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991.
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以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。 相似文献
994.
成体多能干细胞,如来自骨髓和脂肪组织的间充质干细胞等具有多向分化的潜能。虽然自体干细胞移植已经发展成为器官移植的有效代替疗法之一,但是由于移植位点细胞的流失和分化条件的限制等问题使得这种疗法的效率大大降低。本研究目的是将由脂肪干细胞分化而来的类肝细胞制备成具有稳定细胞性状的可移植的肝细胞片。首先在体外分离扩增脂肪干细胞,并通过控制严格地分化条件获得类肝细胞。然后将此细胞接种到聚N-异丙基丙烯酰胺(PNIPAAm)结合的细胞培养皿表面,通过调节培养温度到20oC,使细胞成片脱离培养皿形成细胞片。对细胞片进行了常规HE染色和免疫组化观察,结果显示:这类细胞片中平均含有2~3层细胞,并且保持了细胞外基质的完整。同传统的胰酶消化收集移植用细胞相比,细胞片方法极大地减少了对移植用细胞的细胞膜和细胞外基质的损伤,这将大大促进细胞片和原位组织的相互作用,增加细胞利用效率,从而有望提高治疗效果。 相似文献
995.
Vicky P. Chen Heidi Q. Xie Wallace K. B. Chan K. Wing Leung Gallant K. L. Chan Roy C. Y. Choi Suzanne Bon Jean Massoulié Karl W. K. Tsim 《The Journal of biological chemistry》2010,285(35):27265-27278
Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChET and BChET with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (QN-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or QN-GPI always consist of AChET and BChET homodimers. The dimer formation of AChET and BChET depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChET or BChET homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission. 相似文献
996.
Reductions of carbonyl groups catalyzed by oxidoreductases are involved in all biological processes and are often a class of important biocatalyst. In this article, we report a novel enzyme designated as diketoreductase (DKR) that was able to reduce two carbonyl groups in a diketo ester to corresponding dihydroxy ester with excellent stereoselectivity. The DKR was cloned from Acinetobacter baylyi by reverse genetic method, heterogeneously expressed in Escherichia coli, and purified to homogeneity by two chromatographic steps. This novel enzyme exhibited dual cofactor specificity, with a preference of NADH over NADPH. The dihydroxy ester product catalyzed by the DKR was only 3R,5S-stereoisomer with both diastereomeric excess and enantiomeric excess values more than 99.5%. In addition, some biochemical properties of the enzyme, such as the optimal pH and temperature, were also characterized. Furthermore, sequence analysis indicated that this new enzyme was homologous to bacterial 3-hydroxyacyl coenzyme-A dehydrogenase. More importantly, based on the unique catalytic activity and excellent stereoselectivity, the DKR could be utilized in the synthesis of valuable chiral drug intermediates, such as Lipitor. 相似文献
997.
The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment. 相似文献
998.
Qi Ye Ming Yan Lin Xu Hou Cao Zhenjiang Li Yong Chen Shuya Li Hanjie Ying 《Biotechnology letters》2009,31(4):537-542
An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity
to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction
of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess. 相似文献
999.
1000.
Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells 下载免费PDF全文
Qingyu Zeng Shanshan Qin Hai Zhang Beibei Liu Jiamin Qin Xiaoxue Wang Ruijie Zhang Chunxiao Liu Xiaoqing Dong Shuangquan Zhang Shile Huang Long Chen 《Journal of cellular physiology》2018,233(1):516-529
B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases. 相似文献