Tyrosinase Inhibition and Kinetic Details of Puerol A Having But-2-Enolide Structure from Amorpha fruticosa.

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 µM) and diphenolase (IC50 = 3.8 µM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 µM-1 min-1 and k4 = 0.003 min-1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 µM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 µM.

A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation.

Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with ß1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvß3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.

Dentin wear after simulated toothbrushing with water, a liquid dentifrice or a standard toothpaste.

PURPOSE: To investigate the influence of dentifrices with and without abrasives on the wear and surface topography of human dentin following simulated toothbrushing in vitro. METHODS: 24 dentin specimens were prepared and randomly allocated to a liquid dentifrice (Garglin Gum-Guard), conventional dentifrice (333 Clinic Total Care), and control (distilled water) groups. Specimens were subjected to simulated toothbrushing of 50,000 repeated strokes under a 150 g-load. The dentin surface was profiled in each specimen using a profilometer before and after toothbrushing. The mean surface roughness (Ra) of the specimens was calculated and compared by one-way ANOVA and Tukey's post-hoc test (α = 0.05). The dentin surfaces were further examined by scanning electron microscopy (SEM). RESULTS: The Ra values were similar between the liquid dentifrice and control groups (P > 0.05), and was significantly higher in the conventional dentifrice group (P < 0.001). On SEM examination, patent dentin tubules were observed in the conventional dentifrice and liquid dentifrice groups, but were not observed in the control group.

A transcriptional roadmap to the senescence and differentiation of human oral keratinocytes.

Human epithelial cells undergo morphological and molecular changes leading to terminal differentiation and replicative senescence after a finite number of cell divisions during serial subculture. However, the target genes and their functional significance in the senescence and differentiation in normal human oral keratinocytes have been poorly defined. Here, we demonstrated normal human oral keratinocytes transcriptional signature profiling to senescence and differentiation. Using microarray analysis, our findings indicated that the gene expression profiles induced by serial subculture are distinct classes of gene. The greatest number of these altered genes was identified as being related to biological pathways of transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response, suggesting that the serial subculture is able to induce a multitude of specific gene expression changes during senescence and differentiation. Several highly upregulated genes (IL-1ß, S100A8, S100A9, MMP1, MMP9, IL-8, BHLHB2, HES1, and TWIST1) in response to the serial subculture in normal human oral keratinocytes were observed. In vitro and in vivo studies also exhibited a close relationship between senescence and differentiation of primary oral keratinocytes and expression of inflammatory molecules. These results suggest a new approach to determine the biological events underlying the pathogenesis of oral keratinocyte aging.

Titanium surface coating with a laminin-derived functional peptide promotes bone cell adhesion.

Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration.

Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.

Protein kinase Cα/ß inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cδ.

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/ß. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 µM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/ß signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ.

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3ß1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3ß1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/ß inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3ß1 integrin, and that binding of α3ß1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells.

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo.

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.

Spontaneous tumorigenicity of primary human oral keratinocytes with human papillomavirus negativity and impaired apoptosis.

Although >60% of oral cancer cases are not related to human papillomavirus (HPV) infection, most studies of oral carcinogenesis in human cells in vitro are carried out with human oral keratinocytes immortalized by HPV DNA. To explore whether human oral keratinocytes can spontaneously transform without HPV infection, we attempted to establish spontaneously immortalized and tumorigenic-transformed human oral keratinocytes by serial subculture to the post-mitotic stage. Here we report two spontaneously transformed human oral keratinocyte lines from adult human gingival samples. These lines were obviously immortal (>140 passages) and transformed phenotypes in vitro. One of the lines, Spi-HOK1, remained non-tumorigenic in nude mice, whereas the other line, Spt-HOK80, showed tumorigenicity. These lines showed epithelial origi-nality, but did not contain high-risk types of HPV DNAs. On karyotyping, Spi-HOK1 was aneuploid with a unique stable marker chromosome. Both cell lines revealed a mutation in the p53 gene, loss of p21WAF1/Cip1 and overexpression of p-Rb-Ser807/811. These cell lines were resistant to cisplatin-induced apoptosis by suppressing induction of apoptotic proteins. These results clearly demonstrate that spontaneous immortalization and spontaneous tumorigenic transformation of primary human oral keratinocytes can occur in vitro without HPV infection and are associated with chromosomal alterations, p53 mutation and impaired apoptosis. To our knowledge, this is the first report demonstrating that the Spi-HOK1 and Spt-HOK80 lines are novel cell lines that are spontaneously transformed from primary human oral keratinocytes.

A biologically active sequence of the laminin alpha2 large globular 1 domain promotes cell adhesion through syndecan-1 by inducing phosphorylation and membrane localization of protein kinase Cdelta.

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 alpha2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin alpha2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cdelta (PKCdelta) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCdelta activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 alpha2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCdelta signaling pathway.

alpha3beta1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins.

Laminin-5 and alpha3beta1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/alpha3beta1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 alpha3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes, which is initiated by alpha3beta1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3zeta and sigma, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3zeta with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes is initiated by laminin-5 stimulation via the alpha3beta1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.

Plasma-treated silk fibroin nanofibers for skin regeneration.

Silk fibroin (SF) nanofibers were prepared by electrospinning and treated with plasma in the presence of oxygen or methane gas to modify their surface characteristics. The surface characteristics of the SF nanofibers after plasma treatment were examined using contact angle measurements and XPS analysis. The hydrophilicity of the electrospun SF nanofibers decreased slightly by the CH(4) plasma treatment. On the other hand, the hydrophilicity of the SF nanofibers increased greatly by an O(2) plasma treatment. The O(2)-treated SF nanofibers showed higher cellular activities for both normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) than the untreated ones.