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M L Paine

Publications and source records attributed to M L Paine.

At least 19 recordsLinked to original sources
PubMed2007

Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Amelogenesis↗
PubMed2005

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11).

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Adenoviridae↗
PubMed2005

Controlled failure mechanisms toughen the dentino-enamel junction zone.

STATEMENT OF PROBLEM: The dentino-enamel junction (DEJ) durably unites dissimilar hard brittle enamel and tough flexible dentin. In contrast to artificial bonds between restorations and dentin, the DEJ rarely fails except when it is affected by inherited disorders. Knowledge of DEJ toughening mechanisms is important in understanding inherited disorders, in biomimetic engineering of junctions between artificial restorations and teeth, and in tissue-engineering a DEJ. PURPOSE: The purpose of this study was to identify specific DEJ-zone failure mechanisms and to survey the fracture toughness of the human DEJ zone. MATERIAL AND METHODS: Fracture toughness indentations were made at 3 sites across the DEJ zone of 10 human incisor teeth. Failure modes identified using optical microscopy and fracture toughness (MPa.m(1/2)) were calculated following Vickers microindentation. Site mean values were then calculated and compared using 1-way analysis of variance (alpha=.05). RESULTS: The DEJ did not undergo catastrophic interfacial delamination; instead, damage was distributed over a broad zone. The primary damage mode involved cracking and damage dispersion in the specialized first-formed enamel close to the DEJ. Multiple, somewhat convoluted and sometimes branching, cracks spread and diffused damage over a wide area of adjacent enamel rather than producing catastrophic interfacial failure. Other secondary mechanisms included short microcracks in the DEJ adjacent dentin with possible cracked bridging, as well as plastic deformation of the DEJ without delamination. A DEJ-zone fracture toughness of approximately 0.8 to 0.9 MPa.m(1/2) was calculated. CONCLUSION: DEJ-zone damage occurred primarily within the adjacent layer of specialized first-formed enamel, and the optical DEJ interface resisted delamination.

Biomechanical Phenomena↗
PubMed2005

Tooth developmental biology: disruptions to enamel-matrix assembly and its impact on biomineralization.

Dental enamel is a composite bioceramic material that is the hardest tissue in the vertebrate body, containing long, thin crystallites of substituted hydroxyapatite (HAP). Over a lifetime of an organism, enamel functions under repeated and immense loads, generally without catastrophic failure. Enamel is a product of ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches are now being coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principal structural protein for forming enamel, we have identified two domains that are required for its proper self-assembly into supramolecular structures referred to as nanospheres. Nanospheres are believed to control HAP crystal habit. Other structural proteins of the enamel matrix include ameloblastin and enamelin, but little is known about their biological importance. Transgenic animals have been prepared to investigate the effect of overexpression of wild-type or mutated enamel proteins on the developing enamel matrix. Amelogenin transgenes were engineered to contain deletions to either of the two self-assembly domains and these alterations produced significant defects in the enamel. Additional transgenic animal lines have been prepared and studied and each gives additional insights into the mechanisms for enamel biofabrication. This study summarizes the observed enamel phenotypes of recently derived transgenic animals. These data are being used to help define the role of each of the enamel structural proteins in enamel and study how each of these proteins impact on enamel biomineralization.

Amelogenesis↗
PubMed2002

Ultrastructure of forming enamel in mouse bearing a transgene that disrupts the amelogenin self-assembly domains.

The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.

Amelogenesis↗
PubMed2001

Regulated gene expression dictates enamel structure and tooth function.

Enamel is a complex bioceramic tissue. In its final form, enamel is a reflection of the unique molecular and cellular activities occurring during organogenesis. From the ectodermal origins of ameloblasts, their gene activity and protein expression profiles exist for the sole purpose of producing a mineralized shell, almost entirely devoid of protein, deposited over the 'bone-like' dentine. The interface between enamel and dentine is referred to as the dentine enamel junction and it is also unique in its biology. This review article is narrow in its scope. We restrict our review to selected advances in our understanding of the genetic, molecular and structural aspects of enamel biology. We present a model of enamel formation that relates gene expression to the assembly of an extracellular protein matrix that in turn controls the structural hierarchy and mechanical aspects of enamel and the tooth organ.

Amelogenin↗
PubMed2001

Biological organization of hydroxyapatite crystallites into a fibrous continuum toughens and controls anisotropy in human enamel.

Enamel forms the outer surface of teeth, which are of complex shape and are loaded in a multitude of ways during function. Enamel has previously been assumed to be formed from discrete rods and to be markedly aniostropic, but marked anisotropy might be expected to lead to frequent fracture. Since frequent fracture is not observed, we measured enamel organization using histology, imaging, and fracture mechanics modalities, and compared enamel with crystalline hydroxyapatite (Hap), its major component. Enamel was approximately three times tougher than geologic Hap, demonstrating the critical importance of biological manufacturing. Only modest levels of enamel anisotropy were discerned; rather, our measurements suggest that enamel is a composite ceramic with the crystallites oriented in a complex three-dimensional continuum. Geologic apatite crystals are much harder than enamel, suggesting that inclusion of biological contaminants, such as protein, influences the properties of enamel. Based on our findings, we propose a new structural model.

Anisotropy↗
PubMed2000

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

Ameloblasts↗
PubMed2000

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy.

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.

Amelogenesis Imperfecta↗
PubMed2000

Enamel biomineralization defects result from alterations to amelogenin self-assembly.

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel.

Amelogenesis↗
PubMed1998

Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix.

Enamel crystallites form in a protein matrix located proximal to the ameloblast cell layer. This unique organic extracellular matrix is constructed from structural protein components biosynthesized and secreted by ameloblasts. To date, three distinct classes of enamel matrix proteins have been cloned. These are the amelogenins, tuftelin, and ameloblastin, with recent data implicating ameloblastin gene expression during cementogenesis. The organic enamel extracellular matrix undergoes assembly to provide a three-dimensional array of protein domains that carry out the physiologic function of guiding enamel hydroxyapatite crystallite formation. Using the yeast two-hybrid system, we have surveyed these three known enamel gene products for their ability to direct self-assembly. We measured the capacity of the enamel gene products to direct protein-to-protein interactions, a characteristic of enamel proteins predicated to be required for self-assembly. We provide additional evidence for the self-assembly nature of amelogenin and tuftelin. Ameloblastin self-assembly could not be demonstrated, nor were protein-to-protein interactions observed between ameloblastin and either amelogenin or tuftelin. Within the limits of the yeast two-hybrid assay, these findings constrain the emerging model of enamel matrix assembly by helping to define the limits of enamel matrix protein-protein interactions that are believed to guide enamel mineral crystallite formation.

Amelogenesis↗
PubMed1998

Fluoride use in periodontal therapy: a review of the literature.

Although the relationship between fluoride and dental caries has been widely studied and definitively determined, the relationship between fluoride and periodontal health and disease is not clear. Conflicting studies in the literature either suggest routine fluoride usage as an adjunct to conventional periodontal therapy or argue against topical fluoride use on periodontally involved teeth. This review summarizes the literature that addresses the utility of fluoride in patients with periodontal disease and aims to advance a rational criterion for the use of fluorides in the management of periodontal diseases.

Administration, Oral↗
PubMed1998

Identification of tuftelin- and amelogenin-interacting proteins using the yeast two-hybrid system.

Biomineralization of enamel is a complex process that involves the eventual replacement of an extracellular protein matrix by hydroxyapatite crystallites. To date four different enamel matrix proteins have been identified; the amelogenins, tuftelin, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement. Enamel formation is a complex process and additional proteins are likely to have a role in the assembly of the extracellular matrix. In order to identify additional proteins involved in the assembly process, the yeast two-hybrid system developed by Fields and Song (1989) has been implemented. This system allows for the identification of unknown proteins that interact with proteins of interest. Typically a known protein is used as "bait" to screen a cDNA expression library of interest. In our studies, tuftelin or amelogenin have been used to screen a mouse tooth library produced from one day old pups. A library screening of six million clones with amelogenin as bait resulted in eleven positive clones all of which show high homology to the human leukocyte antigen-B (HLA-B) associated transcript (BAT) family of genes. A library screening of one million clones using tuftelin as the bait identified twenty-one tuftelin-interacting proteins. Ten of these proteins are either keratin K5 or keratin K6, four are constitutively expressed and the remaining seven are novel. Further characterization of the proteins shown to interact with amelogenin or tuftelin may shed additional light on this complex process of enamel matrix assembly.

Amelogenin↗
PubMed1998

Transgene animal model for protein expression and accumulation into forming enamel.

Understanding the cellular and molecular events that regulate the formation of enamel is a major driving force in efforts to characterize critical events during amelogenesis. It is anticipated that through such an understanding, improvements in prevention, diagnosis and treatment-intervention into heritable and acquired diseases of enamel could be achieved. While knowledge of the precise role of an enamel-specific protein in directing the formation of inorganic crystallites remains refractory, progress has been made with other aspects of amelogenesis that can be brought to bear on the subject. One such area of progress has been with the identification of an ameloblast-lineage specific amelogenin gene promoter. This promoter can be used to direct the expression of enamel-specific proteins, as well as the expression of proteins foreign to amelogenesis, into the enamel extracellular matrix where their effect on biomineralization can be ascertained in a prospective manner. The resulting enamel from such animals can be examined by morphologic and biochemical modalities in order to identify the effect of the transgene protein on enamel crystallite formation and subsequent biomineralization. This manuscript outlines such a strategy with the potential for enhancing our understanding of amelogenesis.

Ameloblasts↗
PubMed1997

Protein interactions during assembly of the enamel organic extracellular matrix.

Enamel is the outermost covering of teeth and contains the largest hydroxyapatite crystallites formed in the vertebrate body. Enamel forms extracellularly through the ordered assembly of a protein scaffolding that regulates crystallite dimensions. The two most studied proteins of the enamel extracellular matrix (ECM) are amelogenin and tuftelin. The underlying mechanism for assembly of the proteins within the enamel extracellular matrix and the regulatory role of crystallite-protein interactions have proven elusive. We used the two-hybrid system to identify and define minimal protein domains responsible for supra molecular assembly of the enamel ECM. We show that amelogenin proteins self-assemble, and this self-assembly depends on the amino-terminal 42 residues interacting either directly or indirectly with a 17-residue domain in the carboxyl region. Amelogenin and tuftelin fail to interact with each other. Based upon this data, and advances in the field, a model for amelogenin assemblies that direct enamel biomineralization is presented.

Amelogenin↗
PubMed1996

The murine amelogenin promoter: developmentally regulated expression in transgenic animals.

We are interested in understanding hierarchical regulation pathways that control gene expression in developing teeth. In pursuit of the molecular basis for the regulated expression of amelogenin by developing ameloblasts during tooth formation, we isolated the murine amelogenin promoter. Analysis of this promoter will provide additional details towards the identification of signals generated through instructive-, dissimilar-germ layer interactions that are for responsible for temporal- and spatial-regulation for amelogenin gene expression. Using transgenic mice we demonstrate that a 2263 nucleotide stretch of the murine amelogenin promoter conveys appropriate temporal- and spatial-regulation for amelogenin gene expression in response to instructive-signals. These transgenic animals are useful reagents to further dissect signaling pathways responsible for regulated gene expression by terminally differentiated ameloblasts.

3T3 Cells↗
PubMed1996

Carboxyl-region of tuftelin mediates self-assembly.

Enamel biomineralization relies on a complex series of protein-protein interactions resulting in the formation of an enamel matrix. This protein matrix is subsequently replaced by a fully mineralized crystallite material. The enamel extracellular matrix is comprised principally by two gene products; the amelogenins and enamelins. The enamelins, including the 389 amino-acid, 44 kDa tuftelin, are a group of acidic proteins found in the enamel extracellular matrix. This study has employed the yeast two-hybrid system to investigate the ability of tuftelin to self-assemble and to define protein regions participating in tuftelin self-assembly. We show that for tuftelin the amino-acid residues 252 through 345 contain structurally relevant determinants for self-assembly.

Amino Acid Sequence↗