Phase separation of insulin receptor substrate 1 drives the formation of insulin/IGF-1 signalosomes.

As a critical node for insulin/IGF signaling, insulin receptor substrate 1 (IRS-1) is essential for metabolic regulation. A long and unstructured C-terminal region of IRS-1 recruits downstream effectors for promoting insulin/IGF signals. However, the underlying molecular basis for this remains elusive. Here, we found that the C-terminus of IRS-1 undergoes liquid-liquid phase separation (LLPS). Both electrostatic and hydrophobic interactions were seen to drive IRS-1 LLPS. Self-association of IRS-1, which was mainly mediated by the 301-600 region, drives IRS-1 LLPS to form insulin/IGF-1 signalosomes. Moreover, tyrosine residues of YXXM motifs, which recruit downstream effectors, also contributed to IRS-1 self-association and LLPS. Impairment of IRS-1 LLPS attenuated its positive effects on insulin/IGF-1 signaling. The metabolic disease-associated G972R mutation impaired the self-association and LLPS of IRS-1. Our findings delineate a mechanism in which LLPS of IRS-1-mediated signalosomes serves as an organizing center for insulin/IGF-1 signaling and implicate the role of aberrant IRS-1 LLPS in metabolic diseases.

MTN output feedback tracking control for MIMO discrete-time uncertain nonlinear systems.

An adaptive controller is developed that is based on the multidimensional Taylor network (MTN). This controller is used for multi-input and multi-output (MIMO) uncertain discrete-time nonlinear systems. This newly developed MTN is dissimilar with the neural network, in which only multiplication and addition are needed for this controller. Thus, real-time control is more easily to be achieved. The theoretical analysis shows that the output errors of the system are convergent and the output signals are semi-globally, uniformly and ultimately bounded. To illustrate the validity of MTN-based adaptive controller (MTNAC), a numerical example is given. The simulation data demonstrate that this MNTAC has better real-time performance and higher robustness compared with neural networks.

D11, a novel glycosylated diphyllin derivative, exhibits potent anticancer activity by targeting topoisomerase IIα.

Glycosylated natural products are reliable platforms for the development of anticancer drugs, simply due to the important features added by sugar appendages to the shape and the stereoelectronic properties of natural scaffolds. Herein, we indentified D11, a novel diphyllin glycoside with acetylated D-quinovose sugar moiety, as a potent topoisomerase IIα (Topo IIα) inhibitior. This peculiar sugar moiety endows D11 an optimal conformation with a high binding affinity for Topo IIα via hydrogen bonding to the entrance of ATPase pocket, thereby helping achieve more potent Topo II inhibition activity compared to the aglycon diphyllin. Further biochemical insights manifested that D11 significantly inhibited Topo IIα ATPase-catalyzed ATP hydrolysis in an ATP-dependent, but a DNA-independent manner. All these underlie the consequent superiority of D11 in the in vitro proliferation inhibition, apoptosis induction and the in vivo remarkable antitumor potency in xenograft mouse model. Moreover, D11 treatment also displayed no obvious body weight loss and other apparent toxicities, indicative of the restricted side effects by the virtue of sugar attachment. Taken together, a defined glycosylated manipulation of diphyllin may be a promising alternative approach in the development of novel Topo II inhibitors.

BB, a new EGFR inhibitor, exhibits prominent anti-angiogenesis and antitumor activities.

Aberrant activation of the epidermal growth factor receptor (EGFR) is closely associated with malignant progression of tumors. EGFR inhibitors have been used successfully in clinic in the treatment of solid tumors. In the present study, we revealed that BB, a new synthetic quinonazoline derivative, was a potent EGFR inhibitor. BB selectively inhibited EGFR with a IC(50) value of 50 +/- 37 nM, at least 32-fold more potent than suppressed all other ten tested receptor tyrosine kinases including the same family member ErbB2 (IC(50) = 5.6 +/- 3.2 microM). BB effectively abrogated autophosphorylation of the EGF-stimulated EGFR and phosphorylation of its key downstream signaling molecules ERK and AKT in A549 cells. BB was shown to suppress EGF-stimulated proliferation of A549 cells with an apparently lower IC(50) value (0.33 +/- 0.07 microM) than that (2.7 +/- 0.4 microM) for the serum-stimulated cells. BB also inhibited the EGF-independent proliferation of a panel of tumor cells. In addition, BB exhibited anti-angiogenesis activity, as evidenced by antagonizing EGF-induced HMEC-1 migration in vitro, blocking HMEC-1 tube formation, and inhibiting microvessel sprouting from rat aortic rings. Most importantly, BB prominently inhibited in vivo tumorigenesis of NIH3T3 cells specifically driven by the activation-mutated EGFR genes. As reported, normal NIH3T3 cells lack tumorigenicity in nude mice. NIH3T3 cells transfected with the EGFR gene with activating mutation (A750P or L858R) produced rapidly growing xenografts in nude mice. BB, when given orally at 100 mg/kg consecutively for 2 w, prominently inhibited the growth of the xenografts and reduced the number of microvessels. Taken together, the data indicate that BB is a new selective EGFR inhibitor with potent antitumor activity, revealing its potential as a promising anticancer candidate.

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models.

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.

Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model.

Chemotherapy for cancer is partly limited by the inability of drugs to act on poorly vascularized or avascularized areas of tumors. Tumor-targeting bacteria are capable of preferentially replicating in these poorly perfused regions. Some strains have been combined with chemotherapeutic agents and the results have been promising. However, no systematic work has been carried out to test the effect of bacteria on clinical modes of chemotherapy, such as standard maximum tolerated dose (MTD) and novel low-dose metronomic (LDM) chemotherapy. Here Salmonella typhimurium VNP20009 was combined with cyclophosphamide (CTX) at both MTD and LDM schedules in a murine melanoma model. The results showed that VNP20009 significantly improved the effects of all forms of CTX treatments. The combination of VNP20009 and CTX led to a more significant decrease in tumor microvessel density and serum vascular endothelial growth factor (VEGF) level, compared with either treatment alone. Furthermore, combination therapy remarkably increased the number of bacteria within tumors when compared with bacteria treatment alone. These findings suggest that tumor-targeting bacteria, in conjunction with CTX at standard MTD and LDM regimens, might be of clinical value for the treatment of melanoma.

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity.

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology.

Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the cDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli. Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

TKI-31 inhibits angiogenesis by combined suppression signaling pathway of VEGFR2 and PDGFRbeta.

Tyrosine kinases have been strongly implicated as therapeutic targets that influence the angiogenic process in growing tumors. In this study, we revealed that TKI-31 is a potent broad spectrum tyrosine kinase inhibitor, which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFRbeta) and also inhibits kinases of other class, such as c-Kit and c-Src on molecular base, but showed no activity against vascular endothelial growth factor receptor 1 (VEGFR1) and epidermal growth factor receptor (EGFR). TKI-31 inhibits VEGF-induced phosphorylation of VEGFR2 in endothelial cells as well as PDGF(BB)-induced phosphorylation in fibroblast cells, and leading to the inhibition of down-stream signaling triggered by these receptors such as PI3K/Akt/mTOR, MAPK42/44(ERK) and paxillin. TKI-31 also inhibited VEGF-induced endothelial cells proliferation, migration and their differentiation into capillary-like tube formation. Its anti-angiogenic property was further confirmed by the inhibition of neovascularization on CAM, in vivo. These results collectively highlight the therapeutic potential of this compound for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

An improved strategy for high-level production of human vasostatin120-180.

We previously reported a strategy for expression and purification of human Vasostatin120-180 (VAS), a potent angiogenesis inhibitor in a GST fusion form; however, the yield of 7.2 mg per liter of culture was relatively low. The aim of this study was to develop a more efficient system to improve and facilitate the production of VAS protein in a soluble and native form in Escherichia coli. The VAS gene with optimized condons was cloned into pET28a and overexpressed as a N-terminal His-tagged fusion protein. Between His-tag and VAS, an enterokinase recognition site was introduced to release the intact VAS. Optimal expression of soluble His-VAS was achieved by examining the contribution of chaperone coexpression and lower temperature fermentation. Ammonium sulfate precipitation was first employed to remove nucleic acid and partial host proteins. When further purified by Ni2+ affinity chromatography, 40 mg of His-VAS was isolated with purity over 85% from 1 L of culture. After desalting with Sephadex G15 and digestion with His-EK, followed by the removal of the His-tag and His-EK with Ni(2+)-NTA resin, 21 mg of intact VAS was finally obtained from 1 L of bacterial culture, which was approximately 3-fold the yield we previously obtained via GST fusion expression strategy. The identity of His-VAS and VAS was confirmed by Western blot. Purified VAS displayed distinct anti-angiogenic activity, which was shown by the endothelial cell proliferation inhibition assay and chicken chorioallantoic membrane assay. In sum, we greatly improved the yield of intact and bioactive VAS protein, and using this successful example, we propose a more efficient system for the high-level production of intact functional proteins, especially for low molecule weight peptides.

Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization.

According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods. Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E. coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain. GST-VAS protein approximately accounted for 45% of the total bacterial proteins. Most of target protein existed in inclusion body. To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested. The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased. When purified by GST affinity chromatography, 50 mg GST-VAS was obtained with purity over 85% from 1 L culture. Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography. About 7.2 mg intact homogeneous VAS protein was finally produced from 1L bacterial culture. The identity of GST-VAS and VAS was validated by Western blotting analysis. Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization.

Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of alpha-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His6 encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EKL-His6 was attained by methanol induction and its molecular weight is 43 kD. Because of the existence of His6-tag, EKL-His6 was easily purified from P. pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EKL-His6 demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.

[Identification of a resistance gene to bacterial blight (Xanthomonas oryzae pv. oryzae) in a somaclonal mutant HX-3 of indica rice].

Using the mature embryo of a susceptible rice variety Minghui 63 as the explant, we have obtained a somaclonal mutant HX-3 through selection in vitro, which has showed resistance to bacterial blight. In 8 successive years, the resistance of R1 to R9 generations of HX-3 was identified by ZJ173, a typical bacterial blight strain in Yangtsu River valley, and the results showed that the resistance of HX-3 was stable and heritable. Genetic analysis also indicated that the resistance of HX-3 to bacterial blight was under a dominant gene controlling. Using 32 bacterial blight strains collected in China, Philippines and Japan, the resistance spectrum of HX-3 and other 13 testers with different major dominant resistance genes were tested. Results of 2 years (1999-2000) experiment showed that HX-3 had a broad resistance spectrum, which seemed to be different with those of the other dominant resistance genes identified. Allelic tests were also conducted by crossing HX-3 with IRBB4, IRBB7, CBB12 and IRBB21, and the F2 populations of each of the 4 crosses demonstrated resistant and susceptible plant segregation, indicating that the resistance gene in HX-3 different from Xa-4, Xa-7, Xa-12 and Xa-21. All these results proved that there was a new resistance gene in HX-3. We have designated the new gene as Xa-25(t).