Biomedical subjects

M MacDougall

Publications and source records attributed to M MacDougall.

At least 19 recordsLinked to original sources

PubMed2006

Targeted expression of csCSF-1 in op/op mice ameliorates tooth defects.

OBJECTIVE: The aim of this study was to characterize the tooth phenotype of CSF-1-deficient op/op mice and determine whether expression of csCSF-1 in these mice has a role in primary tooth matrix formation. DESIGN: Ameloblasts and odontoblasts, isolated from wt/wt frozen sections using laser capture microdissection, were analysed for csCSF-1, sCSF-1 and CSF-1R mRNA by RT-PCR. Mandibles, excised from 8 days op/op and wt/wt littermates, were examined for tooth morphology as well as amelogenin and DMP1 expression using in situ hybridisation. op/opCS transgenic mice, expressing csCSF-1 in teeth and bone using the osteocalcin promoter, were generated. Skeletal X-rays and histomorphometry were performed; teeth were analysed for morphology and matrix proteins. RESULTS: Normal dental cells in vivo express both CSF-1 isoforms and CSF-1R. Compared to wt/wt, op/op teeth prior to eruption showed altered dental cell morphology and dramatic reduction in DMP1 transcripts. op/opCS mice showed marked resolution of osteopetrosis, tooth eruption and teeth that resembled amelogenesis imperfecta-like phenotype. At 3 weeks, op/op teeth showed severe enamel and dentin defects and barely detectable amelogenin and DMP1. In op/opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased. Enamel integrity improved in op/opCS, although it was thinner than wt enamel. CONCLUSIONS: Results demonstrate that ameloblasts and odontoblasts are a source and potential target of CSF-1 isoforms in vivo. Expression of csCSF-1 within the tooth microenvironment is essential for normal tooth morphogenesis and may provide a mechanism for coordinating the process of tooth eruption with endogenous matrix formation.

Ameloblasts↗

PubMed2006

Predicting survival in patients with hepatocellular carcinoma: a UK perspective.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a cancer of rising incidence in the UK. The aim of this study was to compare the Okuda, Cancer of the Liver Italian Program (CLIP), and Barcelona Clinic Liver Cancer (BCLC) classifications as predictors of survival in UK patients with HCC. METHODS: Data were analysed from a prospective database maintained in a specialist hepatobiliary unit from 1998 to 2003. Each system was assessed for its discriminatory power, monotonicity of gradient, and independent contribution to prediction of mortality status based on a multivariate model. RESULTS: One hundred and two patients (77 males, 25 females) were identified with a median age of 65 (range, 14-87) years. The overall median survival time was 13 months and the one- and five-year survival rates were 52.9% (95% CI: 43.2%, 62.6%) and 35.3% (95% CI: 26.0%, 44.6%), respectively. All three classification systems had the capacity to differentiate between patient survival times across different stages. The Okuda system was superior in overall discriminatory power and in strength of monotonicity. The BCLC system, however, made the highest independent contribution of all three systems in predicting survival in the Cox regression model. CONCLUSIONS: All three classification systems were effective in predicting survival for patients with HCC in a UK population.

Adolescent↗

PubMed2006

Ultrastructural and immunocytochemical characterization of immortalized odontoblast MO6-G3.

AIM: To investigate an immortalized murine odontoblast cell line as a potential alternative for experimental studies on dentinogenesis. METHODOLOGY: The MO6-G3 cell line was investigated morphologically over 3, 7, 11 and 42 days of culture, using histochemical localization of dentine sialoprotein (DSP), alkaline phosphatase (AP), type I collagen and actin filaments, histoenzymatic staining and biochemical investigation of AP and finally, transmission and scanning electron microscopy. RESULTS: Scanning electron micrographs showed elongated cells. Accordingly, a polarized organization of odontoblasts was observed by transmission electron microscopy, identifying distinct subcellular compartments as described in vivo. The secretion apparatus, which includes cisternae of rough endoplasmic reticulum, Golgi apparatus saccules and secretion vesicles and granules, was longitudinally organized in the supranuclear compartment ending distally in the secretory pole. A cellular process was observed. The investigation of the cytoskeleton network revealed that actin microfilaments were organized in parallel stress fibre oriented depending on the longitudinal axis of the cytoplasm. Immunofluorescent labelling showed a continuous expression of type I collagen, DSP and AP. A unipolar distribution characterized intracellular DSP immunoreactivity. Histoenzymology revealed AP active sites increasing from 3 to 11 days albeit with a moderate level of activity comparatively to the in vivo situation in dental cells. CONCLUSION: This cell line MO6-G3 not only showed the criteria of odontoblast phenotype as previously reported but also the characteristic morphodifferentiation pattern of polarized odontoblasts at the cellular level but with an apparent random distribution.

Actin Cytoskeleton↗

PubMed2005

Mouse amelogenin exons 8 and 9: sequence analysis and protein distribution.

Amelogenin is the major protein of the developing enamel. Two additional exons, termed 8 and 9, have been characterized in the rat. Our aim was: to identify the mouse amelogenin exons 8/9 sequences; to investigate the potential presence of the alternative spliced isoforms of amelogenin exons 8/9; and to immunolocalize proteins containing sequences encoded by exons 8/9 during odontogenesis. RT-PCR analysis with exon 9 anti-sense primer generated 2 major amplicons with the use of a mouse tooth cDNA library and dental cell lines. DNA sequence analysis showed 93% identify with the rat exons 8/9 sequence. Alternative splicing of exon 3 was also found, but only in cDNAs lacking exons 8 and 9. Immunohistochemistry localized exons 8/9-encoded proteins in ameloblasts, young odontoblasts, and stratum intermedium cells. Analysis of our data supports the hypothesis that: (1) AMELX contains 2 additional exons; (2) ameloblasts and odontoblasts synthesize amelogenin 8/9; and (3) amelogenin splice variants may have unique functions during tooth formation.

Alternative Splicing↗

PubMed2003

Expression of amelogenin in odontoblasts.

Amelogenin is the major enamel protein produced by ameloblasts. Its expression has been shown to be down-regulated in ameloblasts of vitamin-D-deficient (-D) rats. The potential expression and localization of amelogenin in odontoblasts and its regulation by vitamin D were investigated in this study. RT-PCR and semi-quantitative Northern blot analyses were performed using the odontoblast cell line MO6-G3 and microdissected dental pulp mesenchyme. Both in vitro and in vivo odontoblasts expressed various alternatively spliced amelogenin transcripts. In situ hybridization studies showed that amelogenin expression was restricted to young odontoblasts during mantle dentin deposition. Electron microscopy studies localized the amelogenin protein in the odontoblast cell process cytoplasm and mantle dentin. Amelogenin immunolabeling was stronger in -D rats, suggesting an inverse regulation by vitamin D in odontoblasts. Furthermore, amelogenin mRNA steady-state levels were significantly increased in -D dental pulp mesenchyme. In addition, a temporal-spatial lengthening of the mantle dentin stage was observed in -D animals, suggesting that developmental perturbations occur in relation to the vitamin D status and/or amelogenin expression. These data show that amelogenin is expressed by odontoblasts selectively during mantle dentin deposition. This developmental regulated expression pattern is enhanced under vitamin-D-deficiency status and in a broader context may play an important role during ameloblast and odontoblast differentiation and function.

Amelogenin↗

PubMed2002

TGF beta-1 downregulates DMP-1 and DSPP in odontoblasts.

Transforming growth factor beta-1 (TGF beta-1) is a multifunctional growth factor that is expressed in numerous cell types. It has been shown to induce secretion of dentin extracellular matrix components associated with primary dentinogenesis and to play a role in tertiary or reparative dentinogenesis. In this study, we investigated the potential transcriptional regulation by TGF beta-1 of two dentin matrix proteins: dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP). In vitro promoter studies were performed using plasmid constructs containing mouse DMP-1 and DSPP promoter sequences fused to the luciferase reporter gene. Constructs were transiently transfected in the mouse odontoblast cell line M06-G3 and cultured in the presence or absence of TGF beta-1. The integrity of the TGF beta-1 signaling pathway was investigated in the M06-G3 cells by identifying known key effectors of TGF beta-1 signal transduction. Transient transfection studies demonstrate for the first time that TGF beta-1 downregulates both DMP-1 and DSPP genes. Our findings indicate that the TGF beta-1 type I receptor ALK5 is expressed by odontoblasts as well as the signal transduction proteins Smad2, Smad3, and Smad4. These results suggest that TGF beta-1 regulates two key dentin proteins involved in matrix mineralization most likely mediated through the type I ALK5 receptor and transduced by Smads 2, 3, and 4.

Activin Receptors, Type I↗

PubMed2002

Cross-talk between Msx/Dlx homeobox genes and vitamin D during tooth mineralization.

Rickets is associated with site-specific disorders of enamel and dentin formation, which may reflect the impact of vitamin D on a morphogenetic pathway. This study is devoted to potential cross-talk between vitamin D and Msx/Dlx transcription factors. We raised the question of a potential link between tooth defects seen in mice with rickets and Msx2 gene misexpression, using mutant mice lacking the nuclear vitamin D receptor as an animal model. Our data showed a modulation of Msx2 expression. In order to search for a functional impact of this Msx2 misexpression secondary to rickets, we focused our attention on osteocalcin as a target gene for both vitamin D and Msx2. Combining Msx2 overexpression and vitamin D addition in vitro, we showed an inhibitory effect on osteocalcin expression in immortalized MO6-G3 odontoblasts. Finally, in the same cells, such combinations appeared to modulate VDR expression outlining the existence of complex cross-regulations between vitamin D and Msx/Dix pathways.

Animals↗

PubMed2002

Genomic organization and localization of mouse Nma/BAMBI: possible implications related to ameloblastoma formation.

Our laboratory has determined the DNA sequence and transcriptional expression pattern of a mouse cDNA clone termed Nma/BAMBI. This clone encodes a highly conserved protein with 89% identity to the human homologue (termed Nma) and 78% similarity to the Xenopus homologue (termed BAMBI) at the predicted amino acid level. Nma/BAMBI encodes a 260-amino-acid transmembrane protein that has homology to the transforming growth factor (TGF) beta type I receptor family. This protein contains an extracellular ligand binding domain, a 24-amino-acid transmembrane domain, and a short intracellular domain that lacks a functional serine/threonine kinase domain. It is believed that Nma/BAMBI is important in the negative regulation of TGF beta signal transduction pathways during development and has implications in tumor progression. We have determined the genomic organization of the mouse Nma/BAMBI gene and confirmed the chromosomal mapping to human chromosome 10 and mouse chromosome 2. Furthermore, we report the production and utilization of an anti-peptide antibody in preliminary immunohistochemical analysis of an ameloblastoma.

Ameloblastoma↗

PubMed2002

Differential epithelial and mesenchymal regulation of tooth-specific matrix proteins expression by 1,25-dihydroxyvitamin D3 in vivo.

Enamel defects have been reported in rickets and related to disturbed expression of amelogenin in ameloblasts. The present study is devoted to amelogenin, enamelin, ameloblastin, and dentin sialophosphoprotein (DSPP) expression in both the epithelium and mesenchyme of vitamin D-deficient rat incisors. Quantitative Northern blotting analysis (relatively to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA steady-state levels) was performed on microdissected cells of rachitic (-D) and control (+D) 56 day old rats. Steady-state levels of amelogenin and enamelin mRNA were significantly reduced in the -D epithelium versus the +D epithelium ones. In contrast, ameloblastin expression was slightly increased in -D epithelium. In the same samples, DSPP mRNA levels remained unchanged in -D dental mesenchyme. Comparative electron microscopy studies between +D and -D animals showed a dramatic decrease of intraprismatic enamel (amelogenin and enamelin immunoreactive) consistent with our molecular results. In conclusion, tooth formation results from the coordinated expression of several matrix proteins that may be controlled by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3].

Animals↗

PubMed2002

Msx1 homeogene antisense mRNA in mouse dental and bone cells.

Msx1 plays a key role in early dental and cranio-facial patterning. A systematic screening of Msx1 transcripts during late postnatal stages of development evidenced not only sense mRNA but also antisense mRNA in the skeleton. Natural antisenses are able to bind their corresponding sense RNAs and block protein expression. Specific reverse-transcription polymerase chain reaction (RT-PCR) Northern-blotting using riboprobes and primer extension analysis allowed to identify and sequence a mouse 2184-base Msx1 antisense transcript. The transcription start site was located in a region including a consensus TATA box. In situ hybridization evidenced an increase in antisense mRNA expression during dental and bone cell differentiation in prenatal (Theiler stages E15.5-18.5) and newborn mice. This upregulation was related to Msx1 protein downregulation in cells expressing Msx1 sense mRNA. In vitro, transient Msx1 sense and antisense mRNA overexpression was performed in MO6-G3 cells, which pertain to the odontoblast lineage (polarization and dentin sialoprotein and phosphoprotein synthesis). The balance between antisense and sense Msx1 mRNAs appeared to control Msx1 protein levels. These data suggest that a bidirectional transcription of Msx1 homeogene may control Msx1 protein levels, and therefore may be critical in cell communication and differentiation during dental and cranio-facial development and mineralization.

Animals↗

PubMed2001

Human ameloblastin gene: genomic organization and mutation analysis in amelogenesis imperfecta patients.

A gene encoding the enamel protein ameloblastin (AMBN) was recently localized to a region on chromosome 4q21 containing a gene for the inherited enamel defect local hypoplastic amelogenesis imperfecta (AIH2). Ameloblastin protein is located at the Tomes processes of secretory ameloblasts and in the sheath space between rod-interrod enamel, and the AMBN gene therefore represents a viable candidate gene for local hypoplastic amelogenesis imperfecta (AI). In this study, the genomic organization of human AMBN was characterized. The gene was shown to consist of 13 exons and 12 introns. An alternatively spliced 45 bp sequence was shown not to represent a separate exon and is most likely spliced by the use of a cryptic splice site. The finding that there were no recombinations between an intragenic microsatellite and AIH2 encouraged us to evaluate this gene's potential role as a candidate gene for local hypoplastic AI. Mutation screening was performed on all 13 exons in 20 families and 8 sporadic cases with 6 different forms of AI. DNA variants were found but none that was associated exclusively with local hypoplastic AI or any of the other variants of AI in the identified Swedish families. This study excludes the coding regions and the splice sites of AMBN from a causative role in the pathogenesis of AIH2.

Amelogenesis Imperfecta↗

PubMed2001

Cloning, characterization, and tissue expression pattern of mouse Nma/BAMBI during odontogenesis.

Degenerate oligonucleotides to consensus serine kinase functional domains previously identified a novel, partial rabbit tooth cDNA (Zeichner-David et al., 1992) that was used in this study to identify a full-length mouse clone. A 1390-base-pair cDNA clone was isolated encoding a putative 260-amino-acid open reading frame containing a hydrophobic 25-amino-acid potential transmembrane domain. This clone shares some homology with the TGF-beta type I receptor family, but lacks the intracellular kinase domain. DNA database analysis revealed that this clone has 86% identity to a newly isolated human gene termed non-metastatic gene A and 80% identity to a Xenopus cDNA clone termed BMP and activin membrane bound inhibitor. Here we report the mouse Nma/BAMBI cDNA sequence, the tissue expression pattern, and confirmed expression in dental cell lines. This study demonstrates that Nma/BAMBI is a highly conserved protein across species and is expressed at high levels during odontogenesis.

Activins↗

PubMed2001

Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis.

Tooth formation is the result of reciprocal instructive interactions between oral epithelium and cranial neural-crest-derived ectomesenchymal tissues. These interactions lead to the cytodifferentiation of highly specialized matrix-forming cell types, the ameloblast, odontoblast, and cementoblast, that produce the mineralized tissues enamel, dentin, and cementum, respectively. Our laboratory has been developing immortalized dental cell lines representative of these various cell types to facilitate studies on gene regulation, cell differentiation, matrix formation, and mineralization. Odontoblasts are solely responsible for the synthesis and secretion of the dentin extracellular matrix bilayer that consists of non-mineralized predentin and mineralized dentin. The mouse immortalized MO6-G3 cell line expresses the major matrix proteins associated with the odontoblast phenotype, producing a matrix that is capable of mineralization. This cell line serves as a useful tool in studies designed to explore the various processes of dentinogenesis. In this paper, we present studies using the mouse odontoblast cell line MO6-G3 as examples of the various research applications. Studies highlighted are: in vitro promoter studies investigating the tooth-specific gene regulation of the major non-collagenous dentin matrix protein, dentin sialophosphoprotein; regulation of tertiary dentin formation by cytokines, such as transforming growth factor-Beta 1; and the utilization of dentally relevant cells in dental material biocompatibility testing.

Ameloblasts↗

PubMed2000

Dental cells express factors that regulate bone resorption.

Odontoblasts and osteoblasts produce similar highly mineralized extracellular matrices. In bone, osteoblasts/stromal cells regulate osteoclast (ocl) formation and bone resorption by producing factors like osteoprotegerin (OPG), osteoclast differentiating factor (ODF/RANKL), and macrophage colony-stimulating factor (M-CSF) that interact with hematopoietic ocl precursor cells. Using odontoblast and pulp cell lines, we detected a constitutive expression of OPG, RANKL, and M-CSF mRNA in both cell types. OPG and RANKL proteins were also detectable. In vivo, RANKL and OPG were localized to odontoblasts, ameloblasts, and pulp cells in developing mouse teeth by immunohistochemistry. In a coculture system, we found the dental cells to be inhibitory to ocl formation from spleen and bone marrow precursors, despite their production of osteoclast stimulatory factors. Our data indicate for the first time that dental cells express factors important in regulation of osteoclastogenesis and bone resorption. Since both stimulatory (RANKL, M-CSF) and inhibitory (OPG) factors are expressed, a balance between positive and negative factors may contribute to regulation of bone resorption.

Animals↗

PubMed2000

Cloning, characterization and immunolocalization of human ameloblastin.

Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

Alternative Splicing↗

PubMed2000

Enamelin maps to human chromosome 4q21 within the autosomal dominant amelogenesis imperfecta locus.

Amelogenesis imperfecta is a group of hereditary enamel defects. Of the autosomal dominant forms, only the local hypoplastic type has been mapped to human chromosome 4q 13-4q21. Enamelin is a large enamel matrix protein secreted by ameloblasts. The purpose of this study was to determine the human chromosomal localization of enamelin to establish an association with various forms of amelogenesis imperfecta. Chromosomal mapping was performed by polymerase chain reaction (PCR) amplification using somatic hybrid and deletion/derivation cell line panels with an enamelin primer set based on 100% conserved regions between pig and mouse cDNAs. Sequence-tagged site content mapping using eight markers within the critical local hypoplastic amelogenesis imperfecta region was then performed using an isolated human enamelin genomic BAC clone. The human enamelin amplicon was confirmed by DNA sequence analysis, revealing 81% and 73% identity to pig and mouse cDNAs, respectively. PCR amplification using a somatic cell hybrid panel placed enamelin on chromosome 4 with analysis of a regional chromosome 4 mapping panel refining the localization to 4q 13.1-q21.23. An identified human enamelin BAC genomic clone was shown to contain markers D4S2604 and D4S2670, as well as the first exon of the human ameloblastin gene, placing enamelin in the critical amelogenesis imperfecta locus between markers HIS1 and D4S2604 at 4q21. Our results suggest that enamelin is a strong candidate gene for this disease. Furthermore, human 4q21 may contain a second cluster of enamel matrix genes located proximally to the identified cluster of dentin and bone genes.

Amelogenesis Imperfecta↗

PubMed2000

Inositol hexasulphate, a casein kinase inhibitor, alters enamel formation in cultured embryonic mouse tooth germs.

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

Ameloblasts↗

PubMed1999

Expression of DLX5 during human embryonic craniofacial development.

Dlx (distal-less gene) homeogenes encode transcription factors that are involved in the patterning of orofacial skeleton derived from cephalic neural crest cells. In order to study the role of DLX genes during embryonic development in human, DLX5 expression pattern was investigated in 6- to 11-week-old human embryos. A DLX5 PCR fragment was amplified from a human dental cDNA library subcloned and used for in situ hybridization investigations. DLX5 gene expression was primarily detected in the mandible at 6 weeks and then, after in the maxilla. DLX5 gene expression became restricted to progenitor cells of developing tooth germs, bones and cartilages of mandible and maxilla. During odontogenesis from bud to late cap stages, DLX5 transcripts were present in both dental epithelium and mesenchyme tissues. DLX5 expression was restricted to few cells in the vestibular aspect of the dental epithelium, while DLX5 mRNA signal was more widely distributed in dental mesenchyme. The observed expression pattern of DLX5 homeogene extends the proposed site-specific combination of homeogene expression in neural crest derived cells to human specific dentition. Furthermore, during the bud and cap stages of tooth morphogenesis, the asymmetric expression of DLX5 in the dental epithelium and dental mesenchyme may contribute to the complex patterning of human tooth shape.

Brain↗