Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

Prevention of brain injury by the nonbacteriolytic antibiotic daptomycin in experimental pneumococcal meningitis

Bacteriolytic antibiotics cause the release of bacterial components that augment the host inflammatory response, which in turn contributes to the pathophysiology of brain injury in bacterial meningitis. In the present study, antibiotic therapy with nonbacteriolytic daptomycin was compared with that of bacteriolytic ceftriaxone in experimental pneumococcal meningitis, and the treatments were evaluated for their effects on inflammation and brain injury. Eleven-day-old rats were injected intracisternally with 1.3 x 10(4) +/- 0.5 x 10(4) CFU of Streptococcus pneumoniae serotype 3 and randomized to therapy with ceftriaxone (100 mg/kg of body weight subcutaneously [s.c.]; n = 55) or daptomycin (50 mg/kg s.c.; n = 56) starting at 18 h after infection. The cerebrospinal fluid (CSF) was assessed for bacterial counts, matrix metalloproteinase-9 levels, and tumor necrosis factor alpha levels at different time intervals after infection. Cortical brain damage was evaluated at 40 h after infection. Daptomycin cleared the bacteria more efficiently from the CSF than ceftriaxone within 2 h after the initiation of therapy (log(10) 3.6 +/- 1.0 and log(10) 6.3 +/- 1.4 CFU/ml, respectively; P < 0.02); reduced the inflammatory host reaction, as assessed by the matrix metalloproteinase-9 concentration in CSF 40 h after infection (P < 0.005); and prevented the development of cortical injury (cortical injury present in 0/30 and 7/28 animals, respectively; P < 0.004). Compared to ceftriaxone, daptomycin cleared the bacteria from the CSF more rapidly and caused less CSF inflammation. This combined effect provides an explanation for the observation that daptomycin prevented the development of cortical brain injury in experimental pneumococcal meningitis. Further research is needed to investigate whether nonbacteriolytic antibiotic therapy with daptomycin represents an advantageous alternative over current bacteriolytic antibiotic therapies for the treatment of pneumococcal meningitis.

Processing of procollagen III by meprins: new players in extracellular matrix assembly?

Meprins ? and ?, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins ? and ? release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin ? is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin ? increases and meprin ? begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Characterization of granite matrix porosity and pore-space geometry by in situ and laboratory methods

Most available studies of interconnected matrix porosity of crystalline rocks are based on laboratory investigations; that is, work on samples that have undergone stress relaxation and were affected by drilling and sample preparation. The extrapolation of the results to in situ conditions is therefore associated with considerable uncertainty, and this was the motivation to conduct the ‘in situ Connected Porosity’ experiment at the Grimsel Test Site (Central Swiss Alps). An acrylic resin doped with fluorescent agents was used to impregnate the microporous granitic matrix in situ around an injection borehole, and samples were obtained by overcoring. The 3-D structure of the porespace, represented by microcracks, was studied by U-stage fluorescence microscopy. Petrophysical methods, including the determination of porosity, permeability and P -wave velocity, were also applied. Investigations were conducted both on samples that were impregnated in situ and on non-impregnated samples, so that natural features could be distinguished from artefacts. The investigated deformed granites display complex microcrack populations representing a polyphase deformation at varying conditions. The crack population is dominated by open cleavage cracks in mica and grain boundary cracks. The porosity of non-impregnated samples lies slightly above 1 per cent, which is 2–2.5 times higher than the in situ porosity obtained for impregnated samples. Measurements of seismic velocities (Vp ) on spherical rock samples as a function of confining pressure, spatial direction and water saturation for both non-impregnated and impregnated samples provide further constraints on the distinction between natural and induced crack types. The main conclusions are that (1) an interconnected network of microcracks exists in the whole granitic matrix, irrespective of the distance to ductile and brittle shear zones, and (2) conventional laboratory methods overestimate the matrix porosity. Calculations of contaminant transport through fractured media often rely on matrix diffusion as a retardation mechanism.

Diagnosing Organizational Identity Beliefs by Eliciting Complex, Multi-Modal Metaphors

The purpose of this article is to extend the organizational development diagnostics repertoire by advancing an approach that surfaces organizational identity beliefs through the elicitation of complex, multimodal metaphors by organizational members. We illustrate the use of such "Type IV" metaphors in a postmerger context, in which individuals sought to make sense of the implications of the merger process for the identity of their organization. This approach contributes to both constructive and discursive new organizational development approaches; and offers a multimodal way of researching organizational identity that goes beyond the dominant, mainly textual modality.

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.