Biomedical subjects

J D Bartlett

Publications and source records attributed to J D Bartlett.

At least 19 recordsLinked to original sources

PubMed2002

Regulation and interactions of MT1-MMP and MMP-20 in human odontoblasts and pulp tissue in vitro.

MT1-MMP is a cell-membrane-bound metalloenzyme that activates other proMMPs such as proMMP-2 and -13. We studied MT1-MMP expression in mature human odontoblasts and pulp tissue, the regulation of MT1-MMP expression by growth factors TGF-beta1 and BMP-2, and the activation of odontoblast-derived MMP-20 by MT1-MMP. MT1-MMP mRNA is expressed by native and cultured mature human odontoblasts and pulp tissue. Western blot analysis of human odontoblasts and pulp tissue detects 65- and 51-kDa pro- and active forms of MT1-MMP, and smaller truncated MT1-MMP forms. BMP-2 down-regulates MT1-MMP expression in odontoblasts and pulp tissue, while TGF-beta1, alone or with BMP-2, decreases MT1-MMP mRNA levels only slightly. We also demonstrate that MT1-MMP is capable of converting proMMP-20 into a form corresponding to the active MMP-20. In conclusion, this study demonstrates the expression and differential regulation of MT1-MMP in human dentin-pulp complex cells, and the activation of MMP-20 by MT1-MMP.

Adolescent↗

PubMed2002

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Absorbable Implants↗

PubMed2001

Hypercapnia invokes an acute loss of contrast sensitivity in untreated glaucoma patients.

BACKGROUND/AIM: It is widely accepted that hypercapnia results in increased retinal, choroidal, and retrobulbar blood flow. Reports of a visual response to hypercapnia appear mixed, with normal subjects exhibiting reduced temporal contrast sensitivity in some studies, while glaucoma patients demonstrate mid-peripheral visual field improvements in others. This suggests that under hypercapnic conditions a balance exists between the beneficial effects of improved ocular blood flow and some other factor such as induced metabolic stress; the outcome may be influenced by the disease process. The aim of this study was to evaluate the contrast sensitivity response of untreated glaucoma patients and normal subjects during mild hypercapnia. METHODS: 10 previously untreated glaucoma patients and 10 control subjects were evaluated for contrast sensitivity and intraocular pressure while breathing room air and then again during mild hypercapnia. RESULTS: During room air breathing, compared with normal subjects, glaucoma patients had higher IOP (p = 0.0003) and lower contrast sensitivity at 3 cycles/degree (cpd) (p = 0.001). Mild hypercapnia caused a significant fall in contrast sensitivity at 6, 12, and 18 cpd (p < 0.05), only in the glaucoma group. CONCLUSION: Glaucoma patients with early disease exhibit central vision deficits as shown by contrast sensitivity testing at 3 cpd. Hypercapnia induces further contrast loss through a range of spatial frequencies (6-18 cpd) which may be predictive of further neuronal damage due to glaucoma.

Case-Control Studies↗

PubMed2001

Gelatinase A (MMP-2) in developing tooth tissues and amelogenin hydrolysis.

Matrix metalloproteinases (MMPs) are thought to play important roles during enamel and dentin biomineralization. Previously, membrane type-1 matrix metalloproteinase (MT1-MMP) was localized to the plasma membranes of ameloblasts and odontoblasts of the developing tooth. The best-characterized function of MT1-MMP is to initiate the activation of gelatinase A (MMP-2). Thus, we hypothesized that gelatinase A may also be expressed by developing tooth tissues. A full-length porcine gelatinase A mRNA was isolated by RT-PCR homology cloning of an enamel-organ-specific cDNA library. Northern blot and in situ hybridization analyses demonstrated gelatinase A expression in developing tooth tissues. Immunohistochemical analysis localized gelatinase A close to the plasma membrane of these tissues. Furthermore, recombinant gelatinase A was demonstrated to cleave recombinant amelogenin into several fragments of differing molecular masses. Thus, gelatinase A is expressed by developing tooth tissues along with its activator MT1-MMP and may, therefore, play an important role during tooth development.

Ameloblasts↗

PubMed2001

Expression and regulation of MMP-20 in human tongue carcinoma cells.

Human matrix metalloproteinase-20 (MMP-20, enamelysin) fragments the enamel-specific protein amelogenin and has been shown to be synthesized exclusively by odontoblasts and ameloblasts and in certain odontogenic tumors. Here we demonstrate, for the first time, the expression of MMP-20 mRNA and protein in two carcinoma cell lines originating from the tongue. Treatment of the SCC-25 and HSC-3 cells with phorbol 12-myristate 13-acetate (10 nmol/L) up-regulated MMP-20 mRNA and protein expression by up to 1.6-fold, but transforming growth factor beta (10 ng/mL) had no effect. The latent proform of recombinant (r) human MMP-20 was converted by tumor-related trypsin-2. Activated rMMP-20 did not degrade type I or type II collagen, but efficiently hydrolyzed fibronectin, type IV collagen, laminin-1 and -5, tenascin-C, and beta-casein. This implies that MMP-20 not only participates in dental matrix remodeling but is also present in tongue carcinoma cells.

Amelogenin↗

PubMed2000

One-step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP-20).

A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP-20, enamelysin) was established by use of a solid-phase monoclonal antibody and a separate peroxidase-labeled monoclonal antibody. The EIA system was shown to be sensitive and quantitative for the detection of MMP-20. As little as 1.0 ng/ml (50 pg/assay) of MMP-20 protein could be reliably detected. The EIA system was linear over a range of 2.5-160 ng/ml (125-8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP-20, active MMP-20, and MMP-20 with COOH-terminal deletions. The EIA system was validated by the successful detection of MMP-20 in the culture medium of Chinese hamster ovary cells (CHO-K1) that were transfected with an MMP-20 expression vector. No MMP-20 was detected in normal human serum, normal saliva, or in selected tumors. However, when recombinant human MMP-20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP-20, indicating that the system will work within the framework of complex in biological fluids.

Adenocarcinoma↗

PubMed2000

Localization of EMSP1 expression during tooth formation and cloning of mouse cDNA.

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.

Amino Acid Sequence↗

PubMed2000

Immunohistochemical detection and distribution of enamelysin (MMP-20) in human odontogenic tumors.

Enamelysin is a tooth-specific protease that was initially isolated from porcine enamel organ and subsequently from human odontoblasts. Since this protease is thought to play important roles in tooth development, the evaluation of enamelysin in odontogenic tumors may aid our understanding of the histogenesis and cell differentiation of such lesions. A monoclonal antibody (203-1C7) was generated against synthesized human enamelysin oligopeptide and was used to assess the immunolocalization of enamelysin in healthy developing tooth germs and various types of odontogenic lesions. In tooth germs, enamelysin expression was detected only in the secretory enamel. Thus, 203-1C7 may serve as an enamel-specific marker in the late stage of enamel matrix development and calcification. In odontogenic lesions, strong enamelysin staining was demonstrated in the immature enamel matrix of ameloblastic fibro-odontomas and odontomas. Furthermore, enamelysin was also detected in globular amyloid masses and calcified foci in calcifying epithelial odontogenic tumors, hyaline droplets, small and large mineralized areas in adenomatoid odontogenic tumors, and a portion of ghost cells in calcifying odontogenic cysts. Positive reactivity was also observed in selected tumor cells in some of these tumors. No intracellular staining for enamelysin was detected in ameloblastomas or the ameloblastic portion of ameloblastic fibro-odontomas. Also, enamelysin was not detected in dentin, dysplastic dentinoid hyaline matrices, and cementum that were present within the tumors examined. Thus, taken together, our results suggest that the enamelysin-specific monoclonal antibody (203-1C7) may be utilized as a marker of early enamel development and that enamelysin may be involved in the pathogenesis of specific odontogenic tumors.

Ameloblastoma↗

PubMed2000

Cloning, characterization, and expression analysis of mouse enamelysin.

Enamelysin is a recently isolated member of the matrix metalloproteinase (MMP) family of extracellular matrix (ECM)-degrading enzymes. Here we describe the isolation and characterization of the mouse enamelysin cDNA. Expression of mouse enamelysin was detectable only in ameloblasts and odontoblasts of developing teeth. Characterization of mouse enamelysin demonstrated that it is highly conserved in both its sequence content and pattern of expression relative to the porcine, human, and bovine homologues previously described.

Amino Acid Sequence↗

PubMed1999

Isolation, characterization, and chromosomal location of the mouse enamelysin gene.

Mouse enamelysin (Mmp20), a member of the matrix metalloproteinase (MMP) family of extracellular matrix degrading enzymes, shows a high degree of homology with other MMPs, particularly those of the stromelysin/collagenase subfamilies. It is expressed exclusively in ameloblasts and odontoblasts. The mouse enamelysin gene (Mmp20) is made up of 10 exons spanning approximately 65 kb within the MMP gene cluster at the centromeric end of chromosome 9.

Animals↗

PubMed1999

Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins.

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.

Amelogenesis↗

PubMed1999

Proteinases in developing dental enamel.

For almost three decades, proteinases have been known to reside within developing dental enamel. However, identification and characterization of these proteinases have been slow and difficult, because they are present in very small quantities and they are difficult to purify directly from the mineralizing enamel. Enamel matrix proteins such as amelogenin, ameloblastin, and enamelin are cleaved by proteinases soon after they are secreted, and their cleavage products accumulate in the deeper, more mature enamel layers, while the full-length proteins are observed only at the surface. These results suggest that proteinases are necessary for "activating" enamel proteins so the parent proteins and their cleavage products may perform different functions. A novel matrix metalloproteinase named enamelysin (MMP-20) was recently cloned from tooth tissues and was later shown to localize primarily within the most recently formed enamel. Furthermore, recombinant porcine enamelysin was demonstrated to cleave recombinant porcine amelogenin at virtually all of the sites that have previously been described in vivo. Therefore, enamelysin is at least one enzyme that may be important during early enamel development. As enamel development progresses to the later stages, a profound decrease in the enamel protein content is observed. Proteinases have traditionally been assumed to degrade the organic matrix prior to its removal from the enamel. Recently, a novel serine proteinase named enamel matrix serine proteinase-1 (EMSP1) was cloned from enamel organ epithelia. EMSP1 localizes primarily to the early maturation stage enamel and may, therefore, be involved in the degradation of proteins prior to their removal from the maturing enamel. Other, as yet unidentified, proteinases and proteinase inhibitors are almost certainly present within the forming enamel and await discovery.

Amelogenesis↗

PubMed1999

The effect of diclofenac sodium on the initial comfort of RGP contact lenses: a pilot study.

BACKGROUND: We conducted a pilot study to assess the effect of 0.1% diclofenac sodium eyedrops on the initial comfort of rigid gas permeable (RGP) contact lenses. METHODS: A double-masked, placebo-controlled, crossover trial was performed on 20 unadapted subjects. In Phase I, subjects used either 0.1% diclofenac or placebo eyedrops four times per day for 3 days before RGP lenses were dispensed. Contact lenses were inserted on Day 4 and worn for 8 continuous hours. Eyedrops were used twice during the 8-hour period. Ocular symptoms were graded on a visual analog scale at six points during the day. Visual acuity and slitlamp examinations were performed at the same six study points. After a 2-week wash-out period, subjects received the opposite treatment in Phase II, following the same protocol used in Phase I. RESULTS: The treatment effect was tested using the Mainland-Gart chi-square test. There was no statistically significant treatment effect (chi-square = 0.20, p = 0.653) on patient symptoms. Biomicroscopy findings were analyzed and, likewise, showed no significant treatment effect (chi-square = 2.10, p = 0.15). CONCLUSIONS: Data from this pilot study showed no statistically significant reduction in symptoms or signs with diclofenac eyedrops.

Adult↗

PubMed1999

Central nervous system and plasma lipid profiles associated with carteolol and timolol in postmenopausal black women.

PURPOSE: Among ocular hypotensive agents, intrinsic sympathomimetic activity (ISA) is unique to carteolol hydrochloride. This study was conducted to evaluate the central nervous system (CNS) and plasma lipid profiles associated with timolol maleate and carteolol hydrochloride in postmenopausal black women with primary open-angle glaucoma (POAG) or ocular hypertension. METHODS: One hundred subjects met the inclusion and exclusion criteria for this randomized, double-masked, multicenter, parallel-group study. After completion of informed consent and complete ophthalmic examination, eligible patients entered a washout period, during which no topical ophthalmic medications were used. Blood samples were obtained for hematology and blood chemistry evaluations. Vital signs, ocular symptoms, Symptom Checklist-90-R (SCL-90-R) evaluation, intraocular pressure (IOP) measurements, and slit-lamp examinations were performed before randomization to treatment with either topical carteolol hydrochloride 1.0% or topical timolol maleate 0.5%. Patients received active medications twice daily and were monitored at 4 weeks and 12 weeks. At the conclusion of treatment, vital signs, ocular symptoms, SCL-90-R evaluation, IOP, slit-lamp examinations, and blood samples were obtained. RESULTS: Compared with baseline, high-density lipoprotein (HDL) cholesterol was significantly decreased (worsened) in the timolol group but did not change significantly in the carteolol group. The between-group difference was statistically significant. Total cholesterol to HDL ratio significantly increased (worsened) in the timolol group compared with baseline but did not change in the carteolol group. The difference between groups was statistically significant. No significant differences were observed between groups in SCL-90-R results for either somatization or depression. CONCLUSIONS: These results suggest that topical carteolol hydrochloride may have a more favorable blood lipid profile than topical timolol maleate in postmenopausal black women with POAG or ocular hypertension. Carteolol and timolol appear to have similar CNS side effect profiles.

Adrenergic beta-Antagonists↗

PubMed1998

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues.

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.

Ameloblasts↗

PubMed1998

Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation.

Dentin sialophosphoprotein [designated DSPP and cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP)], enamelysin and ameloblastin are each expressed in unique fashions during tooth development. It is possible that these components participate in cell differentiation and the conversion of unmineralized matrix into mineralized structures. In order to delineate the timing and the positioning of these three molecules in a physiological context, we compared their expression profiles by performing in situ hybridization experiments on consecutive sections in developing mouse tissues. Hybridization signals were uniquely detected for DSPP mRNA in odontoblasts and preameloblasts, for enamelysin mRNA in odontoblasts and in the facing ameloblast layer, and for ameloblastin mRNA in preodontoblasts, polarizing odontoblasts and ameloblasts. Immunohistochemistry showed that DSP and ameloblastin transcripts were translated into proteins that were deposited at the apical pole of the differentiated cells (odontoblasts and ameloblasts, respectively). The interrelated expression profiles found for these tooth-specific molecules illustrate the importance of a specific molecular network to initiate highly regulated processes such as cytodifferentiation and the subsequent mineralization.

Ameloblasts↗

PubMed1998

Purification, characterization, and cloning of enamel matrix serine proteinase 1.

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.

Amelogenesis↗

PubMed1998

Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis.

The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.

Ameloblasts↗