Streptococcus mutans is a recognized principal etiologic agent in coronal caries. Although S. mutans has the ability to bind collagen and degrade FALGPA, a synthetic peptide mimicking collagen substrate, its role in dental root caries has not yet been fully elucidated. Degradation of collagen fibrils in dentin was attributed to S. mutans, but a collagenase enzyme has not yet been isolated from this organism. Considering the increased incidence of dental root decay among the elderly, an understanding of the role of the pathogenic factors is necessary to the development of preventive measures. The present study has focused on the cloning and analysis of S. mutans collagenase enzyme. Toward this goal, a putative collagenase gene was identified in S. mutans UA159 by genomic analysis and a primer set was designed and used to amplify the corresponding gene in S. mutans GS-5 used as a model organism. The PCR product was cloned into the vector pCR 2.1 TOPO-TA, and the gene sequenced and analyzed. Alignment of the S. mutans GS-5 and UA159 putative collagenase genes showed 99% homology. The gene was next cloned in frame into the inducible expression vector pET100/D TOPO. Induction and expression of recombinant protein in E. coli were confirmed by SDS-PAGE and Western immunoblotting, while biochemical analysis indicated that it was a calcium- dependent metalloproteinase. Enzyme analysis of the recombinant enzyme showed both gelatinolytic and collagenolytic activity. Further analysis of the GS5 gene using databases such as ExPASy, Pfam, and SMART indicated that it was highly homologous to the U32 peptidase family, which includes the PrtC collagenase of Porphyromonas gingivalis, a bacterium causing periodontitis. The present study was the first to unequivocally demonstrate the existence of a collagenase gene in S. mutans, and to identify it as a member of the U32 peptidase family. The obtaining of the S. mutans collagenase gene should help in further investigation of the role of this enzyme in dental root decay and its potential use as a dental root caries vaccine.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.

Flexor tendon injuries in zone II of the hand (i.e. between the distal volar crease and the distal interphalangeal joint) can be costly for both the afflicted individual and society because of the high cost of a long rehabilitation period, complicated by tendon ruptures or scarring with adhesion formation, causing impaired range of motion. The aim of the present thesis was to characterize more fully the deep flexor tendon, the tendon sheath and their response to injury in a rabbit model in order to find potential targets to improve the outcome of repair. The intrasynovial rabbit deep flexor tendon differed from the extrasynovial peroneus tendon in the expression of collagens and transforming growth factor-β1 gene expression. Differences were also found in collagen III and proteoglycans between regions of the flexor tendon subjected to either compressive or tensile load. After laceration and subsequent repair of the flexor tendon, a shift in collagen gene expression from type I to type III occurred. Proteoglycans were generally increased with the notable exception of decorin, a potential inhibitor of the profibrotic transforming growth factor-β1 which was markedly increased during the first two weeks after repair in tendon tissue but remained unaltered in the sheaths. Both vascular endothelial growth factor and basic fibroblast growth factor mRNA levels remained essentially unaltered, whereas insulin-like growth factor-1 increased later in the healing process, suggesting potential beneficial effects of exogenous addition, increasing tendon strength through stimulating tenocyte proliferation and collagen synthesis. Matrix metalloproteinase-13 mRNA levels increased and remained high in both tendon and sheath, whereas there was only a transient increase of matrix metalloproteinase-3 mRNA in tendon. We could also demonstrate a significant increase of the proportion of myofibroblasts, mast cells and neuropeptide containing nerve fibers in the healing tendon tissue, all components of the profibrotic myofibroblast-mast cell-neuropeptide pathway. / Biomolecular aspects of flexor tendon healing

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.<p> Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.

Three protease inhibitor-like proteins (PILPs) identified from Bungarus multicinctus genome are structurally homologous with Kunitz-type proteinase inhibitor. The goal of the present study is to explore whether PILPs exhibit an inhibitory action on matrix metalloproteinase 2 (MMP-2) activity. Unlike PILP-1 and PILP-2, PILP-3 was found to inhibit MMP-2 activity as evidenced by specific substrate assay. Moreover, in vitro migration and invasion assays, and wound-healing assay showed that PILP-3 suppressed the migration and invasion of human neuroblastoma SK-N-SH cells. Pull-down assay and dot blotting-binding assay proved an interaction between PILP-3 and MMP-2. Nevertheless, PILP-3 did not affect either expression or secretion of MMP-2 in SK-N-SH cells. In terms of highly structural similarity between PILP-2 and PILP-3, two chimeric mutants in which amino acids at N-terminus and C-terminus of PILP-3 were substituted by those of PILP-2 were prepared. In contrast to N-terminus chimera, C-terminus mutant of PILP-3 was unable to inhibit MMP-2 activity and showed a reduction in binding with MMP-2. Taken together, our data suggest that PILP-3 may be a useful template for rational designing pharmaceutical agent in inhibiting MMP-2 activity.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 62-77).

Reproduction de : Thèse doctorat : Pharmacie.Biochimie-Biologie moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliogr. p.186-218.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliogr.

Familial history is the strongest risk factor for developing ovarian cancer (OC), and a significant contributor to breast cancer risk. Most hereditary breast cancers and OCs are associated with mutation of the tumor suppressor Breast and Ovarian Cancer Susceptibility Gene 1 (BRCA1). Studying risk-associated BRCA1 truncation mutations, such as the founder mutation 185delAG, may reveal signaling pathways important in OC etiology. Recent studies have shown novel BRCA1 mutant functions that may contribute to breast and OC initiation and progression independent of the loss of wtBRCA1. Previously, we have found that normal human ovarian surface epithelial (HOSE) cells expressing the 185delAG mutant, BRAT ( BRCA1 185delAG Amino Terminal truncated protein), exhibit enhanced chemosensitivity and up-regulation of the OC-associated serpin, maspin. In the current study, I identify an additional target of the BRAT mutation, matrix metalloprotease 1 (MMP1), a key player in invasion and metastasis. BRAT-expressing HOSE cells exhibit increased MMP1 messenger RNA (mRNA) by real time PCR and protein by Western blotting. Pro-MMP1 levels are also higher in conditioned media of BRAT-expressing cells and HOSE cell lines derived from BRAT mutation carriers. c-Jun is critical for BRAT-mediated MMP1 up-regulation, as siRNA knockdown diminishes MMP1 levels. Luciferase reporter constructs reveal that activator Protein 1 (AP1) sites throughout the distal end of the promoter contribute to BRAT-mediated MMP1 expression, and basal activity is mediated in part by an AP1 site at (-72). Reporters containing a single nucleotide polymorphism (SNP) associated with OC risk and progression yield increased activity that is further enhanced in BRAT cells. Interestingly, BRAT-mediated changes in chemosensitivity and gene regulation are not recapitulated in a normal breast epithelial or breast cancer cell model. This suggests tissue-specific mutant BRCA1 functions may contribute to breast and ovarian tissue specificity of BRCA1 mutation-associated cancer risk and also to differential breast and ovarian cancer risk and penetrance associated with specific mutations. Early molecular and cellular changes such as MMP1 up-regulation in the ovarian surface epithelium of BRCA1 mutation carriers may promote OC initiation and progression and represent a step forward on the continuum of cellular malignancy. Further investigation is warranted, as elucidating these early changes will aid in identification of potential screening and treatment strategies.