Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used targeting to generate a knock-in mouse carrying null allele of (Enam) that has lacZ reporter replacing Enam translation initiation site sequences through exon 7. Correct transgene was confirmed by Southern blotting PCR analyses. No protein could be detected Western Enam-null mice. Histochemical...

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting generate a knockin mouse that replaces the Klk4 sequence, starting at translation initiation site, with lacZ reporter gene. Correct of transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining demonstrated expression beta-galactosidase...

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod inter-rod (Uchida et al., 1995). Two porcine proteins, with apparent molecular weights of 13 15 kDa, are characterized by protein sequencing. The primary structures these match a portion derived amino acid sequences clones isolated from organ epithelia-specific cDNA library. RNA messages differ inclusion or deletion 45-nucleotide...

The maturation of dental enamel succeeds the degradation organic matrix. Inhibition studies have shown that this is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding proteinase, we used two degenerate primer approaches to amplify part coding region using polymerase chain-reaction (PCR). First, purified proteinase from porcine transition-stage matrix characterized it partial protein sequencing. enzyme was isolated neutral soluble extract successive...

Dental enamel forms by matrix-mediated biomineralization. The components of the developing matrix are generally specific for that matrix. primary structures three proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)—have been derived from cDNA sequences. Here we report cloning characterization mRNA encoding a fourth protein: enamelin. longest porcine enamelin clone has 3907 nucleotides, exclusive poly(A) tail. structure secreted protein is 1104 amino acids in length. Without...

It has been shown that Emdogain ® Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, growth factors involved in these activities Emd-Gel remain unclear. In this study, was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-β BMP, were examined a specific luciferase reporter gene assay. unfractionated Emd-Gel, TGF-β-like...

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and critical for proper tooth dentin formation. During dentinogenesis, DSPP proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, failure to make these potential cause developmental defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze process DSPP. defined exact catalyzed by proteases during crown formation isolating DSPP-derived proteins...

The potential role of amelogenin phosphorylation in enamel formation is elucidated through vitro mineralization studies. Studies focused on the native 20-kDa porcine proteolytic cleavage product P148 that prominent developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with initial amorphous (ACP). In absence protein, ACP was found to undergo relatively rapid transformation randomly oriented plate-like apatitic crystals. presence non-phosphorylated...

Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate major cleavage products that accumulate during secretory stage of amelogenesis? We isolated from developing pig teeth used them digest tyrosine-rich polypeptide (TRAP), leucine-rich protein (LRAP), 5 fluorescence peptides. characterized digestion by LC-MSMS, SDS-PAGE, C18 RP-HPLC monitored with UV detectors. cleaves sequences after Pro 162 , Ser 148 His 62 Ala 63 Trp 45 . These cleavages all in...

Transforming growth factor-beta (TGF-β) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-β porcine pulp, odontoblasts dentin. The mRNA latent TGF-β1 TGF-β3 predominantly expressed odontoblasts, whereas expression level TGF-β2 high a major isoform TGF-β, TGF-β1, synthesized primarily activated by matrix metalloproteinase 11 (MMP11). Activated enhances levels MMP20 full-length dentin sialophosphoprotein (DSPP) pulp cells,...

We report here a novel biomimetic approach to the regeneration of human enamel. The combines use inorganic pyrophosphate (PP

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and critical for proper dentin formation. DSPP believed to be processed into only two structural/functional domains: sialoprotein (DSP) phosphoprotein (DPP). Here we report the isolation characterization third domain DSPP, designated glycoprotein (DGP). DGP was isolated from guanidine/EDTA extract porcine tooth by ion exchange, hydroxyapatite affinity, size exclusion, RP-HPL chromatography. Endoproteinase lysine C...

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in enamel matrix of developing teeth. AMBN N-terminal accumulate sheath space between rods, while C-terminal localize within rods. We tested hypothesis that MMP-20 is protease cleaves AMBN. Glycosylated recombinant porcine (rpAMBN) was expressed human kidney 293F cells, and enamelysin (rpMMP-20) bacteria. The purified were incubated together at an enzyme:substrate ratio 1:100. sequencing digestion determined...

Amelogenin’s capacity to regulate enamel formation is related its conserved N- and C-terminal domains, ability self-assemble, stabilize amorphous calcium phosphate (ACP) – a enhanced by amelogenin phosphorylation. This in vitro study provides further insight into function, using variations of the Leucine-Rich Amelogenin Peptide (LRAP), an alternative splice product comprised solely amelogenin’s domains. self-assembly was studied dynamic light-scattering transmission electron microscopy...

Background and Objective The periodontal ligament ( PDL ) is characterized by rapid turnover, high remodeling capacity inherent regenerative potential compared with other connective tissues. Periostin, which highly expressed in the fibroblasts , has been widely discussed relation to collagen fibrillogenesis . Recently, several reports have indicated periostin cell migration. aim of this study was examine whether human hPDLF s) levels expression promote migration bone marrow mesenchymal stem...

Bioceramic materials like bioactive glass (BG) are widely used in endodontics due to their bioactivity and osteoinductive properties. This study characterized fluorescence-labeled porcine immortalized dental pulp cells (DsRed-PPU7 cells) contact with BG-based cement (BG-C) advance the development of novel BG-C formulations. discs were prepared on titanium discs, DsRed-PPU7 cultured them. The effects bone morphogenetic protein-2, transforming growth factor beta-1, inhibitors (LDN-193189...

Kallikrein‐4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig was bacteria with without propeptide, two eukaryotic systems. Recombinant secreted as zymogen by ‘293’ cells purified. The proKLK4 activated vitro thermolysin enamelysin, but not KLK4. These results were confirmed using fluorescent peptide analog propeptide–enzyme junction. Native appears...

Amelogenin is the major organic component in enamel matrix of developing teeth and plays an important role biomineralization. has been reported to be a specific secretory product ameloblasts. In this study, we examined amelogenin gene expression various cell layers prepared from porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). amplification products were detected only ameloblast layer after 20 cycles PCR. After 30 PCR, maturation-stage ameloblasts...

The initiation of enamel crystals at the dentino-enamel junction is associated with expression dentin sialophosphoprotein (<i>DSPP,</i> a gene normally linked formation), three ‘structural’ proteins – amelogenin <i>(AMELX),</i> enamelin <i>(ENAM),</i> and ameloblastin <i>(AMBN)</i> matrix metalloproteinase, enamelysin <i>(MMP20).</i> Enamel formation proceeds steady elongation mineralization front just beneath ameloblast distal...