Dok5 is substrate of TrkB and TrkC receptors and involved in neurotrophin induced MAPK activation

Tropomyosin-related kinase (Trk) family receptors are a group of high affinity receptors for neurotrophin growth factors, which have pivotal functions in many physiological processes of nervous system. Trk receptors can dimerize and autophosphorylate upon neurotrophin stimulation, then recruit multiple adaptor proteins to transduct signal. In this report, we identified Dok5, a member of Dok family, as a new substrate of TrkB/C receptors. In yeast two-hybrid assay, Dok5 can interact with intracellular domain of TrkB and TrkC receptor through its PTB domain, but not with that of TrkA receptor. The interaction was then confirmed by GST pull-down assay and Co-IP experiment. Dok5 co-localized with TrkB and TrkC in differentiated PC12 cells, providing another evidence for their interaction. By using mutational analysis, we characterized that Dok5 PTB domain bound to Trk receptor NPQY motif in a kinase-activity-dependent manner. Furthermore, competition experiment indicated that Dok5 competed with N-shc for binding to the receptors at the same site. Finally, we showed that Dok5 was involved in the activation of MAPK pathway induced by neurotrophin stimulation. Taken together, these results suggest that Dok5 acts as substrate of TrkB/C receptors and is involved in neurotrophin induced MAPK signal pathway activation.     

99mTc-labeled cyclic RGDfK dimer: initial evaluation for SPECT imaging of glioma integrin alphavbeta3 expression

This report describes the evaluation of biodistribution properties of three radiotracers, [(99m)Tc(SQ168)(EDDA)], [(99m)Tc(SQ168)(tricine)(PDA)], and [(99m)Tc(SQ168)(tricine)(TPPTS)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; EDDA = ethylenediamine-N,N'-diacetic acid; PDA = 2,5-pyridinedicarboxylic acid; TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate), and their potential to image the glioma integrin alpha(v)beta(3) expression in BALB/c nude mice bearing the U87MG human glioma xenografts. It was found that all three radiotracers were able to localize in glioma tumors with a relatively high tumor uptake and long tumor retention time by binding to the integrin alpha(v)beta(3) expressed on both tumor cells and endothelial cells of tumor neovasculature. It seems that the coligand has minimal effect on integrin alpha(v)beta(3) targeting capability of the (99m)Tc-labeled RGDfK dimer, but it has a significant impact on their biodistribution properties. For example, the complex [(99m)Tc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake and the highest metabolic stability in normal BALB/c nude mice. Results from SPECT imaging studies show that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. Among the three radiotracers evaluated in this study, [(99m)Tc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.     

Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.     

A real time quantitative PCR-based method for the detection and quantification of simian virus 40.

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.     

EDNRB基因编码区点突变及多态性在浙江地区散发性先天性巨结肠症中的检测与分析

本实验应用聚合酶链反应-单链构象多态性(single strand confbrmation polymorphism analysis of polymerase chain reaction products,PCR-SSCP)和DNA直接测序技术,对75例浙江地区散发性先天性巨结肠病例EDNRB基因编码区的全部7个外显子,进行了点突变与单核苷酸多态性的检测与分析,探讨浙江地区先天性巨结肠患者EDNRB基因的突变特征,阐明EDNRB基因与散发性先天性巨结肠症发病之间可能存在的关系。结果有6例患者在第4外显子上检测到密码子277位点 CTG→CTA的置换,导致亮氨酸的同义突变(L277L),属于单核苷酸多态性,发生率为8％(6/75)。有 2例患者在第2外显子上检测到密码子185位点GTG→ATG的置换,导致缬氨酸到蛋氨酸的错义突变(V185M),此突变型未在国内外文献中报道过,认为是新的基因突变型,突变率2．7％(2/75)。研究结果表明,浙江地区先天性巨结肠群体可发生EDNRB基因的杂合性突变,提示EDNRB基因与先天性巨结肠症的发病存在一定程度的关联。     

Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2

The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71?454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5-7.5. The K(m) values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 μM. The corresponding V(max) are 504.0, 1910.0, 117.4 and 159.0 μM min(-1) mg(-1). In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility

Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with beta1 and beta3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using alphaIIbbeta3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.     

Mast cells promote atherosclerosis by releasing proinflammatory cytokines

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Atheromata from compound mutant Ldlr(-/-) Kit(W-sh)(/W-sh) mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-alpha-deficient mast cells restored atherogenesis to Ldlr(-/-)Kit(W-sh/W-sh) mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-gamma-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr(-/-)Kit(W-sh/W-sh) mice resulted from the absence of mast cells and mast cell-derived IL-6 and IFN-gamma. Compared with wild-type or TNF-alpha-deficient mast cells, those lacking IL-6 or IFN-gamma did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell-derived IL-6 and IFN-gamma promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell-derived IL-6 and IFN-gamma in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease.