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Heat-shock protein 90 (Hsp 90) acts as a molecular chaperone that maintains protein stability and regulates cell proliferation, survival, differentiation and apoptosis. The present study investigated the effect of Hsp90 inhibition on human acute myeloid leukemia (AML) cells using the novel small-molecule inhibitor SNX-2112. We found that SNX-2112 more potently inhibited KG-1a cell growth than the classical Hsp90 inhibitor 17-(2-dimethylaminoethyl)amino­17-demethoxygeldanamycin as determined by CCK-8 assay. Flow cytometry was used to examine the cell cycle, differentiation, and apoptosis, and western blotting and qRT-PCR were used to analyze the underlying mechanism. The results revealed that low concentrations of SNX-2112 arrested the cells in the G2/M phase and induced their differentiation and apoptosis, possibly by suppressing Akt and inhibitor of κB kinase, a component of the nuclear factor (NF)-κB signaling pathway. We also found that SNX-2112 increased the expression of the differentiation transcription factors PU.1 and CCAAT­enhancer-binding protein-α. Thus, SNX-2112 induced KG-1a cell differentiation, cell cycle arrest and apoptosis via modulation of Akt and NF-κB signaling, suggesting that it is a promising therapeutic agent for the treatment of AML.

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OBJECTIVE: To investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP. MESULTS: Eighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP. RESULTS: Sequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex. CONCLUSIONS: The mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.

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OBJECTIVE: To compare the volatile constituents of Plumeriae Flos from different origins. METHODS: Water distillation method according to the Chinese Pharmacopeia was used to extract the volatile constituents of fresh Plumeriae Flos samples (red flower and white flower) and dried samples. GC-MS method combined with NIST MS Search 2.0 data base was carried out to identify the volatile constituents and to calculate the relative percentage content. RESULTS: 55 peaks were detected in the GC-MS spectrum. Among of them, 26 volatile constituents were confirmed and calculated, which were mainly fatty alcohols and esters. The relative percentage content of geranyl benzoate in fresh Plumeriae Flos samples was significantly higher than the dried samples. CONCLUSION: The compositions of volatile constituents in Plumeriae Flos have no obvious correlation with the color of flowers, but are related to the flower fresh or dried.

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A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k(L)a value of the Tubespin at standard conditions was 24.3 h(-1), while that of a spinner flask was only 2.7 h(-1). The maximum cell density in the Tubespin bioreactor reached 6 × 10(6) cells mL(-1), which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.

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Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFR alpha-Fc fusion protein expressed in static CHO-k(1) cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFR alpha-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFR alpha-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFR alpha-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expression in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies.

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In order to obtain the recombinant VPAC2 agonist efficiently by intein-mediated single column purification, a gene encoding 32-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-ROM was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the rMROM-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the rMROM with the homogeneity over 95% was released from the chitin-bound intein tag. The recombinant linear rMROM competitively displaced [125I] PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 60 +/- 5 nM, whereas the IC50 of rMROM at human VPAC1 was observed up to 10 microM and no binding was detected at PAC1. rMROM stimulated the cAMP accumulation in Chinese hamster ovary (CHO) cells expressing the human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.6 nM, which was 500-fold less potent at VPAC1and had no activity on PAC1. An efficient production procedure of a novel recombinant VPAC2-selective agonist was established.

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Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).

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AIM: To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15(pREP4) respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami(DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED(50) of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 microg/L and 2.2 microg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15(pQE-HBscFv) or Origami(pQE-HBscFv). But the supernatant of Origami(pQE-HBscFv) lysate displayed weak bioactivity and its counterpart from M15(pQE-HBscFv) did not display any bioactivity. The soluble HBscFv in Origami(pQE-HBscFv) was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62 x 10(7) mol/L. The yield of native HBscFv refolded from inclusion body in M15(pQE-HBscFv) was 30-35 mg/L and the affinity constant was 1.98 x 10(7) mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

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AIM: To obtain high-level expression of nonfusion recombinant human basic fibroblast growth factor (rhbFGF). METHODS: hbFGF cDNA was prepared from the total RNA of embryonic brain tissue. As a template, the obtained gene was used to clone nonfusion rhbFGF. New primers were employed to alter the translation initiation region (TIR) and reduce the G+C content through nucleotide change. Using pET-3C as vector, the cloned rhbFGF was expressed in BL21 (DE3). RESULTS: rhbFGF was expressed in E coli up to 30 % of the total cellular protein. Cation exchange and heparin affinity chromatography were employed to purify the target protein from the supernatant of bacteria lysate. The bioactivity of the purified rhbFGF was identical with the standard bFGF. CONCLUSION: Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli.

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