Dentin slice model of dental stem cells in a fibrin-agarose construct for dental pulp regeneration / Modelo de trozo de dentina de células madre dentales en una construcción de fibrina-agarosa para la regeneración de la pulpa dental

Objectives: To implement a dentin slice model of mesenchymal stem cells derived from dental tissues in a fibrin-agarose construct for dental pulp regeneration. Material and Methods: MSCs derived from different oral cavity tissues were combined with a fibrin-agarose construct at standard culture conditions. Cell viability and proliferation tests were assayed using a fluorescent cell dye Calcein/Am and WST-1 kit. The proliferation assay was evaluated at 24, 48, 72, and 96 hours. Also, we assessed the dental pulp stem cells (DPSCs) cell morphology inside the construct with histological stains such as Hematoxylin and Eosin, Masson's trichrome, and Periodic acid­Schiff. In addition, we elaborated a tooth dentin slice model using a culture of DPSC in the fibrin­agarose constructs co-adhered to dentin walls. Results: The fibrin-agarose construct was a biocompatible material for MSCs derived from dental tissues. It provided good conditions for MSCs' viability and proliferation. DPSCs proliferated better than the other MSCs, but the data did not show significant differences. The morphology of DPSCs inside the construct was like free cells. The dentin slice model was suitable for DPSCs in the fibrin-agarose construct. Conclusion: Our findings support the dentin slice model for future biological use of fibrin-agarose matrix in combination with DPSCs and their potential use in dental regeneration. The multipotency, high proliferation rates, and easy obtaining of the DPSCs make them an attractive source of MSCs for tissue regeneration.

Objetivos: Implementar un modelo de dentina con células madre mesenquimales derivadas de tejidos dentales en una constructo de fibrina-agarosa para la regeneración de la pulpa dental. Material y Métodos: Las MSC derivadas de diferentes tejidos de la cavidad oral se combinaron con una construcción de fibrina-agarosa en condiciones de cultivo estándar. Las pruebas de viabilidad y proliferación celular se ensayaron utilizando un kit de colorante celular fluorescente Calcein/Am y WST-1. El ensayo de proliferación se evaluó a las 24, 48, 72 y 96 horas. Además, evaluamos la morfología celular de las células madre de la pulpa dental (DPSC) dentro de la construcción con tinciones histológicas como hematoxilina y eosina, tricrómico de Masson y ácido peryódico de Schiff. Además, elaboramos un modelo de rebanadas de dentina dental utilizando un cultivo de DPSC en las construcciones de fibrina-agarosa coadheridas a las paredes de la dentina. Resultados: La construcción de fibrina-agarosa fue un material biocompatible para las MSC derivadas de tejidos dentales. Proporcionó buenas condiciones para la viabilidad y proliferación de las MSC. Las DPSC proliferaron mejor que las otras MSC, pero los datos no mostraron diferencias significativas. La morfología de las DPSC dentro de la construcción era como la de las células libres. El modelo de corte de dentina fue adecuado para DPSC en la construcción de fibrina-agarosa.Conclusión: Nuestros hallazgos respaldan el modelo de corte de dentina para el futuro uso biológico de la matriz de fibrina-agarosa en combinación con DPSC y su uso potencial en la regeneración dental. El multipotencial, las altas tasas de proliferación y la fácil obtención de las DPSC las convierten en una fuente atractiva de MSC para la regeneración de tejidos.

Purification and characterization of phytase with a wide pH adaptation from common edible mushroom Volvariella volvacea (Straw mushroom).

A novel phytase with a molecular mass of 14 kDa was isolated from fresh fruiting bodies of the common edible mushroom Volvariella volvacea (Straw mushroom). The isolation procedure involved successive chromatography on DEAE-cellulose, CM-cellulose, Affi-gel blue gel, Q-Sepharose and Superdex-75. The enzyme was a monomeric protein and was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, but was adsorbed on Q-Sepharose. The enzyme was purified 51.6-fold from the crude extract with 25.9% yield. Its N-terminal amino acid sequence GEDNEHDTQA exhibited low homology to the other reported phytases. The optimal pH and temperature of the purified enzyme was 5 and 45oC, respectively. The enzyme was quite stable over the pH range of 3.0 to 9.0 with less than 30% change in its activity, suggesting that it can be used in a very wide pH range. The enzyme exhibited broad substrate selectivity towards various phosphorylated compounds, but lacked antifungal activity against tested plant pathogens.

Disease-specific Proteins from Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a chronic inflammatiory disease that mainly destroys cartilages or bones at the joints. This inflammatory disorder is initiated by self-attack using own immune system, but the detail of pathological mechanism is unclear. Features of autoantigens leading to autoimmune disease are also under veil although several candidates including type II collagen have been suggested to play a role in pathogenesis. In this report, we tried to identify proteins responding to antibodies purified from RA patients and screen proteins up-regulated or down-regulated in RA using proteomic approach. Fibronectin, semaphorin 7A precursor, growth factor binding protein 7 (GRB7), and immunoglobulin mu chain were specifically associated with antibodies isolated from RA synovial fluids. In addition, some metabolic proteins such as adipocyte fatty acid binding protein, galectin-1 and apolipoprotein A1 precursor were overexpressed in RA synovium. Also, expression of peroxiredoxin 2 was up-regulated in RA. On the contrary, expression of vimentin was severely suppressed in RA synoviocytes. Such findings might give some insights into understanding of pathological mechanism in RA.

Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle.

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.

Biosorption and elution of chromium from immobilized Bacillus coagulans biomass.

Bacillus coagulans, a tannery wastewater isolate, previously shown to bind dissolved Cr(VI), retained its ability to biosorb Cr(VI) in different matrices. Polymeric materials like agar, agarose, calcium alginate and polyacrylamide were screened. Agarose emerged as the suitable candidate for biomass immobilization mainly due to its higher stability and integrity in acidic pH. Aptness of agarose as the matrix for B. coagulans biomass was revealed during Cr(VI) biosorption from natural wastewater.

Evidence for clustered mannose as a new ligand for hyaluronan- binding protein (HABP1) from human fibroblasts.

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.

An enzymatic method for the detection of human serum albumin

Albumin is the most abundant protein in human serum. A dye-binding method is commonly used in clinical laboratories for its estimation using different types of dyes. However, all these dye methods were interfered by a variety of compounds. Here we present a method for the detection of albumin in human serum and other biological fluids. The principle is based on the fact that lactate dehydrogenase isoenzyme-5 (LDH-5) binds specifically to Dextran-Blue (DB). Albumin inhibits the binding of LDH-5 with DB. Absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa.