Mingkai Xu

Effects of chlorimuron-ethyl application with or without urea fertilization on soil ammonia-oxidizing bacteria and archaea.

Chlorimuron-ethyl (CE) has been widely used in modern agriculture, but little is known regarding the influence of CE on ammonia-oxidizing bacteria (AOB) and archaea (AOA) populations in soils. In this study, microcosm incubation of aquic brown soil was conducted for 60 d. Associated changes in the population sizes of AOB and AOA in response to CE application with or without urea fertilization were examined via quantitative real-time PCR (qPCR) assays of the ammonia monooxygenase gene (amoA). The half-life of CE ranged from 11.80 d to 14.54 d in the tested soil. Compared to the untreated control, the application of CE alone had no strong effects on soil pH, and urea fertilization temporarily increased soil pH in the first 7 days. The abundance of the AOA amoA gene was greater than the abundance of the AOB amoA gene in all treatments, but both were significantly suppressed by CE application in a dose-dependent manner. Urea fertilization generally increased AOB and AOA amoA gene abundances, except that the AOA amoA gene level was slightly reduced at the early stage of the incubation period. AOB and AOA preferred different N levels for growth, with AOB only growing significantly at high NH4(+) levels and AOA growing substantially at low NH₄(+) levels. The stimulation effects of urea fertilization on AOA and AOB amoA gene abundances were strongly suppressed by the CE application. This study indicated that the CE application substantially suppressed soil nitrification via inhibiting the AOB and AOA population regardless of urea fertilization, which resulted in significant changes in the soil NH₄(+)-N and NO₃(-)-N levels. Furthermore, AOB and AOA inhabiting separate ecological niches with different NH₄(+) levels played various roles in N cycling.

An engineered superantigen SEC2 exhibits promising antitumor activity and low toxicity

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vβ domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immuntherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.

Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2.

The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vβ domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.

Biological characterization of the zinc site coordinating histidine residues of staphylococcal enterotoxin C2.

The bacterial toxin staphylococcal enterotoxin C2 (SEC2) can cause staphylococcal toxic shock syndrome and food poisoning. Although the previously determined crystal structure of SEC2 revealed that some histidine residues (His47, His118 and His122) contribute to the binding of zinc ions, little is known about their biological roles in SEC2. This prompted us to investigate the role of the zinc site coordinating histidine residues in the biological activities of SEC2. The mutants with substitutions at positions 118 and 122 all retained T-cell stimulatory activity, whereas the histidine mutants at position 47 were defective in the ability to stimulate T-cell proliferation. Further toxicity assays in vivo indicated that mutants SEC2-H118A and SEC2-H122A were defective in emetic and febrile activities. However, mutant SEC2-H47A could cause significant emetic and febrile responses in comparison with the other two histidine mutants. These findings suggested that the zinc-coordinating histidine residues play significant roles in superantigen and toxic activities of SEC2 and further implied that superantigen and febrile activities could be separable in staphylococcal enterotoxins. The results also show that it should be possible to design new SEC2 immunotherapeutic agents that have superantigen activity and low toxicity.

Increased T‐cell stimulating activity by mutated SEC2 correlates with its improved antitumour potency

Aims:  To investigate the improved antitumour activity of SAM‐3 compared with recombinant staphylococcal enterotoxins C2 (rSEC2).

Improved stability and enhanced efficiency to degrade chlorimuron-ethyl by the entrapment of esterase SulE in cross-linked poly (γ-glutamic acid)/gelatin hydrogel.

Free enzymes often undergo some problems such as easy deactivation, low stability, and less recycling in biodegradation processes, especially in soil condition. A novel esterase SulE, which is responsible for primary degradation of a wide range of sulfonylurea herbicides by methyl or ethyl ester de-esterification, was expressed by strain Hansschlegelia sp. CHL1 and entrapped for the first time in an environment-friendly, biocompatible and biodegradable cross-linked poly (γ-glutamic acid)/gelatin hydrogel (CPE). The activity and stability of CPE-SulE were compared with free SulE under varying pH and temperature condition by measuring chlorimuron-ethyl residue. Meanwhile, the three-dimensional network of CPE-SulE was verified by scanning electron microscopy (SEM). The results showed that CPE-SulE obviously improved thermostability, pH stability and reusability compared with free SulE. Furthermore, CPE-SulE enhanced degrading efficiency of chlorimuron-ethyl in both soil and water system, especially in acid environment. The characteristics of CPE-SulE suggested the great potential to remediate chlorimuron-ethyl contaminated soils in situ.

TNF-α produced by SEC2 mutant (SAM-3)-activated human T cells induces apoptosis of HepG2 cells.

Staphylococcal enterotoxins C2 (SEC2) is a classical model of superantigens (SAg), which has the powerful ability to activate T cells as well as induce massive cytokine production. This property makes SEC2 and its mutants well concerned as a potential new immune-regulatory agent for cancer therapy. We previously constructed a SEC2 mutant named SAM-3, which had prominently antitumor activity in BALB/c mice model. But, the underlying molecular mechanism for stimulation of human peripheral blood mononuclear cells (PBMCs) and antitumor effect on human tumor cells induced by SAM-3 is not clear. Here, we showed that SAM-3 could activate human TCR Vβ 12, 13A, 14, 15, 17, and 20 CD8+ subgroup T cells, which secreted the cytokines IL-2, IFN-γ, and TNF-α, and exhibit stimulation activity in a dose-dependent manner. TNF-α secreted from activated T cells could induce apoptosis and G1-phase arrest and lead to the antitumor effect in HepG2 cells. Meanwhile, SAM-3 upregulated the expression of tumor necrosis factor receptor 1 (TNFR1) mRNA and activity of caspase-3 and caspase-8. We also found that the antitumor activity and activity of caspase-3 and caspase-8 were decreased when the neutralizing TNF-α monoclonal antibody presented. These data suggest that TNF-α secreted by SAM-3-activated T cells is an important factor in inducing apoptosis in HepG2 cells.

T-cell proliferation and antitumour activities of a truncated mutant of staphylococcal enterotoxin C2 with decreased cytokine secretion

As a superantigen, staphylococcal enterotoxin C2 (SEC2) has commonly been used as an antitumour immunotherapy agent in China. However, the clinical application of SEC2 has been hampered by its pyrogenic toxicity and the presence of neutralizing antibody in patients. Thus, an improvement in its superantigen-based immunotherapy is highly needed. In this study, a truncated SEC2 mutant, SEC(14-128), was constructed without the N-terminal 13 and C-terminal 111 aa. This mutant retained T-cell proliferation and antitumour activities in in vitro experiments. However, it induced a significantly decreased release of the main inflammatory cytokines inerleukin-2 and gamma interferon. Moreover, SEC(14-128) exhibited reduced toxicity and affinity to anti-SEC2 IgG compared with native SEC2. Based on the considerable antitumour activity and low toxicity, it is proposed that the mutant SEC(14-128) could be a potential candidate for cancer treatment.

SEC2-induced superantigen and antitumor activity is regulated through calcineurin

Once the TCR-SAg-MHC II ternary complex is established, it triggers a variety of intracellular signal transduction pathways, which provoke extreme responses in the immune system. However, the signaling events that involved in SAg-induced immune activation are not well understood. In this study, we demonstrated that the Ca2+/calcineurin (CaN)/nuclear factor of activated T cells (NFAT) signaling pathway was involved in SEC2-induced immune activation, and selective blockade of CaN by its inhibitor cyclosporine A (CsA) can completely inhibited the SEC2-induced T-cell stimulating potency. In addition, we selected an engineered SEC2 mutant named SAM-1 based on a series of biological activity tests, and our further studies on it not only confirmed that the CaN activity and gene transcription of its key substrates were proportional to the SEC2/SAM-1-induced T-cell stimulating potency, but also suggested that intensified Ca2+/CaN/NFAT signaling transduction induced by SAM-1 resulted in enhanced T-cell stimulating potency, production of cytokines and cytotoxicity, which finally elicit the improved antitumor activity of SAM-1 in vivo.