ABSTRACT
X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.
Subject(s)
Familial Hypophosphatemic Rickets/genetics , Genetic Diseases, X-Linked/genetics , PHEX Phosphate Regulating Neutral Endopeptidase/genetics , Tooth Demineralization/genetics , Adolescent , Calcium/analysis , Cells, Cultured , Child , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Humans , Immunoblotting , Immunohistochemistry , Microscopy, Electron, Scanning , Osteopontin/metabolism , Phenotype , Phosphoproteins/metabolism , Polymerase Chain Reaction , Spectrum Analysis, Raman , Tissue Scaffolds , Tooth, Deciduous , X-Ray MicrotomographyABSTRACT
Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.
Subject(s)
Dental Pulp/cytology , Pulpotomy , Regeneration , Tissue Engineering/methods , Animals , Cell Proliferation , Dentin, Secondary/physiology , Extracellular Matrix Proteins/analysis , Hydrogels , Integrin-Binding Sialoprotein/analysis , Phosphoproteins/analysis , Sialoglycoproteins/analysis , Staining and Labeling , Swine , Swine, Miniature , X-Ray MicrotomographyABSTRACT
X-linked hypophosphatemia (XLH) is a rare genetic skeletal disease where increased phosphate wasting in the kidney leads to hypophosphatemia and prevents normal mineralization of bone and dentin. Here, we examined the periodontal status of 34 adults with XLH and separated them according to the treatment they received for hypophosphatemia. We observed that periodontitis frequency and severity were increased in adults with XLH and that the severity varied according to the hypophosphatemia treatment. Patients who benefited from an early and continuous vitamin D and phosphate supplementation during their childhood presented less periodontal attachment loss than patients with late or incomplete supplementation. Continued hypophosphatemia treatment during adulthood further improved the periodontal health. Extracted teeth from patients with late or incomplete supplementation showed a strong acellular cementum hypoplasia when compared with age-matched healthy controls. These results show that XLH disturbs not only bone and dentin formation but also cementum and that the constitutional defect of the attachment apparatus is associated with attachment loss.
Subject(s)
Familial Hypophosphatemic Rickets/complications , Familial Hypophosphatemic Rickets/drug therapy , Periodontitis/prevention & control , Phosphates/therapeutic use , Vitamin D/therapeutic use , Adult , Case-Control Studies , Familial Hypophosphatemic Rickets/diagnostic imaging , Female , Humans , Immunohistochemistry , Male , Middle Aged , Periodontitis/diagnostic imaging , Prospective Studies , Radiography, Panoramic , Treatment OutcomeABSTRACT
Mutations in phosphate-regulating gene (PHEX) lead to X-linked hypophosphatemic rickets (XLH), a genetic disease characterized by impaired mineralization in bones and teeth. In human XLH tooth dentin, calcospherites that would normally merge as part of the mineralization process are separated by unmineralized interglobular spaces where fragments of matrix proteins accumulate. Here, we immunolocalized osteopontin (OPN) in human XLH teeth, in a three-dimensional XLH human dental pulp stem cell-collagen scaffold culture model and in a rat tooth injury repair model treated with acidic serine- and aspartate-rich motif peptides (ASARM). In parallel, matrix extracellular phosphoglycoprotein (MEPE) immunolocalization and alkaline phosphatase (ALP) activity were assessed in XLH teeth. OPN was expressed by odontoblasts in the XLH models, and localized to the abnormal calcospherites of XLH tooth dentin. In addition, ALP activity and MEPE localization were abnormal in human XLH teeth, with MEPE showing an accumulation in the unmineralized interglobular spaces in dentin. Furthermore, XLH odontoblasts failed to form a well-polarized odontoblast layer. These data suggest that both MEPE and OPN are involved in impaired tooth mineralization associated with XLH, possibly through different effects on the mineralization process.
Subject(s)
Calcification, Physiologic/physiology , Extracellular Matrix Proteins/metabolism , Familial Hypophosphatemic Rickets/metabolism , Glycoproteins/metabolism , Odontoblasts/cytology , Osteopontin/metabolism , Phosphoproteins/metabolism , Adolescent , Animals , Cell Differentiation/physiology , Familial Hypophosphatemic Rickets/genetics , Female , Humans , Rats , Tooth/cytology , Tooth/metabolismABSTRACT
Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.
Subject(s)
Ameloblasts/metabolism , Amelogenesis/physiology , Collagen Type I/analysis , Ameloblasts/cytology , Amelogenin/analysis , Animals , Cell Differentiation/physiology , Collagen Type I, alpha 1 Chain , Dental Enamel/cytology , Dental Enamel/metabolism , Dentinogenesis/physiology , Enamel Organ/anatomy & histology , Metamorphosis, Biological/physiology , Microscopy, Electron, Transmission , Models, Animal , Odontoblasts/cytology , Odontoblasts/metabolism , Odontogenesis/physiology , Pleurodeles , Tooth Germ/anatomy & histologyABSTRACT
Many of our assumptions concerning oral implant osseointegration are extrapolated from experimental models studying skeletal tissue repair in long bones. This disconnect between clinical practice and experimental research hampers our understanding of bone formation around oral implants and how this process can be improved. We postulated that oral implant osseointegration would be fundamentally equivalent to implant osseointegration elsewhere in the body. Mice underwent implant placement in the edentulous ridge anterior to the first molar and peri-implant tissues were evaluated at various timepoints after surgery. Our hypothesis was disproven; oral implant osseointegration is substantially different from osseointegration in long bones. For example, in the maxilla peri-implant pre-osteoblasts are derived from cranial neural crest whereas in the tibia peri-implant osteoblasts are derived from mesoderm. In the maxilla, new osteoid arises from periostea of the maxillary bone but in the tibia the new osteoid arises from the marrow space. Cellular and molecular analyses indicate that osteoblast activity and mineralization proceeds from the surfaces of the native bone and osteoclastic activity is responsible for extensive remodeling of the new peri-implant bone. In addition to histologic features of implant osseointegration, molecular and cellular assays conducted in a murine model provide new insights into the sequelae of implant placement and the process by which bone is generated around implants.
Subject(s)
Dental Implants , Models, Animal , Osseointegration , Animals , Bone Remodeling , Cell Survival , Humans , Maxilla/diagnostic imaging , Maxilla/pathology , Mesoderm/pathology , Mice , Neural Crest/diagnostic imaging , Neural Crest/pathology , Osteocytes/pathology , Radiography , Tibia/diagnostic imaging , Tibia/pathology , Wound HealingABSTRACT
Several genetic disorders affecting bone mineralization may manifest during dentin mineralization. Dentin and bone are similar in several aspects, especially pertaining to the composition of the extracellular matrix (ECM) which is secreted by well-differentiated odontoblasts and osteoblasts, respectively. However, unlike bone, dentin is not remodelled and is not involved in the regulation of calcium and phosphate metabolism. In contrast to bone, teeth are accessible tissues with the shedding of deciduous teeth and the extractions of premolars and third molars for orthodontic treatment. The feasibility of obtaining dentin makes this a good model to study biomineralization in physiological and pathological conditions. In this review, we focus on two genetic diseases that disrupt both bone and dentin mineralization. Hypophosphatemic rickets is related to abnormal secretory proteins involved in the ECM organization of both bone and dentin, as well as in the calcium and phosphate metabolism. Osteogenesis imperfecta affects proteins involved in the local organization of the ECM. In addition, dentin examination permits evaluation of the effects of the systemic treatment prescribed to hypophosphatemic patients during growth. In conclusion, dentin constitutes a valuable tool for better understanding of the pathological processes affecting biomineralization.
Subject(s)
Calcification, Physiologic/genetics , Dentin/pathology , Genetic Diseases, Inborn/pathology , Tooth/pathology , Bone and Bones/pathology , Genetic Diseases, Inborn/diagnostic imaging , Genetic Diseases, Inborn/therapy , Humans , RadiographyABSTRACT
Disulone (dapsone + ferrous oxalate) is a sulphone marketed in France since 1958 and authorized in P. Carinii prophylaxis in HIV+ cotrimoxazole intolerant patients, bullous dermatosis, leprosy and polychondritis. Between 1983 and 1998, 249 adverse reactions were reported to French pharmacovigilance centres and Aventis, the manufacturer. Every side-effect was reviewed and the causal relationship was assessed on the basis of the French method for causality assessment. Main side-effects were divided as follows: 117 blood dyscrasias (generally neutropenia and agranulocytosis, rarely methaemoglobinaemia, haemolysis, macrocytosis, anaemia, aplastic anaemia, haemochromatosis and sulphaemoglobinaemia); 29 hypersensitivity syndrome; 39 cutaneous reactions, generally rash; 27 liver injuries (cholestatic, cytolytic and mixed hepatitis); 27 neurological and psychiatric side-effects including 7 axonal neuropathy; 10 gastrointestinal effects, generally nausea and vomiting. Five deaths were reported (4 septicaemia including one case not due to dapsone and 1 digestive bleeding due to underlying disease). In the other cases the outcome was favourable. The results were compared with the published references. It would seem to be important to reinforce information to prescribers about the possible serious adverse reactions with dapsone, particularly hypersensitivity syndrome and agranulocytosis, that can cause death if the drug is not stopped in time.
Subject(s)
AIDS-Related Opportunistic Infections/drug therapy , Adverse Drug Reaction Reporting Systems , Anti-Infective Agents/adverse effects , Pneumonia, Pneumocystis/drug therapy , Adverse Drug Reaction Reporting Systems/statistics & numerical data , France , HumansABSTRACT
The telo-box, an interstitial telomere motif, was shown to regulate gene expression in root meristems, in synergy with a cis-acting element involved in the activation of expression of plant eEF1A genes, encoding the translation elongation factor EF1A, and of several ribosomal protein genes. We demonstrate here that the telo-box is also required for transcription activation by two other cis elements present within the promoter of genes encoding the acidic ribosomal protein rp40 and the proliferating cell nuclear antigen respectively. The control of gene expression by telo-boxes during cell cycle progression in Arabidopsis root meristems is discussed. A parallel is drawn with the function of telomeric sequences in Saccharomyces cerevisiae.
Subject(s)
Arabidopsis/genetics , Meristem/genetics , Plant Roots/genetics , Regulatory Sequences, Nucleic Acid/genetics , Telomere/genetics , Base Sequence , DNA, Plant/genetics , Gene Expression Regulation, Plant , Glucuronidase/genetics , Glucuronidase/metabolism , Peptide Elongation Factor 1/genetics , Plants, Genetically Modified , Proliferating Cell Nuclear Antigen/genetics , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
The tef box, a cis-acting element identified in promoters of several plant genes encoding components of the translation apparatus, is involved in the activation of gene expression in cycling cells. In vitro, this element mediates the formation of two protein complexes called C1 and C2. A tef-like box is also found within the intergenic transcribed spacer of several plant rRNA genes. In radish this sequence has already been described as a protein-binding site putatively involved in the regulation of rDNA expression and is sufficient for formation of C1 complexes. By using mutated tef boxes, we show that tef-dependent activation of transcription is correlated with formation of both C1 and C2 complexes in a context-dependent manner. In transient expression experiments, the activation of a minimal promoter-GUS gene fusion is associated with the formation of C2 complexes. In contrast, the ability to form C1 complexes appears to allow activation of reporter gene expression in root meristems of transgenic Arabidopsis. SDS-PAGE analysis of purified protein fractions containing either the C1 or the C2 activity indicates a complex heteromeric structure for these potential regulators. Thus, the tef box seems to be a central component of the regulation of gene transcription in distinct and overlapping developmental programs, and could be involved in co-regulation of transcription by RNA polymerases I and II.
Subject(s)
DNA, Ribosomal/genetics , Plant Proteins/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Arabidopsis/genetics , Base Sequence , Cells, Cultured , DNA Primers , Gene Expression Regulation, PlantABSTRACT
The promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo-box, associated with an activating sequence, the tef-box. Database searches indicated the presence of telo-boxes in the 5' region of numerous genes encoding components of the translational apparatus. By using several promoter constructs we demonstrate that the telo-box is required for the expression of a beta-glucoronidase gene in root primordia of transgenic Arabidopsis. This effect was observed when a telo-box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef-box. These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression. South-western screening of a lambdaZAP library with a double-stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Puralpha. The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant , Glucuronidase/genetics , Telomere/genetics , Amino Acid Sequence , Animals , Arabidopsis/physiology , Chromosomes/genetics , Drosophila/genetics , Glucuronidase/chemistry , Glucuronidase/metabolism , Humans , Meristem/physiology , Molecular Sequence Data , Plant Roots/physiology , Plants, Genetically Modified , Promoter Regions, Genetic , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In Arabidopsis thaliana, EF-1 alpha proteins are encoded by a multigene family of four members. Three of them are clustered at the same locus, which was positioned 24 cM from the top of chromosome 1. A region of DNA spanning 63 kb around these locus was sequenced and analyzed. One main characteristic of the locus is the mosaic organization of both genes and intergenic regions. Fourteen genes were identified, among which only four were already described, and other unidentified are most likely present. Functionally diverse genes are found at close intervals. Exon and intron distribution is highly variable at this locus, one gene being split into at least 20 introns. Several duplications were found within the sequenced segment both in coding and noncoding regions, including two gene families. Moreover, a sequence corresponding to the 5' noncoding region of the EF-1 alpha genes and harboring a 5' intervening sequence is duplicated and found upstream of several genes, suggesting that noncoding regions can be shuffled during evolution.
Subject(s)
Arabidopsis/genetics , Genes, Plant/genetics , Multigene Family/genetics , Oxidoreductases , Peptide Elongation Factors/genetics , Plant Proteins/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Artificial, Yeast , Glutaredoxins , Molecular Sequence Data , Peptide Elongation Factor 1 , Plants, Toxic , Proteins/genetics , Ricinus/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.
Subject(s)
Arabidopsis/genetics , DNA, Complementary/genetics , Databases, Factual , Genes, Plant , Genetic Markers , Amino Acid Sequence , Base Sequence , Conserved Sequence , Gene Expression , Gene Library , Molecular Biology/trends , Molecular Sequence Data , Oryza/genetics , Reproducibility of Results , Sequence Alignment , Sequence Analysis, DNA , Sequence HomologySubject(s)
Arabidopsis/genetics , Genes, Plant , Plant Proteins/genetics , Receptors, Laminin/genetics , Animals , DNA, Complementary/metabolism , Databases, Factual , Drosophila , Humans , Mice , Molecular Sequence Data , Plant Proteins/biosynthesis , Receptors, Laminin/biosynthesis , Sequence Homology, Amino AcidABSTRACT
The activity of the Arabidopsis thalana A1 EF-1 alpha gene promoter was analyzed in transgenic Arabidopsis plants. The 5' upstream sequence of the A1 gene and several promoter deletions were fused to the beta-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5' non-coding region (5'IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1 alpha cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5'IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1 alpha upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1 alpha upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-alpha promoter but has no significant effect when fused to an enhancer-less 35S promoter.
Subject(s)
Arabidopsis/genetics , Peptide Elongation Factors/genetics , Plant Proteins/genetics , Base Sequence , Gene Expression Regulation , Molecular Sequence Data , Peptide Elongation Factor 1 , Plants, Genetically Modified , Promoter Regions, Genetic , Regulatory Sequences, Nucleic AcidABSTRACT
A cDNA clone encoding a small GTP-binding protein, the ADP-ribosylation factor (ARF) was isolated from a cDNA library of Arabidopsis thaliana cultured cells. The predicted amino acid sequence was highly homologous to the known yeast, bovine and human ARF sequences. Southern analysis of Arabidopsis genomic DNA suggested the existence of at least two copies of ARF genes. The level of ARF mRNA was found to be nearly constant during all cell growth stages in suspension cultures.
Subject(s)
Arabidopsis/genetics , GTP-Binding Proteins/genetics , Plant Proteins/genetics , ADP-Ribosylation Factors , Amino Acid Sequence , Arabidopsis/chemistry , Cells, Cultured , Cloning, Molecular , DNA , GTP-Binding Proteins/biosynthesis , Molecular Sequence Data , Plant Proteins/biosynthesis , Sequence AlignmentABSTRACT
In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene promoter.