1B) In this manner, ROS generation in nodavirus-infected cells

1B). In this manner, ROS generation in nodavirus-infected cells

was examined to determine oxidative stress. As well, free radical species were effectively reduced by the NAC antioxidant. These observations suggested that nodavirus infection selleck chemicals may induce oxidative stress in cells. During nodavirus infection, one of the mechanisms to alleviate cellular oxidative stress associated cytotoxicity may involve reaction with tyrosine amino acid to form dityrosine. The potent initial step in dityrosine formation is the creation of a tyrosine radical, which promotes the intermolecular cross-linking of two monomeric tyrosine containing proteins to generate dityrosine as a stable end product. Evidence of in vivo generation can be demonstrated by the presence of dityrosine in cells from nodavirus-infected Adriamycin clinical trial tissues. Thus, the detection of dityrosine can serve as an indicator of ROS. More exuberant expression of dityrosine was found in nodavirus-infected injured

eye and brain tissues ( Fig. 2A and B). Dityrosine staining was observed mainly in the eye, with a diffuse distribution pattern throughout the retina. Notably, the expression pattern of crystallin was similar in both healthy and nodavirus-infected grouper tissues ( Fig. 2C and D). Serial biopsy sections obtained from nodavirus-infected grouper confirmed that all dityrosine-labeling retina coexpressed crystallin. In contrast, histological and immunohistochemical examinations of eye and brain tissue from nodavirus-infected grouper revealed immunoreactivity to coat protein in eye and brain tissues ( Fig. 2E and F). A higher magnification

view clearly revealed brain vacuolization ( Fig. 2G and H). To summarize, no significant difference in the retina staining score DCLK1 of crystallin between healthy and nodavirus-infected grouper was found, but dityrosine formation was significantly different compared with that obtained in eye tissue with non-viral healthy grouper. In all cases studied the majority of dityrosine-expressing cellular region, which were distributed throughout the retina of nodavirus infected groupers, were located in close proximity to nodavirus antigen-expressing eye tissue. ROS-mediated dityrosine formation of crystallin protein was markedly induced during nodavirus infection, and appeared to be associated with the histological severity of viral nervous necrosis (VNN). Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were used to demonstrate and analyze the expression of crystallins in grouper lenses. Abundant protein spots detected at the 20–31 kDa range (pH 6–9) appeared likely to represent crystallins. Peptide mass fingerprinting of 24 of these spots demonstrated that 12 were crystallins (Table 1).

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