The in vitro analysis of migration and polarity of blastema cells in the extracellular matrix derived from bovine mesenteric in the presence of fibronectin / 대한해부학회지

Cell migration is an essential process in embryonic development, wound healing, and pathological conditions. Our knowledge of cell migration is often based on the two dimentional evaluation of cell movement, which usually differs from what occurred in vivo. In this study, we investigated cellular migration from blastema tissue toward bovine decellularized mesentery tissue. In this regard, fibronectin (FN) was assessed to confirm cell migration. Therefore, we established a cell migration model using blastema cells migration toward the extracellular matrix derived from bovine mesenteric tissue. A physiochemical decellularization method was utilized based on freeze-thaw cycles and agitation in sodium dodecyl sulfate and Triton X-100 to remove cells from the extracellular matrix (ECM) of bovine mesenteric tissue. These types of matrices were assembled by the rings of blastema tissues originated from the of New Zealand rabbits pinna and cultured in a medium containing FN in different days in vitro, and then they are histologically evaluated, and the expression of the Tenascin C gene is analyzed. By means of tissue staining and after confirmation of the cell removal from mesenteric tissue, polarity, and migration of blastema cells was observed in the interaction site with this matrix. Also, the expression of the Tenascin C gene was assessed on days 15 and 21 following the cell culture process. The results showed that the three dimentional model of cellular migration of blastema cells along with the ECM could be a suitable model for investigating cell behaviors, such as polarity and cell migration in vitro.

Efficient media for high lipase production: one variable at a time approach

Background: lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using "one variable at a time" strategy

Methods: for this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated

Results: maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup [1600+/-69.1u/mg], 1% of fish powder [1238+/-36.7 u/mg] and olive oil [1407+/-2.1 u/mg] as low cost carbon and nitrogen sources and substrate, respectively

Conclusion: our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme

Inactivation of aprE gene in bacillus subtilis 168 by homologus recombination

One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis [B. Subtilis]. One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate [FITC]-labeled casein-substrate. The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase