Expression, purification, and characterization of recombinant Metarhizium anisopliae acid trehalase in Pichia pastoris

The mature peptide of Metarhizium anisopliae acid trehalase (ATM1) (EC3.2.1.28) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant ATM1 (reATM1) was secreted into culture medium. After 48-h 0.5% methanol induction, the activity of reATM1 in the culture supernatant reached the peak, 5.35 U/mg. Enzyme with a histidine sequence appended to the C terminus was still active and was purified using metal-chelate affinity chromatography. The yield of purified reATM1 was 2.5 mg from 1L supernatant. The purified reATM1 exhibited a molecular mass of approximately 170 kDa on SDS-PAGE. The optimum temperature and pH of reATM1 were 30 degrees C and 6.0, respectively, and the K(m) and V(max) values for reATM1 were 2.6 mM and 0.305 mmol/min/mg, respectively. Studies showed that the enzymatic properties of reATM1 were similar to those of the native ATM1.     

Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.     

Expression, purification, and in vitro refolding of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. Here, a recombinant form of the extracellular domain of the TRAIL (sTRAIL) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded sTRAIL was purified by anion-exchange chromatography. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 19.2 kDa, which equalled the theoretically expected mass. N-terminal sequencing of refolding sTRAIL showed the sequence which corresponded to the designed protein. The renatured protein displayed its immunoreactivity with the antibodies to TRAIL protein by Western blotting. The purified sTRAIL had a strong cytotoxic activity against human cervical cancer HeLa cells with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

Curcumin induces apoptosis through mitochondrial hyperpolarization and mtDNA damage in human hepatoma G2 cells

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.     

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

Background

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.

Results

We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.

Conclusion

About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.