Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Enzyme catalyzed synthesis of some vinyl drug esters in organic medium

A series of vinyl drug esters was synthesized using acyclovir and chloramphenicol with different carbon chain length acyl donors by alkaline protease from Bacillus subtilis and Lipozyme respectively, in non-aqueous medium. The corresponding vinyl drug derivatives were confirmed by nuclear magnetic resonance and infrared spectrometry. The influences of different organic solvents, reaction time, temperature, and content of water on synthesis of vinyl chloramphenicol esters were studied.     

绵羊Myostatin基因敲除载体的构建

根据Myostatin基因突变可导致肌肉量激增而产生"双肌"表型的特点,构建绵羊Myostatin基因置换型敲除栽体.利用LA-PCR技术成功地扩增得到绵羊Myostatin基因同源臂序列,其中同源长臂4.9kb,包括全部的exonl,intronl,exon2及部分启动子和大部分intron2;同源短臂1.1kb,包括部分exon3和31非翻译区序列,将二者连入PloxpⅡ正负筛选敲除骨架载体,利用骨架载体上Neo基因替代Myostatin基因的exon3,从而成功构建专门针对Myostatin第3外显子区域缺失的置换型敲除载体PloxpⅡ-OVIS-MSTN.酶切和测序鉴定证明载体构建正确,为后续获得绵羊Myostatin基因缺失型体细胞株奠定试验基础.     

稻株含氮量和密度对褐飞虱存活、发育和生殖特性的影响

对褐飞虱种群在不同含氮量稻株和若虫密度条件下的反应进行了研究。结果表明,若虫密度对褐飞虱存活的抑制作用随若虫密度提高而增强,但随寄主含氮量的增加而显著下降,表现为低含量的寄主植物可以增强对种群调节的负反馈作用。在低氮稻株上的饲养代数也明显影响若虫存活率,而在高氮稻株上饲养的不同代别褐飞虱之间则无显著差异。若虫率与稻株含氮量呈极显著的负相关,即在高若虫密度下寄主含氮量的增加可显著缩短褐飞虱若虫的发育历期。与高含氮量稻株上的褐飞虱种群相比,饲养在低含氮量稻株上的褐飞虱种群的若虫发育时间在高若虫密度下显著延长。在每盆40头褐飞虱若虫的密度下,成虫性比与稻株含氮量呈极显著的正相关,而在不同若虫密度下,随着若虫密度的增加雌性成虫比例显著下降。在每盆160头的若虫密度时低氮稻株上褐飞虱种群的性比低于0.3,显著低于在高氮稻株上的褐飞虱种群的性比0.85。在含氮量低的稻株上的雌成虫体重随若虫密度的增加极显著减少,连续取食第2代的雌成虫又比取食第1代时的轻。在所研究的所有若虫密度下,取食高含氮量稻株的褐飞虱种群的雌成虫寿命均为取食低含氮量稻株褐飞虱种群雌成虫寿命的3倍左右,差异极为显著。稻株含氮量和若虫密度对褐飞虱生殖力的作用最大,特别是在低氮稻株上若虫密度对褐飞虱生殖力的作用更为突出。在高含氮量稻株上的卵孵化率均随若虫密度的增加而有所下降,但在相同含氮量稻株上卵孵化率的差异均不显著。结果推测由于施用氮肥较多的水稻可以承受高密度的褐飞虱,提高了它们的迁出临界密度,减少了褐飞虱在克服逆境过程中的种群损失,从而造成更高的密度和更重的田间危害程度。     

局部pUDKH基因治疗对实验性犬缺血肢体的某些生理、生化变化的影响

目的：探讨携带人肝细胞生长因子基因的真核表达质粒pUDKH在治疗犬肢体缺血时对其生理、生化的影响。方法：建立犬左下肢血管完全闭塞性血管病模型,一次性局部肌肉注射不同剂量pUDKH,并在不同时间点检测犬生理、生化指标。结果：转染pUDKH组,能使损伤的血管功能恢复,股动脉血流量恢复,脉搏搏动有力,下肢活动正常,肌电图指标无明显改变。血液生化指标在正常值范围内波动。结论：局部pUDKH基因治疗犬肢体缺血后对其生理、生化无明显影响。     

miR-135b promotes proliferation and metastasis by targeting APC in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of microRNA-135b (miR-135b) in TNBC. A real-time polymerase chain reaction assay was used to quantify miR-135b expression levels in 90 paired TNBC tissue and adjacent normal tissue samples. Wound-healing and transwell assays were performed to evaluate the effects of miR-135b expression on the migration and invasion of TNBC cells. Luciferase reporter and western blot analyses were used to verify whether the mRNA encoding APC is a major target of miR-135b. In the current study, we found that miR-135b was highly expressed in TNBC tissue and cells, and the expression levels were correlated with lymph node status and TNM stage. In TNBC cells, the ectopic expression of miR-135b promoted cell proliferation and invasion in vitro. In addition, our study proved that the overexpression of miR-135b significantly suppressed APC expression by targeting the 3′-untranslated region of APC, whereas enhanced APC expression could partially abrogate the miR-135b-mediated promotion of carcinogenic traits in TNBC cells. Taken together, our study demonstrated that miR-135b expression promoted the proliferation and invasion of TNBC by downregulating APC expression, indicating that miR-135b may serve as a promising target for the treatment of TNBC patients.     

In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts

Porcine transgenic cloning has potential applications for improving production traits and for biomedical research purposes. To produce a transgenic clone, kidney fibroblasts from a newborn Guangxi Bama mini-pig were isolated, cultured, and then transfected with red and green fluorescent protein genes using lipofectamine for nuclear transfer. The results of the present study show that the kidney fibroblasts exhibited excellent proliferative capacity and clone-like morphology, and were adequate for generation of somatic cell nuclear transfer (SCNT)-derived embryos, which was confirmed by their cleavage activity and blastocyst formation rate of 70.3% and 7.9%, respectively. Cells transfected with red fluorescent protein genes could be passed more than 35 times. Transgenic embryos cloned with fluorescent or blind enucleation methods were not significantly different with respect to cleavage rates (92.5% vs. 86.8%, p?>?0.05) and blastocyst-morula rates (26.9% vs. 34.0%, p?>?0.05), but were significantly different with respect to blastocyst rates (3.0% vs. 13.2%, p?p?>?0.05), blastocyst (14.1%, 16.1% vs. 23.1%, p?>?0.05) and morula/blastocyst rates (43.5%, 47.0% vs. 57.6%, p?>?0.05) were not significantly different between the groups of transgenic cloned embryos, cloned embryos, and parthenogenetic embryos. This indicates that long-time screening by G418 caused no significant damage to kidney fibroblasts. Thus, kidney fibroblasts represent a promising new source for transgenic SCNT, and this work lays the foundation for the production of genetically transformed cloned Guangxi Bama mini-pigs.     

Mesothelial cells differentiate into fibroblast-like cells under the scirrhous gastric cancer microenvironment and promote peritoneal carcinomatosis in vitro and in vivo

Peritoneal metastases are one reason for the poor prognosis of scirrhous gastric cancer (SGC), and myofibroblast provides a favorable environment for the peritoneal dissemination of gastric cancer. The aim of this study was to determine whether myofibroblast originates from peritoneal mesothelial cells under the influence of the tumor microenvironment. Immunohistochemical studies of peritoneal biopsy specimens from patients with peritoneal lavage cytological (+) status demonstrate the expression of the epithelial markers cytokeratin in fibroblast-like cells entrapped in the stroma, suggesting that these cells stemmed from local conversion of mesothelial cells. To confirm this hypothesis in vitro, we co-incubated mesothelial cells with SGC or non-SGC to investigate morphology and function changes. As we expected, mesothelial cells undergo a transition from an epithelial phenotype to a mesenchymal phenotype with loss of epithelial morphology and decrease in the expression of cytokeratin and E-cadherin when exposed to conditioned medium from HSC-39, and the induction of mesothelial cells can be abolished using a neutralizing antibody to transforming growth factor-beta1 (TGF-β1) as well as by pre-treatment with SB431542. Moreover, we found that these mesothelial cells-derived cells exhibit functional properties of myofibroblasts, including the ability to increase adhesion and invasion of SGC. In summary, our current data demonstrated that mesothelial cells are a source of myofibroblasts under the SGC microenvironment which provide a favorable environment for the dissemination of gastric cancer; TGF-β1 produced by autocrine/paracrine in peritoneal cavity may play a central role in this pathogenesis.     

Characterization and fine mapping of a novel rice albino mutant low temperature albino 1

Albino mutants are useful genetic resource for studying chlorophyll biosynthesis and chloroplast development and cloning genes involved in these processes in plants.Here we report a novel rice mutant low temperature albino 1(lta1) that showed albino leaves before 4-leaf stage when grown under temperature lower than 20℃,but developed normal green leaves under temperature higher than 24℃or similar morphological phenotypes in dark as did the wild-type(WT).Our analysis showed that the contents of chlorophylls and chlorophyll precursors were remarkably decreased in the ltal mutant under low temperature compared to WT.Transmission electron microscope observation revealed that chloroplasts were defectively developed in the albino lta1 leaves,which lacked of well-stacked granum and contained less stroma lamellae.These results suggested that the lta1 mutation may delay the light-induced thylakoid assembly under low temperature.Genetic analysis indicated that the albino phenotype was controlled by a single recessive locus.Through map-based approach,we finally located the Lta1 gene to a region of 40.3 kb on the short arm of chromosome 11.There are 8 predicted open reading frames(ORFs) in this region and two of them were deleted in lta1 genome compared with the WT genome.The further characterization of the Ltal gene would provide a good approach to uncover the novel molecular mechanisms involved in chloroplast development under low temperature stress.