Biomedical applications of nisin.

Nisin is a bacteriocin produced by a group of Gram-positive bacteria that belongs to Lactococcus and Streptococcus species. Nisin is classified as a Type A (I) lantibiotic that is synthesized from mRNA and the translated peptide contains several unusual amino acids due to post-translational modifications. Over the past few decades, nisin has been used widely as a food biopreservative. Since then, many natural and genetically modified variants of nisin have been identified and studied for their unique antimicrobial properties. Nisin is FDA approved and generally regarded as a safe peptide with recognized potential for clinical use. Over the past two decades the application of nisin has been extended to biomedical fields. Studies have reported that nisin can prevent the growth of drug-resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Enterococci and Clostridium difficile. Nisin has now been shown to have antimicrobial activity against both Gram-positive and Gram-negative disease-associated pathogens. Nisin has been reported to have anti-biofilm properties and can work synergistically in combination with conventional therapeutic drugs. In addition, like host-defence peptides, nisin may activate the adaptive immune response and have an immunomodulatory role. Increasing evidence indicates that nisin can influence the growth of tumours and exhibit selective cytotoxicity towards cancer cells. Collectively, the application of nisin has advanced beyond its role as a food biopreservative. Thus, this review will describe and compare studies on nisin and provide insight into its future biomedical applications.

Anoikis mediators in oral squamous cell carcinoma.

Anoikis - apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts - is dysregulated in many chronic debilitating and fatal diseases. Mechanisms rendering tumor cells resistant to anoikis, although not completely understood, possess significant therapeutic promise. In death receptor-mediated anoikis mechanisms, focal adhesion kinase (FAK) and receptor-interacting protein (RIP) dissociate, leading to association of RIP with Fas, formation of the death-inducing signaling complex (DISC), activation of caspase-3, and propagation of anoikis. In contrast, anoikis resistance is accomplished through constitutive activation of survival pathways that include integrin-dependent activation of FAK and extracellular-signal-regulated kinase (ERK). In addition, FAK and RIP association confers anoikis resistance by inhibiting the association of RIP with Fas and formation of the death signaling complex, which allows cells to escape anoikis. Up-regulation of CD44 also contributes to survival signals and promotes anoikis resistance. This review will focus on the roles of death receptors, prosurvival pathways, and the molecular players involved in anoikis escalation and resistance in oral squamous cell carcinoma.

Proapoptotic fibronectin fragment induces the degradation of ubiquitinated p53 via proteasomes in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: The extracellular matrix (ECM) plays a key role in signaling necessary for tissue remodeling and cell survival. However, signals from the ECM altered by disease, e.g. inflammatory diseases such as periodontitis and arthritis, may lead to apoptosis or programmed cell death of resident cells. Previously, we found that a disease-associated fibronectin fragment triggers apoptosis of primary human periodontal ligament cells via a novel apoptotic pathway in which the tumor suppressor, p53, is transcriptionally downregulated. MATERIAL AND METHODS: We used immunofluorescence, transfection assays, western blotting and ELISAs to show that p53 is degraded by a proteasomal pathway in response to a proapoptotic disease-associated fibronectin fragment. RESULTS: We found that in these apoptotic conditions, p53 is further downregulated by post-translational ubiquitination and subsequent targeting to proteasomes for degradation. Pretreatment of cells with the proteasomal inhibitors MG132 and lactacystin rescued the cells from apoptosis. The p53 levels in cells transfected with ubiquitin small interfering RNA were resistant to degradation induced by the proapoptotic fibronectin fragment, showing that ubiquitination is important for the proapoptotic fibronectin fragment-induced degradation of p53. CONCLUSION: These data show that a proapoptotic fibronectin matrix induces ubiquitination and degradation of p53 in the proteasome as part of a novel mechanism of apoptosis associated with inflammatory diseases.

TNF-alpha promotes an odontoblastic phenotype in dental pulp cells.

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

NG2, a novel proapoptotic receptor, opposes integrin alpha4 to mediate anoikis through PKCalpha-dependent suppression of FAK phosphorylation.

Disruption of cell-matrix interactions can lead to anoikis - apoptosis due to loss of matrix contacts. Altered fibronectin (FN) induces anoikis of primary human fibroblasts by a novel signaling pathway characterized by reduced phosphorylation of focal adhesion kinase (FAK). However, the receptors involved are unknown. FAK phosphorylation is regulated by nerve/glial antigen 2 (NG2) receptor signaling through PKCalpha a point at which signals from integrins and proteoglycans may converge. We found that an altered FN matrix induced anoikis in fibroblasts by upregulating NG2 and downregulating integrin alpha4. Suppressing NG2 expression or overexpressing alpha4 rescued cells from anoikis. NG2 overexpression alone induced apoptosis and, by reducing FAK phosphorylation, increased anoikis induced by an altered matrix. NG2 overexpression or an altered matrix also suppressed PKCalpha expression, but overexpressing integrin alpha4 enhanced FAK phosphorylation independently of PKCalpha. Cotransfection with NG2 cDNA and integrin alpha4 siRNA did not lower PKCalpha and pFAK levels more than transfection with either alone. PKCalpha was upstream of FAK phosphorylation, as silencing PKCalpha decreased FAK phosphorylation. PKCalpha overexpression reversed this behavior and rescued cells from anoikis. Thus, NG2 is a novel proapoptotic receptor, and NG2 and integrin alpha4 oppositely regulate anoikis in fibroblasts. NG2 and integrin alpha4 regulate FAK phosphorylation by PKCalpha-dependent and -independent pathways, respectively.

Disease-associated fibronectin matrix fragments trigger anoikis of human primary ligament cells: p53 and c-myc are suppressed.

Inflammation in periodontal disease is characterized by the breakdown of the extracellular matrix. This study shows that an inflammation-associated matrix breakdown fragment of fibronectin (FN) induces anoikis of human periodontal ligament (PDL) cells. This 40 kDa fragment was identified in human inflammatory crevicular fluid and is associated with disease status. Previously, we reported that a similar recombinant FN fragment triggered apoptosis of PDL cells by an alternate apoptotic signaling pathway that requires transcriptional downregulation of p53 and c-myc. Thus, to determine whether the physiologically relevant 40 kDa fragment triggers apoptosis in these cells, the 40 kDa fragment was generated and studied for its apoptotic properties. The 40 kDa fragment induces apoptosis of PDL cells, and preincubation of cells with intact vitronectin, FN, and to a limited extent collagen I, rescue this apoptotic phenotype. These data suggest that the 40 kDa fragment prevents PDL cell spreading, thereby inducing anoikis. The signaling pathway also involves a downregulation in p53 and c-myc, as determined by Western blotting and real time quantitative PCR. These data indicate that an altered FN matrix as is elaborated in inflammation induces anoikis of resident cells and thus may contribute to disease progression.

Coordinate induction of collagenase-1, stromelysin-1 and urokinase plasminogen activator (uPA) by the 120-kDa cell-binding fibronectin fragment in fibrocartilaginous cells: uPA contributes to activation of procollagenase-1.

Specific fibronectin (Fn) fragments found in synovial fluid of arthritic joints potentially contribute to the loss of cartilage proteoglycans by inducing matrix metalloproteinase (MMP) expression. However, whether or not the Fn fragment-modulated changes in expression of MMPs result in a net increase in matrix-degradative activity through alterations in the balance between MMP activation and inhibition has not been established. To understand the mechanisms by which proteolytic Fn fragments may contribute to joint degeneration, conditioned medium from fibrocartilaginous cells exposed to Fn, its 30-kDa fragment containing the collagen/gelatin-binding domain, its 120-kDa fragment containing the central cell-binding domain, and the RGD peptide were assayed for MMPs, and MMP activators and inhibitors. We found that the 120-kDa fragment of Fn (but not intact Fn), the 30-kDa fragment, and the RGD peptide, dose-dependently induced procollagenase-1 and prostromelysin-1 and decreased levels of the tissue inhibitor of metalloproteinases (TIMPs) -1 and -2. The alpha5beta1 integrin was implicated in the induction of collagenase by the 120-kDa Fn fragment, since collagenase induction was abrogated in the presence of blocking antibody to this integrin. Conditioned medium from cells exposed to the 120-kDa Fn fragment also demonstrated increased levels of the activated collagenase-1, which resulted in significantly elevated collagen degradative activity. That the urokinase plasminogen activator (uPA) was involved in the activation of procollagenase-1 was suggested by findings that the 120-kDa Fn fragment induced uPA coordinately with procollagenase-1, and the activation of procollagenase-1 was dose-dependently inhibited in the presence of plasminogen activator inhibitor-1. These data demonstrate that the 120-kDa cell-binding fragment of Fn induces a net increase in matrix-degradative activity in fibrocartilaginous cells by concomitantly inducing MMPs and their activator, uPA, while decreasing TIMPs.

Mutations in the heparin binding domain of fibronectin in cooperation with the V region induce decreases in pp125(FAK) levels plus proteoglycan-mediated apoptosis via caspases.

Intact fibronectin (FN) protects cells from apoptosis. When FN is fragmented, specific domains induce proteinase expression in fibroblasts. However, it is not known whether specific domains of FN can also regulate apoptosis. We exposed fibroblasts to four recombinant FN fragments and then assayed for apoptosis using criteria of cellular shape change, condensed nuclear morphology, and DNA fragmentation. The fragments extended from the RGD-containing repeat III10 to III15; they included (V(+)) or excluded (V(-)) the alternatively spliced V region and contained either a mutated (H(-)) or an unmutated (H(+)) heparin binding domain. Only the V(+)H(-) fragment triggered decreases in pp125(FAK) levels and apoptosis, which was rescued by intact FN and inhibitors of caspase-1 and caspase-3. This apoptotic mechanism was mediated by a chondroitin sulfate proteoglycan, since treating cells with chondroitin sulfate or chondroitinase reversed the apoptotic cell shape changes. The alpha4 integrin receptor may also be involved, since using a blocking antibody to alpha4 alone induced apoptotic cell shape changes, whereas co-treatment with this antibody plus V(+)H(+) reversed these effects. These results demonstrate that the V and heparin binding domains of FN modulate pp125(FAK) levels and regulate apoptosis through a chondroitin sulfate proteoglycan- and possibly alpha4 integrin-mediated pathway, which triggers a caspase cascade.

The response of periodontal ligament cells to fibronectin.

Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.

The high affinity heparin-binding domain and the V region of fibronectin mediate invasion of human oral squamous cell carcinoma cells in vitro.

Fibronectin is an extracellular matrix molecule composed of repeating subunits that create functional domains. These domains contain multiple binding sites for heparin and for various cell-surface receptors that modulate cell function. To examine the role that the high affinity heparin-binding region and the alternatively spliced V region of fibronectin play in tumor invasion, we expressed and purified four complementary recombinant fibronectin proteins. These proteins either included or excluded the alternatively spliced V region and contained either a mutated, non-functional high affinity heparin-binding domain (Hep-) or an unmutated heparin-binding domain (Hep+). Cultured oral squamous cell carcinoma cells were assayed for invasion into a Matrigel/collagen matrix supplemented with these four purified recombinant proteins, and for spreading and motility on plastic. Increased invasion was observed in gels supplemented with the V-Hep+ protein when compared with the V-Hep- protein. Inclusion of the V region in the proteins enhanced the invasion and migration associated with both Hep+ and Hep- proteins, whereas cell spreading was enhanced with the Hep+ recombinant proteins. These data demonstrate that both the high affinity heparin-binding domain and the V region of fibronectin play important roles in invasion, motility, and spreading of oral squamous cell carcinoma cells.

Cocaine-associated rapid gingival recession and dental erosion. A case report.

This case report chronicles the clinical presentation and unusual response to treatment of a patient with rapid gingival recession and dental erosion secondary to local cocaine application. The initial clinical diagnosis was necrotizing ulcerative periodontitis; only after several years of therapy did the patient voluntarily inform one of the therapists that cocaine had been regularly applied to the affected gingival sites. This case illustrates the importance of including cocaine application to gingival tissues in a differential diagnosis in cases of rapid gingival recession and dental erosion of unknown etiology.

Fibronectin and fibronectin fragments modulate the expression of proteinases and proteinase inhibitors in human periodontal ligament cells.

Fragments of the matrix molecule fibronectin (FN) have been shown to modulate tissue remodeling activity by inducing matrix metalloproteinases (MMPs) in synovial fibroblasts. These molecules could contribute to the tissue degradation that occurs during periodontal disease if they also modulate the expression of proteinases in cells of the periodontal ligament (PDL). We tested the hypothesis that FN and specific FN fragments induce the expression of specific proteinases in PDL cells. Using substrate zymograms, reverse zymograms and Western immunoblots, we found that PDL cells constitutively express 72 kDa gelatinase, urokinase-type plasminogen activator (uPA) and at least three inhibitors whose molecular masses correspond to those of the tissue inhibitors of metalloproteinases (TIMPs). A fourth, previously uncharacterized, proteinase inhibitor of approximately 22 kDa was also observed in some cell isolates. PDL cells, when exposed to a 120 kDa proteolytic FN fragment containing the cell-binding domain, were induced to express collagenase and stromelysin and also demonstrated an increased secretion of the serine proteinase uPA. Expression of collagenase increased with increasing concentrations (0.001 microM-1 microM) of the 120 kDa FN fragment. This fragment also induced the expression of a 20 kDa inhibitor, but not of the higher-molecular-mass inhibitors, in PDL cells. The observed alterations in proteinases were associated specifically with the 120 kDa FN fragment, since similar responses were not seen when PDL cells were exposed to either a 60 kDa heparin-binding FN fragment or a 45 kDa collagen/gelatin-binding FN fragment. PDL cells exposed to intact FN did not express the proteinases induced by the 120 kDa fragment but did express 92 kDa gelatinase and the 20 kDa proteinase inhibitor. These data suggest that FN and specific FN fragments can differentially induce the expression of proteinases in PDL cells. Thus, functional regions of FN may modulate many of the functions of PDL cells that contribute to periodontal disease, wound healing and maintenance of extracellular matrix in periodontal tissues.