Constitutive activity of endogenous receptors by inducible Gq overexpression

We have developed an inducible cell line that transiently expresses Gq alpha G protein subunits in response to doxycycline. HEK293/Tet-On pBI(Gq alpha) cells worked consistently, achieving high and tightly regulated levels of Gq alpha overexpression (38-fold increase compared with non-induced cells). We investigated the possibility of using an inducible system to increase the proportion of constitutively active endogenously expressed G protein-coupled receptors (GPCRs) by overexpressing Gq alpha. Not only did we observe an increase in basal activity following doxycycline treatment, but also increased intrinsic activity of agonists such as carbachol, endothelin, lysophosphatidic acid (LPA), and bradykinin. Furthermore, carbachol and LPA potency increased following Gq alpha overexpression, as did the intrinsic activity of the partial agonist pilocarpine, observations indicative of constitutive activity. An inducible cell line, transiently expressing G proteins, can therefore be employed to induce constitutive activity of endogenously expressed GPCRs. This model system could be used to identify clinically important inverse agonists.     

Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells

Overexpression of the ErbB2 receptor in one-third of human breast cancers contributes to the transformation of epithelial cells and predicts poor prognosis for breast cancer patients. We report that the overexpression of ErbB2 inhibits IGF-I-induced MAPK signaling. IGF-I-induced MAPK phosphorylation and MAPK kinase activity are reduced in ErbB2 overexpressing MCF-7/HER2-18 cells relative to control MCF-7/neo cells. In SKBR3/IGF-IR cells, reduction of ErbB2 by antisense methodology restores the IGF-I-induced MAPK activation. The inhibition of IGF-I-induced MAP kinase activation in ErbB2 overexpressing breast cancer cells is correlated with decreased IGF-I-induced Shc tyrosine-phosphorylation, leading to a decreased association of Grb2 with Shc and decreased Raf phosphorylation. However, IGF-I-induced tyrosine-phosphorylation of IGF-I receptor and IRS-I and AKT phosphorylation were unaffected by ErbB2 overexpression. Consistent with these results, we observed that the proportion of IGF-I-stimulated proliferation blocked by the MAPK inhibitor PD98059 fell from 82.6% in MCF-7/neo cells to 41.2% in MCF-7/HER2-18 cells. These data provide evidence for interplay between the IGF-IR and ErbB2 signaling pathways. They are consistent with the view that the IGF-IR mediated attenuation of trastuzumab-induced growth inhibition we recently described is dependent on IGF-I-induced PI3K signaling rather than IGF-I-induced MAPK signaling.     

Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli

An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.     

Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration

An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder. The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E. coli cellular protein as r-fGH. The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment. The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration. More than 47% of the r-fGH was recovered from the E. coli cell lysates and the purity of recovered r-fGH was 98%. The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 μg r-fGH g⁻¹ fish body weight, resulted in significant increases both in weight and length. These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.     

Cloning of the Gene Encoding FNR Domain of FNR of Cyanobacterium Synechococcus sp. PCC 7002 and its Overexpression in Escherichia coli

The petHL of Synechnococcus sp. PCC 7002 encoding FNR domain (FNRD) of FNR was amplified by PCR, and cloned into expressing vector pET-3a. Ovemxpression of petHL was achieved with E. coli BL21 (DE3). The recombinant FNRD was purified to homogeneity by DEAE-Sephadex A-50 and Sephadex G-100 chromatography. N-terminal amino acid sequencing showed that rFNRD was encoded by petHL and initial Met was not posttranslationally removed, rFNRD had the same absorption spectrum, optimal pH and optimal temperature as those of rFNR. rFNRD could catalyze photosynthetic electron transport from PT00 to NADP+ in vitro.     

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.     

miR-21的高表达与胰腺癌预后关系的meta分析

为评价miR-21的高表达与胰腺癌预后的相关性,通过全面检索Pub Med,EMBASE,Google scholar,维普,CNKI等数据库,收集已公开发表的关于miR-21的高表达与胰腺癌预后相关性的文献,按meta分析的要求对原始文献的质量进行评估,采用STATA V12.0软件对各研究的效应量进行统计分析。结果发现共纳入4篇文献(共300个病例),合并总生存率(OS)HR为1.26(95%CI:1.08~1.47,P0.05)。由此可以推断胰腺癌预后的一个危险因素为miR-21的高表达。