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Grimontia hollisae collagenase (Ghcol) exhibits high collagen‐degrading activity. To explore its catalytic mechanism, its substrate (Gly‐Pro‐Hyp‐Gly‐Pro‐Hyp, GPOGPO)‐complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active‐site zinc ion. Since this water was not observed in the product (GPO)‐complexed Ghcol, it was hypothesized that the GPOGPO‐complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active‐site geometry and site‐directed mutagenesis of the active‐site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3‐L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3‐L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3‐L1 cell migration via stimulating the nuclear translocation of yes‐associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high‐fat diet fed mice. Increased expression of YAP in the cells cultured on col I‐coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small‐interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro. [ABSTRACT FROM AUTHOR]

Amylin and amyloid-β (Aβ) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic β-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and β (ERα and ERβ) and generation of ROS/RNS in amylin/Aβ1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aβ1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aβ1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aβ1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aβ1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aβ1-42 -treated INS-1 cells. [ABSTRACT FROM AUTHOR]

The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C‐terminal segment that includes the bacterial pre‐peptidase C‐terminal domain. Here, we produced a recombinant C‐terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen‐binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C‐terminal segment binds to collagen only when the collagen is in its triple‐helical conformation. Moreover, the C‐terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C‐terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate‐labeled type I collagen to show that the C‐terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Collagen peptides have been widely used as a food supplement. After ingestion of collagen peptides, oligopeptides containing hydroxyproline (Hyp), which are known to have some physiological activities, are detected in peripheral blood. However, the effects of collagen‐peptide administration on immune response are unclear. In the present study, we tested the effects of collagen‐peptide ingestion on allergic response and the effects of collagen‐derived oligopeptides on CD4+ T‐cell differentiation. Methods: BALB/c mice fed a collagen‐peptide diet were immunized with ovalbumin (OVA), and their serum IgE and IgG levels, active cutaneous anaphylaxis, and cytokine secretion by splenocytes were examined. Naive CD4+ T cells were stimulated with anti‐CD3 and anti‐CD28 in the presence of collagen‐derived oligopeptides, and the expression of IFN‐γ, IL‐4, and Foxp3 was analyzed. Results: In an active anaphylaxis model, oral administration of collagen peptides suppressed serum OVA‐specific immunoglobulin E (IgE) production and diminished anaphylaxis responses. In this model, the ingestion of collagen peptides skewed the pattern of cytokine production by splenocytes toward T‐helper (Th) type 1 and regulatory T (Treg) cells. In vitro T‐helper cell differentiation assays showed that Hyp‐containing oligopeptides promoted Th1 differentiation by upregulating IFN‐γ‐induced signal transducer and activator of transcription 1 (STAT1) signaling. These oligopeptides also promoted the development of Foxp3+ Treg cells in response to antigen stimulation in the presence of TGF‐β. Conclusions: Collagen‐peptide ingestion suppresses allergic responses by skewing the balance of CD4+ T cells toward Th1 and Treg cells and seems to be a promising agent for preventing allergies and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3049-3055, 2014. [ABSTRACT FROM AUTHOR]

Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1737-1743, 2014. [ABSTRACT FROM AUTHOR]

ABSTRACT Limited information is available about conformational differences between the abnormal isoform of prion protein (PrPSc) and cellular prion protein (PrPC) under native conditions. To clarify conformational differences between these two isoforms, PrP-deficient mice were immunized with brain homogenates of normal and scrapie-infected animals. All mice generated anti-PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrPSc consisting of QGSPGGN (PrP41-47) at the N-terminus. This study demonstrated a conformational dissimilarity at the N-terminus between PrPSc and PrPC, a finding that may provide novel information about conformational features of PrPSc. [ABSTRACT FROM AUTHOR]

To examine the sensitivity of a commercially available bovine spongiform encephalopathy ( BSE) kit ( NippIBL) for the detection of ovine scrapie, 50 scrapie-positive ovine samples from the UK, and 54 scrapie-negative ovine samples from Japan were obtain and tested using this kit. The sensitivity and specificity of NippIBL for ovine samples were 96% and 100%, respectively. The detection limit of the abnormal isoform of prion protein ( PrPSc) of NippIBL was examined using diluted scrapie-positive samples. The sensitivity of NippIBL to ovine scrapie was 3-10 times superior to that of another commercial BSE diagnosis kit. Thus, the NippIBL kit proved more effective for the detection of ovine scrapie. [ABSTRACT FROM AUTHOR]

To investigate the pathophysiological roles of matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinases (TIMP)-1 in the differentiation of keratinocytes, we used human keratinocytes in culture to examine the gelatinolytic activity of MMP-2 and MMP-9 by zymographic analysis, MMP-9 mRNA by reverse transcriptase-polymerase chain reaction, and TIMP-1 mRNA by Northern blot analysis. Adding of calcium (1 mM) to the culture medium increased the activity of MMP-9 and MMP-9 mRNA without affecting MMP-2 activity or the expression of TIMP-1 mRNA. These results suggest that keratinocytes regulate the expressions of MMP-9 and TIMP-1 differently during the process of differentiation. [ABSTRACT FROM AUTHOR]