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M MacDougall

Publications and source records attributed to M MacDougall.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genetic linkage of the dentinogenesis imperfecta type III locus to chromosome 4q.

Dentinogenesis imperfecta type III (DGI-III) is an autosomal-dominant disorder of dentin formation which appears in a tri-racial southern Maryland population known as the "Brandywine isolate". This disease has suggestive evidence of linkage to the long arm of human chromosome 4 (LOD score of 2.0) in a family presenting with both juvenile periodontitis and DGI-III. The purpose of this study was to screen a family presenting with only DGI-III to determine if this locus was indeed on chromosome 4q. Furthermore, we wanted to determine if DGI-III co-localized with dentinogenesis imperfecta type II (DGI-II), which has been localized to 4q21-q23. Therefore, a large kindred from the Brandywine isolate was identified, oral examination performed, and blood samples collected from 21 family members. DNA from this family was genotyped with 6 highly polymorphic markers that span the DGI-II critical region of chromosome 4q. Analysis of the data yielded a maximum two-point LOD score of 4.87 with a marker for the dentin matrix protein 1 (DMP1) locus, a gene contained in the critical region for DGI-II. Our results demonstrated that the DGI-III locus is on human chromosome 4q21 within a 6.6 cM region that overlaps the DGI-II critical region. These results are consistent with the hypothesis that DGI-II is either an allelic variant of DGI-III or the result of mutations in two tightly linked genes.

Aggressive Periodontitis↗
PubMed1999

Comparative study of MSX-2, DLX-5, and DLX-7 gene expression during early human tooth development.

Msx and Dlx family transcription factors are key elements of craniofacial development and act in specific combinations with growth factors to control the position and shape of various skeletal structures in mice. In humans, the mutations of MSX and DLX genes are associated with specific syndromes, such as tooth agenesis, craniosynostosis, and tricho-dento-osseous syndrome. To establish some relationships between those reported human syndromes, previous experimental data in mice, and the expression patterns of MSX and DLX homeogenes in the human dentition, we investigated MSX-2, DLX-5, and DLX-7 expression patterns and compared them in orofacial tissues of 7.5- to 9-wk-old human embryos by using in situ hybridization. Our data showed that MSX-2 was strongly expressed in the progenitor cells of human orofacial skeletal structures, including mandible and maxilla bones, Meckel's cartilage, and tooth germs, as shown for DLX-5. DLX-7 expression was restricted to the vestibular lamina and, later on, to the vestibular part of dental epithelium. The comparison of MSX-2, DLX-5, and DLX-7 expression patterns during the early stages of development of different human tooth types showed the existence of spatially ordered sequences of homeogene expression along the vestibular/lingual axis of dental epithelium. The expression of MSX-2 in enamel knot, as well as the coincident expression of MSX-2, DLX-5, and DLX-7 in a restricted vestibular area of dental epithelium, suggests the existence of various organizing centers involved in the control of human tooth morphogenesis.

Animals↗
PubMed1999

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts.

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.

Ameloblasts↗
PubMed1998

Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein.

Our laboratory has reported that two major noncollagenous dentin proteins, dentin sialoprotein and dentin phosphoprotein, are specific cleavage products of a larger precursor protein termed dentin sialophosphoprotein (MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J. Q., and Gu, T. T. (1997) J. Biol. Chem. 272:835-842). To confirm our single gene hypothesis and initiate in vitro promoter studies, we have characterized the structural organization of the mouse dentin sialophosphoprotein gene. This gene has a transcription unit of approximately 9.4 kilobase pairs and is organized into 5 exons and 4 introns. Exon 1 contains a noncoding 5' sequence, and exon 2 contains the transcriptional start site, signal peptide, and first two amino acids of the NH2 terminus. Exons 3 and 4 contain coding information for 29 and 314 amino acids, respectively. The remainder of the coding information and the untranslated 3' region are contained in exon 5. Chromosomal mapping localized the gene to mouse chromosome 5q21 in close proximity to other dentin/bone matrix genes. Computer analysis of the promoter proximal 1.6-kilobase pair sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements of AP-1, AP-2, Msx-1, serum response elements, SP-1, and TCF-1. In vitro studies showed that the DSPP promoter is active in an odontoblast cell line, MO6-G3, with basal activity mapped to -95 bp. Two potential enhancer and suppresser elements were identified in the regions between -1447 and -791 bp and -791 and -95 bp, respectively. The structural organization of the dentin sialophosphoprotein gene confirms our finding that both dentin sialoprotein and dentin phosphoprotein are encoded by a single gene with a continuous open reading frame.

Amino Acid Sequence↗
PubMed1998

Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci.

Dentinogenesis imperfecta type II and dentin dysplasia type II are diseases resulting in abnormal dentin formation, which have been mapped to overlapping regions of human chromosome 4q defined by markers D4S2691 and D4S2692 (6.6 cM) and D4S3291 and SPP1 (14.1 cM), respectively. Recently, two of the major non-collagenous proteins of dentin, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP, phosphophoryn) have been shown to be encoded by a single gene, termed dentin sialophosphoprotein (DSPP), which has been mapped to human chromosome 4. The purpose of this study was to perform refined mapping of DSPP related to these disease loci by gene content mapping, as well as to place the DSPP gene on the physical map of human chromosome 4 by sequence tagged site (STS) content mapping. Human genomic DSPP clones were isolated, and gene content mapping performed with specific primers for dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (secreted phosphoprotein 1, SPP1). STS content mapping was then performed with flanking STS markers to these dentin/bone gene loci. Our results demonstrate that the DSPP and DMP1 genes are within a maximum distance of 110 kb. Both DSPP and DMP-1 have been placed on the physical map of human chromosome 4 within the interval defined by markers D4S564 and D4S1292. DSPP is thereby strengthened as a candidate gene for both DGI-II and DD-II.

Amino Acid Sequence↗
PubMed1998

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA.

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.

Amino Acid Sequence↗
PubMed1998

Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues.

Dentin matrix protein 1 (Dmp1) is an acidic phosphoprotein first identified by cDNA cloning from a rat tooth library. Northern blot hybridization of a variety of tissues detected Dmp1 mRNAs only in odontoblasts, suggesting that this protein was odontoblast specific. In situ hybridization studies showed expression of Dmp1 in odontoblasts with transient expression in secretory ameloblasts. The purpose of this study was to isolate and characterize a mouse Dmp1 cDNA and determine its spatial expression pattern related to other mineralizing tissues. A mouse molar cDNA library was screened with a 32P-labeled Dmp1 polymerase chain reaction amplification product in order to isolate a full-length clone. DNA sequence analysis of the largest mouse Dmp1 cDNA (2802 base pairs [bp]) revealed an open reading frame of 1509 nucleotides encoding a 503 amino acid protein with a single polyadenylation signal. Comparison with rat and bovine Dmp1 sequence showed high homology and the identification of a 45 bp (15 amino acid) insert, representing an alternative spliced mRNA. This 45 bp segment was shown to represent a small exon by DNA analysis of a mouse genomic Dmp1 clone. In situ hybridization studies revealed a much broader Dmp1 tissue expression pattern than previously reported. Dmp1 transcripts were detected in the odontoblast and ameloblasts, osteoblasts, and cementoblasts. Our data indicate that Dmp1 is alternatively spliced, and the primary full-length transcript contains a 45 bp insert which is encoded by a small exon. Therefore, Dmp1 is not a tooth-specific protein but rather is expressed in a number of mineralizing tissues including enamel, bone, and cementum.

Ameloblasts↗
PubMed1998

Identification and characterization of a cDNA for mouse ameloblastin.

Ameloblastin was first identified as one of the most abundant novel transcripts from a random screening of a rat incisor cDNA library. In situ hybridization experiments have shown ameloblastin expression to be specific to ameloblasts, with highest levels in secretory and maturation stage ameloblasts and cells of the epithelial root sheath. Ameloblastin has been identified as a candidate gene for the local hypoplastic form of autosomal dominant amelogenesis imperfecta, by virtue of it's location within the critical disease locus. The purpose of this study was to isolate a full length mouse ameloblastin cDNA and determine its temporal expression pattern during odontogenesis. A newborn mouse molar cDNA library was screened using a rat ameloblastin cDNA probe. Positive clones were confirmed by PCR analysis with ameloblastin-specific primers, and their size determined with vector-specific primers. Phage clones were rescued to phagemid using Exassist helper phage and the nucleotide sequence determined. We report here the identification of two clones, exhibiting alternative splicing of the putative open reading frame, and use of multiple polyadenylation signals. Nucleotide sequence analysis indicated a high degree of similarity to rat ameloblastin, rat amelin 1 and 2 and porcine sheathlin. Reverse transcriptase-PCR analysis using mouse first and second mandibular molar mRNA indicated initial expression at E-14. This is one day after the initial expression of tuftelin (E-13) and one day prior to that of amelogenin (E-15).

Amino Acid Sequence↗
PubMed1998

Developmental regulation of dentin sialophosphoprotein during ameloblast differentiation: a potential enamel matrix nucleator.

The two major dentin matrix proteins, dentin sialoprotein and dentin phosphoprotein have been shown to be expressed as a single large transcript termed dentin sialophosphoprotein (DSPP). These non-collagenous matrix proteins, identified biochemically by their unique physical-chemical properties, are specific cleavage products of a large parent acidic phosphorylated protein (pI 4.0). Previous studies have shown expression of dentin sialoprotein at the protein level by ameloblasts. The purpose of this study was to determine the temporal-spatial pattern of DSPP expression during amelogenesis. In situ hybridization and immunohistochemistry were performed on sections of developing mouse molars. These data were correlated with RT-PCR analysis of in vitro enamel organ epithelium monolayer cell cultures enriched for ameloblasts. Our data indicates initial expression of the DSPP transcripts and protein during early ameloblast differentiation prior to the secretory phase when the majority of the enamel matrix is formed. Ameloblasts appear to tightly down-regulate DSPP transcription as enamel matrix formation is up-regulated. These data demonstrate DSPP expression during amelogenesis is under highly controlled developmental regulation. Therefore, DSPP may have a primary role in the initial mineralization events of both enamel and dentin, acting as a potential nucleator of hydroxyapatite crystal formation.

Ameloblasts↗
PubMed1998

Murine enamelin: cDNA and derived protein sequences.

Enamelin is the largest enamel protein. Recently we reported the characterization of a cDNA clone encoding porcine enamelin. The secreted protein has 1104 amino acids--over 6 times the length of amelogenin (173 amino acids) and almost 3 times the lengths of sheathlin (395 amino acids) and tuftelin (389 amino acids). Immunohistochemistry has shown that uncleaved porcine enamelin concentrates at the growing tips of the enamel crystallites while its cleavage products localize to rod and interrod enamel. Here we report the isolation and characterization of cDNA encoding murine amelogenin and demonstrate the tooth specificity of porcine enamelin. The murine clone is 4154 nucleotides in length and encodes a protein of 1274 amino acids. In the absence of post-translational modifications murine enamelin has an isotope averaged molecular mass of 137 kDa and an isoelectric point of 9.4. Multiple tissue Northern blot analyses detect porcine enamelin mRNA in developing teeth but not in liver, heart, brain, spleen, skeletal muscle and lung. Mouse and porcine enamelin share 61% amino acid identity and 75% DNA sequence identity. Mouse enamelin has 14 tandemly arranged copies of an 11 amino acid segment that is found only once in porcine enamelin.

Amino Acid Sequence↗
PubMed1998

Immortalized mouse odontoblast cell line MO6-G3 application for in vitro biocompatibility testing.

PURPOSE: This study was designed to determine the usefulness of an established stable immortalized mouse odontoblast cell line (MO6-G3) for dental material biocompatibility testing. Using a standard toxicity assay based on cell respiratory activity, the response to MO6-G3 cells was compared to the mouse fibroblastic cell line, L929, presently used for dental materials testing. The dental resin monomer TEGDMA was used as the dental material for the assay. MATERIALS AND METHODS: Cell lines (1 x 10(3)/well) were plated in 96 well culture plates and grown in DMEM supplemented with 10% FCS, 100 units/ml each of penicillin and streptomycin, and 50 micrograms/ml ascorbic acid in an atmosphere of 95% air and 5% CO2. Cells were exposed to TEGDMA resin monomer covering a dose range of 1 x 10(-6) to 0.5 x 10(-3) M. Unexposed control cells, as well as cells exposed to the DMSO vehicle in which the TEGDMA was dissolved, were included in all assays. Cytotoxicity was evaluated by determining cell respiratory activity spectrophotometrically using the tetrazolium compound WST-1. RESULTS: Statistical analysis by ANOVA using Tukey's method for pair wise comparisons as the post hoc test indicated toxic effects of TEGDMA at 1 x 10(-5) M in the odontoblast cell line MO6-G3. By contrast, the monomer produced no toxic effects on the L929 fibroblast cell line after 24 hours of exposure, over the entire concentration range tested. Furthermore, MO6-G3 cells exposed to a concentration of 0.5 x 10(-3) M were unable to recover from the effects of the exposure 48 hours after removal of the resin. MO6-G3 cells exposed to 1 x 10(-4) and 0.5 x 10(-4) TEGDMA recovered 40-50% and 75-80% of control respiratory activity respectively, 48 hours after removal of the resin. Respiratory activity by L929 cells exposed to all TEGDMA concentrations tested was not different from the vehicle control 48 hours after removal of the resin.

Analysis of Variance↗
PubMed1997

Elucidation of the sequence and the genomic organization of the human dentin matrix acidic phosphoprotein 1 (DMP1) gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II.

The dentin matrix acidic phosphoprotein 1 (DMP1) gene has been mapped to human chromosome 4q21 and shown to exhibit no recombination with the autosomal dominant disorder of dentin formation, dentinogenesis imperfecta type II. In the current study, sequencing of DMP1 cDNA and genomic clones has indicated that the human gene contains an open reading frame of 1539 bp, which predicts a highly acidic, serine-rich protein of 513 amino acids. Comparison of the human DMP1-coding sequence with that of the rat, mouse, and cow indicated that the predicted protein contains a conserved hydrophobic signal peptide sequence and an Arg-Gly-Asp cell attachment sequence. The gene is encoded by six exons, the splicing phase of which is type 0, the first exon containing solely 5' untranslated sequence. Sequencing of each of the coding exons in individuals affected by dentinogenesis imperfecta type II failed to reveal any disease-specific mutations, suggesting that mutations in DMP1 are not causative of this condition at least in the two families examined in this study.

Amino Acid Sequence↗
PubMed1997

Ameloblastin gene (AMBN) maps within the critical region for autosomal dominant amelogenesis imperfecta at chromosome 4q21.

Amelogenesis imperfecta (AI) is a broad group of hereditary enamel defects that is characterized by a high degree of clinical diversity. Recently, the local hypoplastic form of autosomal dominant AI (AIH2) has been mapped to human chromosome 4q in a 17.6-cM region. This locus has been further refined to a 4-Mb interval between D4S2421 and Albumin. Recently, a cDNA clone for an enamel matrix protein, ameloblastin (AMBN), has been isolated. In this report, we have isolated a PAC human genomic clone containing the human AMBN gene. The AMBN was mapped by two color fluorescence in situ hybridization using two P1 genomic clones for sequence tagged site (STS) markers, D4S400 and D4S409, which flank the critical AIH2 region. Our results place AMBN at 4q21 between D4S409 (4q13) and D4S400 (4q21). Furthermore, the AMBN PAC genomic clone was shown to contain three STS markers, D4S2604, D4S2670, and D4S2609, which are contained within the critical region defined by six Swedish families with AIH2. AMBN is therefore a strong candidate gene for AIH2.

Amelogenesis Imperfecta↗
PubMed1997

Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination.

Dentin is the major mineralized extracellular matrix of the tooth. The organic components of dentin consist of type I collagen (90%) with 10% noncollagenous proteins, which are also components of bone. Two dentin proteins, dentin sialoprotein and dentin phosphoprotein, have been shown to be tooth-specific being expressed mostly by odontoblast cells. In this study, we screened a mouse molar tooth library for dentin sialoprotein and dentin phosphoprotein cDNA clones. Analysis of the clones resulted in characterization of a 4420-nucleotide cDNA that contained a 940-amino acid open reading frame. The signal peptide and NH2-terminal sequence was 75% homologous to the cDNA sequence of rat dentin sialoprotein. The continued open reading frame, however, contained a RGD sequence followed by a region of repeated aspartic acid and serine residues. This portion of the protein codes for amino acid sequence consistent with that of dentin phosphoprotein. The noncoding region contains three potential polyadenylation signals, two of which were shown to be utilized. Northern blot analysis indicated the presence of two major transcripts of 4.4 and 2.2 kilobases in odontoblasts. Chromosomal mapping localized the gene to human chromosome 4. These data suggest that the previously identified dentin extracellular matrix proteins, dentin sialoprotein and dentin phosphoprotein, are expressed as a single cDNA transcript coding for a protein that is specifically cleaved into two smaller polypeptides with unique physical-chemical characteristics. Therefore, we propose that the gene be named dentin sialophosphoprotein. The location of the human dentin sialophosphoprotein gene on chromosome 4 suggests that this gene may be a strong candidate gene for the genetic disease dentinogenesis imperfecta type II.

Amino Acid Sequence↗
PubMed1997

Osteocalcin expression in young and aged dental pulps as determined by RT-PCR.

Dental pulps were obtained from third molars of young adults (17-25 yr) or from molar teeth of individuals > 50 yr of age and examined for the expression of osteocalcin (OC) mRNA by RT-PCR. OC was selected as a determinant of pulp vitality, because it has long been associated with the production of hard tissue matrix in teeth and bone. For comparative purposes, the expression of OC in each pulp was normalized relative to its housekeeping gene-product GAPDH by the establishment of a OC/GAPDH ratio. This study demonstrated that OC expression, presumably by cells of odontoblast lineage, does not diminish relative to the extant cell population. Our findings suggest, despite a reduction in volume and cell numbers, that the pulps of aging teeth retain a capacity for dentin deposition and a potential for caries and trauma resistance.

Adolescent↗
PubMed1997

Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo.

Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance ofdentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the "DSP" and "DPP" regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.

Animals↗
PubMed1997

Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Amino Acid Sequence↗