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31.
为制备用地高辛精(Digoxigenin,Dig)标记的酶氨酸羟化酶(TyrosineHydroxylase,TH)RNA探针,本研究用分子生物学技术重组质粒PGEMTHI,即分别将携带T7和Sp6启动子的PGEM-3zf质粒和携带TH基因的PKSTH质粒用限制性内切酶消化并行分离纯化,得到带T7和sp6启动子的DNA片段和TH基因片段;经T4DNA连接酶连接后转入大肠杆菌,得到pGEMTH1重组克隆。经小量提取质粒井用酶切分析检测,证实其确有TH基因且方向正确。将此质粒再经限制性内切酶消化则得到线状DNA片段,用T7RNA聚合酶转录合成带有Dig标记的高比活度的单链RNA探针;经斑点杂交试验证实该探针具有较高的可靠性。这将为基因治疗帕金森氏病动物模型的疗效检测提供有效手段。 相似文献
32.
Determination of the structure of the DNA binding domain of gamma delta resolvase in solution. 下载免费PDF全文
T. Liu E. F. DeRose G. P. Mullen 《Protein science : a publication of the Protein Society》1994,3(8):1286-1295
The DNA binding domain (DBD) of gamma delta resolvase (residues 141-183) is responsible for the interaction of this site-specific DNA recombinase with consensus site DNA within the gamma delta transposable element in Escherichia coli. Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309-16315). The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 phi and chi 1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180. The DBD consists of 3 alpha-helices comprising residues D149-Q157, S162-T167, and R172-N183. Helix-2 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif. The conformation of the NH2-terminal residues, G141-R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by gamma delta resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases. 相似文献
33.
Dr. Shuichi Ueda Toshikuni Tanabe Norihiko Ihara Yutaka Sano 《Cell and tissue research》1989,256(3):457-463
Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue. 相似文献
34.
目的构建大肠埃希菌强毒力岛(HPI)全岛缺失突变株,为进一步评价大肠埃希菌HPI的功能打下基础。方法根据已知大肠埃希菌HPI基因序列设计PCR敲除引物,引物5′端有50 bp的拟敲除基因的同源臂,3′端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,利用pKD46的λ重组系统替换E.coli ZL基因组上的毒力岛全岛基因,再利用表达Flp重组酶的质粒pCP20,可将FRT位点之间的氯霉素抗性基因删除,用鉴定引物进行鉴定并测序。结果构建的全岛缺失株与预期一致。结论成功构建了禽致病性大肠埃希菌强毒力岛(HPI)全岛缺失突变株。 相似文献
35.
Jespersen T Gavillet B van Bemmelen MX Cordonier S Thomas MA Staub O Abriel H 《Biochemical and biophysical research communications》2006,348(4):1455-1462
In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5. 相似文献
36.
Bäckesjö CM Vargas L Superti-Furga G Smith CI 《Biochemical and biophysical research communications》2002,299(3):510-515
Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates. 相似文献
37.
Summary Immunohistochemical properties of the terminal nerve network in the rat heart were assessed by use of the elution-restaining method. The colocalization of the enzymes involved in catecholamine synthesis (tyrosine hydroxylase — TH, dopamine--hydroxylase — DBH) as well as the respective distributions of the neuropeptides associated with the adrenergic nervous system (neuropeptide tyrosine — NPY, C-terminal flanking peptide of neuropeptide Y — C-PON) were studied in series of serial sections throughout the interatrial septum and the atrioventricular junction. Our data suggest that ganglion cells of sulcus terminalis as well as the epicardial ganglia enclosed between the superior vena cava and ascending aorta are VIP- and TH-negative, but neuropeptide Y- and DBH-immunoreactive. They give rise to three intraseptal nerves directed towards the specialised structures of the atrioventricular junction. These nerve fascicles contain abundant, thick TH-immunoreactive nerve fibres and scarce, thin NPY- and DBH-immunoreactive fibres. The cell bodies of the intramural ganglion cells localized between the right and left branches of the bundle of His (Moravec and Moravec 1984) are strongly TH- and DBH-immunoreactive. They are innervated by thick nerve fibres having the same immunohistochemical properties (NPY- and DBH-immunoreactivities) as those of a subpopulation of the epicardial ganglion cells and seem to supply some of the TH-immunoreactive nerve fibres directed via the intraseptal nerves to the epicardial ganglia. The existence of a multicomponent nerve network, characterized by a reciprocal innervation of the sinus node and atrioventricular node areas, is suggested by our immunohistochemical data. 相似文献
38.
Yi Gao XiaoWu Fan WeiNa Li Wei Ping Yu Deng XiangNing Fu 《Biochemical and biophysical research communications》2014
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib are clinically effective treatments for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, therapeutic effect is ultimately limited by the development of acquired TKI resistance. MicroRNAs (miRNAs) represent a category of small non-coding RNAs commonly deregulated in human malignancies. The aim of this study was to investigate the role of miRNAs in gefitinib resistance. We established a gefitinib-resistant cell model (PC9GR) by continually exposing PC9 NSCLC cells to gefitinib for 6 months. MiRNA microarray screening revealed miR-138-5p showed the greatest downregulation in PC9GR cells. Re-expression of miR-138-5p was sufficient to sensitize PC9GR cells and another gefitinib-resistant NSCLC cell line, H1975, to gefitinib. Bioinformatics analysis and luciferase reporter assay showed that G protein-coupled receptor124 (GPR124) was a direct target of miR-138-5p. Experimental validation demonstrated that expression of GPR124 was suppressed by miR-138-5p on protein and mRNA levels in NSCLC cells. Furthermore, we observed an inverse correlation between the expression of miR-138-5p and GPR124 in lung adenocarcinoma specimens. Knockdown of GPR124 mimicked the effects of miR-138-5p on the sensitivity to gefitinib. Collectively, our results suggest that downregulation of miR-138-5p contributes to gefitinib resistance and that restoration of miR-138-5p or inhibition GPR124 might serve as potential therapeutic approach for overcoming NSCLC gefitinib resistance. 相似文献
39.
Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation. 相似文献
40.
SeongJae Jang Katsuki Ohtani Atsushi Fukuoh Kenichiro Mori Takayuki Yoshizaki Noritoshi Kitamoto YounUck Kim Yasuhiko Suzuki Nobutaka Wakamiya 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014