Effects of cyclophosphamide on antioxidative and immune functions of Nile tilapia (Oreochromis Niloticus) via the TLR-NF-κB signaling pathway.

Intensive aquaculture often results in immunosuppression in fish, which may cause a series of diseases. In this study, to investigate the immunosuppressive mechanisms in fish, tilapia were intrapleural injected cyclophosphamide (CTX) at the doses of 10, 25, 50, 75 and 100 mg·kg-1 to induce immunosuppression. We determined the viability of immune cells, the content of lysozyme (LZM) and immunoglobulin M (IgM), the levels of nitric oxide (NO) and antioxidant parameters. Meanwhile, the mRNA levels of complement C3 (c3), igm and the genes associated with the TLR-NF-κB signaling pathway in the head kidney (HK) and spleen were also determined. The results showed that CTX had a significant cytotoxic effect on peripheral blood leukocytes, HK macrophages and spleen cells in a dose-dependent manner. The protein and mRNA levels of C3 and IgM were down-regulated with the increase of CTX concentrations in serum, HK and/or spleen. The NO and LZM contents decreased significantly in HK and spleen after CTX treatments with 75 and 100 mg·kg-1. CTX treatments with 50, 75 and/or 100 mg·kg-1 markedly decreased the antioxidant ability and enhanced lipid peroxidation in HK and spleen. Furthermore, qPCR data showed that CTX treatments with 50-100 mg·kg-1 clearly down-regulated the mRNA levels of tlr2, myd88, irak1, traf6, nfκb1, nfκb2, il-6, il-10 and tnf-α in the HK and/or spleen. Overall results suggested that CTX treatment had a cytotoxic effect on immune cells, induced lipid peroxidation, decreased the antioxidant capacity and inhibited immune function. The immunosuppressive mechanisms of CTX may be associated with the TLR-NF-κB signaling pathway.

A novel design of Rogowski coil for measurement of nanosecond-risetime high-level pulsed current.

In pulsed power systems, pulsed currents with risetimes from nanosecond to microsecond can be effectively measured by self-integrating Rogowski coils. Appropriate design of the structure and the integrating resistor is crucial to the high-frequency response of a coil. In this paper, several novel designs of Rogowski coil's integrating resistors were proposed and tested. Experimental results showed that the optimized coil could response square waves with fronts of ∼1.5 ns and had a sensitivity of ∼0.75 V/kA. The maximal peak current was designed as 100 kA.

Pyranonaphthoquinone lactones: a new class of AKT selective kinase inhibitors alkylate a regulatory loop cysteine.

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.

Discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of AKT/PKB: mechanistic involvement of AKT catalytic activation loop cysteines.

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC(50), 0.149 +/- 0.045 micromol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors.

[Cloning and expression of Oreochromis aurea gonadotropin-releasing hormone cDNA in Escherichia coli and its immunogenicity for mice].

To study the function of the GnRH protein, the recombinant pMAL-GnRH was constructed and expressed in TB1 E. coli. The cDNA encoding gonadotropin-releasing hormone (GnRH) and GnRH associated peptide (GAP) was amplified from total RNA of O. aurea pituitary glands by reverse transcription polymerase chain reaction (RT-PCR), and then blasted against other GnRH cDNA sequences in the GenBank. The analysis of the sequence data indicated that the coding region of the cDNA fragment, which encoded 89 amino acid residues, was about 400 bp in size. The amplified cDNA fragment was cloned into the prokaryotic expression vector, pMAL-c2x, to produce the expression vector pMAL-GnRH. The recombinant plasmid was transformed into E. coli TB1. GnRH-MBP fusion protein was obtained after the addition of IPTG into the growth media. SDS-PAGE analysis revealed that the GnRH-MBP was expressed after induction with IPTG for 4 h. A protein band of 56 kD appeared on SDS-PAGE gel and was proved by Western blot. The mass production of the recombinant protein was about 41.6% of total bacteria protein. After purification and cleavage of the fusion protein purified GnRH protein could be obtained. Then the fusion protein was used to immunise some ICR mice to produce anti-GnRH antibody. This fusion protein could significantly elicit specific antibody response in immunized mice compared with the blank groups, and the titers against GnRH reached peak 0.707 +/- 0.320 at the 5th week after immunization. These results demonstrated that recombinant protein could induce high GnRH antibody responses in laboratory animals.

Pyrimido[1,2-b]-1,2,4,5-tetrazin-6-ones as HCMV protease inhibitors: a new class of heterocycles with flavin-like redox properties.

The synthesis and biological activity of pyrimidotetrazin-6-ones against HCMV protease is described. The mechanism of action for these inhibitors is the oxidation of several cysteine residues to generate cross-linked enzyme.