RESUMO
The study aimed to examine the impact of increasing environmental temperatures on physiological changes, oxidative stress, nitric oxide production, total antioxidant capacity, and blood cell viability in American bullfrog crossbreeds. Frogs and frog blood cells were exposed to temperature ranges of 25-33 °C and 25-37 °C, respectively. Physiological parameters (body temperature, pulse rate, ventilation rate, and oxygen saturation) and biochemical parameters (total antioxidant power, hydrogen peroxide, malondialdehyde, nitric oxide, and mitochondrial activity) were measured at every 2 °C increment. Results showed that body temperature rose with increased environmental temperature (P < 0.05). Pulse rates at 33 °C were higher than those at 25-31 °C (P < 0.05). Ventilation rates at 31 °C exceeded those at 25 °C and 27 °C (P < 0.05). Oxygen saturation levels remained stable at 25-33 °C (P > 0.05). Total antioxidant power at 25 °C was greater than at 27-37 °C (P < 0.05). Hydrogen peroxide levels at 27 °C were higher compared to 25 °C and 31-37 °C (P < 0.05). Malondialdehyde levels at 25-33 °C were higher than at 35 °C and 37 °C (P < 0.05). Nitric oxide levels at 37 °C were higher than at 25-33 °C (P < 0.05), and at 35 °C were higher than at 25-31 °C (P < 0.05). Blood cell viability at 25-31 °C was higher than at 37 °C (P < 0.05). These results suggest that at an environmental temperature of 33 °C, the frogs' body temperature approached 31 °C or higher, and were likely to be harmful to the frogs. Finally, the environmental temperature that caused frog blood cell death was 37 °C.
RESUMO
Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.
