Background: Human brain endothelial cells (HBECs) are part of the blood-brain barrier (BBB). BBB acts as a barrier to control the passage of molecules or materials from the blood into the brain. Identification of specific proteins changes in their expressions that are related to disease state is important in order to understand the disease mechanism involving brain vasculature. To achieve that, the techniques involve in identifying the proteins of interest must be optimized prior to further investigation. Methodology: In this study, identification of Claudin-5 in HBEC lysates was tested using different sample preparation techniques such as; 1) reducing with Dithiothreitol (DTT) and non-reducing conditions; 2) denaturing by heating at 95°C for 5 minutes or 70°C for 20 minutes and 3) protein loading at 3 and 4 µg. The samples were then subjected to an automated capillary-based immunoassay, Jess. Results and Discussion: The results showed that HBEC samples loaded at 4 µg and heated for 5 minutes at 95°C with DTT produced clearer and intense bands for Claudin-5 identification compared to the other set ups. As reducing condition and denaturing by heated at 95°C for 5 minutes conditions demonstrated good results, the conditions were used to identify ICAM-1 expression at different protein loading (3 and 4 µg). The result demonstrated that HBEC samples heated for 5 minutes at 95°C with DTT and loaded at 4 µg produced a good detection for ICAM-1. Conclusion: These optimized conditions could be served as a standard procedure for further identification of Claudin-5 and ICAM-1 proteins in HBEC using a capillary immunoassay instrument.
Zingiber zerumbet has been traditionally used as an anti-inflammation and antioxidant agent. The present study
investigates the neuroprotective effects of ethyl acetate extract of Z. zerumbet against oxidative stress on paraquat
(PQ)-induced Parkinsonism in rats. Forty male Sprague-Dawley rats were divided into five groups: Negative control
(normal saline), positive control (N-acetylcysteine, NAC 20 mg/kg + PQ 10 mg/kg), PQ only, 200 mg/kg Z. zerumbet +
PQ and 400 mg/kg Z. zerumbet + PQ. The extract was given orally for 19 consecutive days and PQ was administered
intraperitoneally on day 8-12th of the treatment regime. Both serum and fresh brains containing substantia nigra (SN)
region were taken for biochemical and histological analysis. Administration of both 200 and 400 mg/kg ethyl acetate
Z. zerumbet extracts to the PQ-treated groups have resulted in: Decreased levels of MDA and PC in the SN homogenates;
and increased SOD, GPx; and CAT activities in the SN and serum. Overall, ethyl acetate extract of Z. zerumbet reduced
oxidative stress in the SN of PQ-induced neuronal damages, therefore, has the potential to be developed as a preventive
agent for neurodegenerative disorders caused by environmental toxins.
Standard fibroblast culture medium usually contains fetal bovine serum (FBS). In theory, unknown risks of infection from bovine disease or immune reaction to foreign proteins may occur if standard culture method is used for future human tissue-engineering development. Human serum (HS) theoretically would be another choice in providing a safer approach and autologous clinically reliable cells.