Thesis

Study of effects of xylanase on the archenteron morphology of sea urchin embryos

The extracellular matrix is a tissue that is present around the cell which provides structural support to the cell. The extracellular matrix also plays an important role in growth and development; it consists of a set of components including proteoglycans, proteolipids, at fibers such as collagen, elastin, fibronectin etc., which play a primary role in the cell-cell adhesion process. Cell-cell adhesion is an important process for cell development and function. In several studies, glycans have been found to play an important role in cell- cell adhesion process (Kleene et al., 2004; Varki et al., 2009). Here, I evaluate the effect of commercially available glycosidase. Sea urchin embryos were used as a model organism and were treated with various concentration of thermostable xylanase. Sea urchin embryos were designated by NIH as model organism for biological studies, because of their availability, transparency and external fertilization. They share development and evolutionary relationships with humans, as reflected by homology of DNA sequences and similarities in several biochemical pathways. I am trying to understand the effects of xylanase on, archenteron organization/ elongation/ attachment to the blastocoel roof in the sea urchin embryos during embryogenesis. The hypothesis for this study was that Xylanase may inhibit the archenteron growth of sea urchin embryo. Enzyme activity was found with a known substrate. SDS PAGE analysis showed no contamination in the enzyme. The Azocoll test showed very minute protease contamination. I plated the Lytechinus pictus sea urchin embryos at the early gastrulation stage in a quantitative dose response manner of enzyme ranging from 0.12-31.81U/ml, xylanase to assess the morphological characteristics. One unit will liberate 1.0μmole of reducing sugar which is measured as xylose equivalents from xylan per minute at pH 4.5 at 30°C. Averages of 4000-7900 embryos were observed for each treatment/ concentration of xylanase. The different morphologies for each treatment were compared with the control, using MANOVA analysis. The p values, which are less than 0.05, indicate a significant difference compared to controls. There were significant differences between all of the concentrations in comparison to the controls. According to univariate F-tests and wilki’s lambda results, 0.99U/ml, 0.49U/ml, 0.24U/ml, and 0.12U/ml concentrations had much significant effect to the whole model. The results suggest that glycans are involved in cellular interactions, and was confirmed that xylan moieties are involved in archenteron development in the embryonic stages of sea urchin embryos. However, because the highest concentrations of xylanase had less effect on archenteron development than some lower concentrations, a dose-response effect was not established.

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