A2M and C4 loci are mapped in the same region not linked to any of the class I or class II major histocompatibility complex (Mhc) loci. These observations are interpreted as supporting the origin of the A2M family of genes by tandem duplications, followed by the dispersal of the copies to different chromosomes. It is also argued that the association of C4 with the class I/II loci in tetrapods is accidental and without functional significance.

Clustering of c-type lectin natural killer receptor-like loci in the bony fish Oreochromis niloticus

The genome of the cichlid (teleost) fish Oreochromis niloticus contains a set of genes which encode group V C‐type lectin proteins homologous to the mammalian NKG2/CD94 family of natural killer (NK) cell receptors. To determine the genomic organization of these killer cell‐like receptor (KLR) genes, an O. niloticus BAC library was screened with a cDNA probe derived previously from an expressed sequence tag of the related cichlid species Paralabidochromis chilotes. Four distinct KLR‐bearing BAC clones were analysed, three of which could be assembled into a contig. One of the clones was sequenced in its entirety, whereas the others were partially sequenced to identify the KLR loci borne by them. Altogether, 28 distinct KLR loci were identified, of which at least 26 occupy a single chromosomal region, the KLR complex. One half of the loci appear to be occupied by pseudogenes. Compared to the human NK cell receptor complex, the Oreochromis KLR complex is more compact and, apart from transposons, appears to contain only KLR loci. The gene density of the complex is one KLR locus per 18 kb of sequence. All the KLR loci constituting the complex are derived from a single most recent common ancestor, which is estimated to have existed 7.7 million years ago. The 180 kb of the determined sequence is a mosaic of blocks of similar segments reflecting a complex history of duplications, deletions and rearrangements. The transposons found in the sequenced part belong to the TC1, Xena, CR1 and TX1 families.

Identification and characterization of ameloblastin gene in an amphibian, Xenopus laevis

Ameloblastin (AMBN) is an enamel sheath protein that presumably has a role in determining the prismatic structure of growing enamel crystals. To investigate the relationship between the molecular evolution of the AMBN gene and development of enamel prismatic structures, it is considered to be of great significance in the identification of homologues of the AMBN genes in nonmammals whose teeth lack an enamel prismatic structure. Several clones containing AMBN cDNA were isolated from an African clawed toad tooth cDNA library by screening with a polymerase chain reaction (PCR) method. Sequence analysis of the clones revealed that they were derived from different genes (toad-A and toad-B), which were found to contain ORFs encoding 408- and 352-amino-acid proteins, respectively. The N-terminal part of the toad AMBN proteins and the phosphorylation motif for casein kinase II, as well as several features, were found to be highly conserved throughout the evolution of tetrapods. Exon-intron boundaries were shared by toad and caiman genes with the exception of exons 6, 7 and 10 while human and caiman genes shared them exclusive of exons 8 and 9 which have been found only in the human. As for exon 7, it was absent in both toad genes. Moreover, the AMBN genes were transcribed only in the upper jaw, presumably in teeth. These results may provide useful information for investigation of the evolution of enamel.

Phex Mutation Causes the Reduction of Npt2b mRNA in Teeth

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.

Biglycan-Like Extracellular Matrix Genes of Agnathans and Teleosts

Abstract. Biglycan and decorin are two members of a family of small extracellular matrix proteoglycans characterized by the presence of 10 leucine-rich repeats and one or two attachment sites for glucosaminoglycans. Both have thus far been described only from tetrapod species, mainly mammals. Because the extracellular matrix has played an important part in the evolution of Metazoa, the phylogeny of its components is of considerable interest. In this study, biglycan-like (BGL) cDNA sequences have been obtained from two teleost (Oreochromis cichlid and zebrafish) and two lamprey species. The analysis of the sequences suggests that, like tetrapods, the lampreys possess two types of proteoglycans, both of which are biglycan-like; decorin-like proteoglycans could not be identified in these species. The genes specifying these two types apparently arose by duplication in the lamprey lineage after its divergence from gnathostomes. The two teleost species possess a BGL proteoglycan and a bona fide decorin. The BGL proteoglycan is highly divergent from the tetrapod biglycan and related to the BGL proteoglycans of the lamprey. Hence, although the duplication generating the ancestors of biglycan and decorin genes occurred after the divergence of agnathans but before the emergence of teleosts, only decorin acquired its characteristic properties in the bony fishes. The BGL gene presumably turned into a typical biglycan only in the tetrapod lineages. The presumed acquisitions of new functions appear to have been accompanied by changes in the evolutionary rate.