Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.     

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21^Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21^Cip1/Waf1. Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.     

Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress,Dysfunction of Calcium Signalling,and Inhibition of Autophagy–Lysosomal Pathway

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-d-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic–lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.     

Impact of the C-terminal loop of histidine triad nucleotide binding protein1 (Hint1) on substrate specificity

Although highly sequence similar, human histidine triad nucleotide binding protein (hHint1) and E. coli hinT (echinT) exhibit significant differences in their phosphoramidase substrate specificity and lysyl-adenylate hydrolytic activity. Observing that the C termini of each enzyme are highly dissimilar, we created two chimeric Hint's: one in which the C terminus of hHint1 was replaced with the C terminus of echinT (Hs/ec) and the other in which the C terminus of echinT was replaced with the C terminus of hHint1 (ec/Hs). The Hs/ec chimera exhibited nearly identical specificity constants (kcat/Km) to those found for echinT, whereas the specificity constants of the ec/Hs chimera were found to approximate those for hHint1. In particular, as observed for echinT, the Hs/ec chimera does not exhibit a preference for phosphoramidates containing d- or l- tryptophan, while the ec/Hs chimera adopts the human enzyme preference for the l configuration. In addition, the studies with each chimera revealed that differences in the ability of hHint1 and echinT to hydrolyze lysyl-AMP generated by either E. coli or human lysyl-tRNA synthetase were partially transferable by C-terminal loop exchange. Hence, our results support the critical role of the C-terminal loop of human and E. coli Hint1 on governing substrate specificity.