Involvement of gamma interferon inducible lysosomal thiol reductase in the innate immune responses of red swamp crayfish, Procambarus clarkii.

The Gamma interferon inducible lysosomal thiol reductase (GILT) plays a key biological role in the immune responses and involves in the processing of class II MHC-restricted antigen by stimulating disulfide bond reduction in mammals. To determine the biological function of GILT in the innate immune system of crustaceans, we sequenced and cloned GILT gene from red swamp crayfish, Procambarus clarkii (Pc-GILT). The deduced amino acid sequence of Pc-GILT contained the putative conserved structures of the GILT family proteins: the GILT signature (CQHGX2ECX2NX4C) sequence and the active site (CXXS) motif. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis suggested that a recombinant Pc-GILT protein was successfully expressed in Escherichia coli (E. coli). Quantitative real-time PCR analysis showed that Pc-GILT transcript level was highest in the hepatopancreas followed by the gut, heart and muscles. Additionally, we analyzed the transcription level of Pc-GILT gene in hepatopancreas of red swamp crayfish under biotic stress conditions. The expression of Pc-GILT gene upregulated after viral (poly I:C) and bacterial (peptidoglycan, lipopolysaccharide) infection. The suppression of Pc-GILT by double stranded RNA influenced the transcript levels of various immune-related genes. These observations indicate that the Pc-GILT probably plays a key biological role in the innate immune responses of red swamp crayfish, since it modulates the expression of genes associated with immune pathways.

Peroxiredoxin 6 modulates Toll signaling pathway and protects DNA damage against oxidative stress in red swamp crayfish (Procambarus clarkii).

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.

Essential role of the peroxiredoxin 4 in Procambarus clarkii antioxidant defense and immune responses.

Peroxiredoxin (Prx) family members play a key role in host defense against oxidative stress, and modulate immune responses following microbial infection. Here, we cloned and characterized Procambarus clarkii Prx4 (Peroxiredoxin 4) cDNA, a regulator of oxidative stress and its expression analysis upon lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C) infection. The cDNA fragment of PcPrx4 was 744 bp in length, encoding a putative protein of 248 amino acid residues. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PcPrx4 was expressed in all the examined tissues, and it was highest in the hepatopancreas followed by the hemocytes and gill. The challenge with LPS and Poly I:C significantly up-regulated the expression of PcPrx4 in hepatopancreas, hemocytes and gill when compared with the control. Recombinant PcPrx4 protein was used to investigate the antioxidant function in vitro by mixed-function oxidase assay. The results demonstrated a dose-dependent inhibition of DNA damage by rPcPrx4 protein. Altogether, our results imply that PcPrx4 is implicated in defense against microbial pathogens and oxidants in P. clarkii.

A role of cathepsin L gene in innate immune response of crayfish (Procambarus clarkii).

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In the present study, cathepsin L gene from the red crayfish Procambarus clarkii, named PcCTSL, was cloned and characterized. The cDNA fragment of PcCTSL was 1026 bp in length, which encoded a putative protein of 341 amino acid residues with a molecular weight of 37.884 kDa. The theoretical isoelectric point was 5.218. The prepro-cathepsin L was comprised of a typical signal peptide (Met1-Ala18), a prodomain proregion peptide (Trp29-Phe89) and a mature peptide (Leu124-Leu340). Homology analysis indicated that PcCTSL exhibited 53.2%-87.1% identity to other selected species. The recombinant protein of PcCTSL was successfully expressed in Escherichia coli and rabbit anti-PcCTSL polyclonal antibodies were prepared. Real-time quantitative reverse transcription-PCR (qPCR) analysis revealed that the PcCTSL was expressed in all examined tissues, while the greatest mRNA level was observed in hepatopancreas. The expression of PcCTSL mRNA was clearly up regulated in hepatopancreas after challenge by lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C). RNA interference of PcCTSL affected the gene expression of members of the Toll pathway. Our results suggest that the PcCTSL may play an important role to defend P. clarkii against the pathogens infection.

[Effect of qingxin kaiqiao formula and saponin on learning and memory abilities and expression of apoptosis signal transducers Abeta and betaAPP in AD rat brain].

OBJECTIVE: To study the effect of qingxin kaiqiao formula and saponin on the learning and memory ability and the expression of the apoptosis signal transducers Abeta and betaAPP in AD rat brain. METHOD: The comparative observation method was adopted for the animal test. Forty male SD rats were randomly divided into five groups, namely the normal group, the model group, the aricept group, the qingxin kaiqiao formula group and the saponin group, with eight rats in each group. Abeta(25-35) (10 g x L(-1)) was injected into their bilateral amygdala to establish the AD rat model. Since the next day, they were intragastrically administered with Aricept (1.67 mg x kg(-1)), Qingxin Kaiqiao decoction (12.67 mL x kg(-1)), saponin (6.30 mg x kg(-1)) and double distilled water filling for 2 weeks to observe their spatial memory ability in a Morris water maze and study the expression of Caspase-3, Abeta and betaAPP in brain tissues by immunohistochemistry. RESULT: Each traditional Chinese medicine groups showed significant improvement in the learning and memory ability of AD rats and notable differences (P < 0.05, P < 0.01) compared with the control group. The qingxin kaiqiao formula group and the saponin group showed a decrease in the expressions of Caspase-3, Abeta and betaAPP in cerebral cortex and hippocampus area, displaying notable differences (P < 0.01, P < 0.05) compared with the control group. CONCLUSION: qingxin kaiqiao formula and saponin can obviously improve the learning and memory ability of AD rats with by decreasing the expression of Caspase-3, Abeta and betaAPP in cortex and hippocampus.