Expression of CYP107Z13 in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK54 catalyzes the oxidation of avermectin to 4″-oxo-avermectin

Streptomyces ahygroscopicus ZB01 has strong catalytic activity for the regiospecific oxidation of 4″-OH of avermectin to form 4″-oxo-avermectin. A cytochrome P450 gene from S. ahygroscopicus ZB01, cyp107z13, was cloned into pKC1139 to generate pKCZ1 and was transformed into Streptomyces lividans TK54, which does not have the ability to catalyze the conversion of avermectin. CYP107Z13, under the control of an ermE* promoter, was actively expressed in the TK54 recombinant strain as determined by a reduced CO difference spectrum analysis of the crude protein. Analysis of whole-cell biocatalytic activity by high-performance liquid chromatography revealed the recombinant to be able to oxidize avermectin regiospecifically to 4″-oxo-avermectin and CYP107Z13 to be a regioselective oxidase of avermectin. In addition, the whole-cell reaction conditions of the recombinant were optimized. Growth on medium ISP-2 at pH 6 was more conducive for the expression of CYP107Z13 than on medium PYG1 or at pH 7, and active cells of the recombinant strain had higher biocatalytic activity than resting cells.     

利用DNA免疫技术制备SAX蛋白多克隆抗体

骨形态发生蛋白(BMP)属于细胞因子和转化生长因子β(TGF-β)超家族,在胚胎形态发生和器官形成中起重要作用,但目前缺乏BMP信号通路I型受体SAX的各类抗体。本文利用果蝇胚胎提取出总RNA,反转录得到cDNA,将其作为模板通过PCR得到sax基因片段,将片段连接到pCAGGS-P7上,构建成重组质粒pCAGGS-P7-sax。采用质粒DNA免疫技术,免疫了BALB/C小鼠,制备了果蝇SAX蛋白多克隆抗体。进一步分析表明,构建的真核表达重组质粒pCAGGS-P7-saxDNA具有良好的免疫原性,在免疫小鼠后所获得的抗血清抗体效价达1∶100。Western blot和果蝇胚胎免疫荧光检测表明,抗血清能特异型地识别SAX蛋白。果蝇SAX抗体的成功制备为进一步研究sax基因及BMP信号在果蝇心脏发育中的作用奠定了基础。     

60Co-γ辐照对越南油茶种子发芽及幼苗形态指标的影响

为了研究⁶⁰Co-γ辐照对越南油茶Camellia drupifera种子发芽和表观形态指标的影响,选取越南油茶璠龙3号‘Fanglong 3’和璠龙5号‘Fanglong 5’种子分别进行0、40 Gy、50 Gy、60 Gy、70 Gy ⁶⁰Co-γ辐照并测定种子发芽率和出苗率以及幼苗生长表观形态指标(苗高、地径、叶长、叶宽、叶面积)。结果表明,⁶⁰Co-γ辐照对越南油茶种子发芽率影响小,对其出苗率、苗高、地径以及叶片生长有明显抑制作用,尤其是高剂量(60 Gy、70 Gy)辐照对越南油茶种子损伤较大,严重影响其出苗及幼苗生长。高剂量辐照明显延迟越南油茶的出苗时间,抑制幼苗生长,导致苗木瘦弱、生长缓慢,植株呈现矮化现象。高剂量亦抑制越南油茶叶片生长,导致叶片瘦小,生长缓慢。璠龙3号种子⁶⁰Co-γ辐照半致死剂量为64 Gy,璠龙5号为57 Gy。     

中耳术后并发耳廓软骨膜炎致耳廓畸形——1例病例报告及文献回顾

目的:通过报道1例中耳术后并发耳廓软骨膜炎致耳廓畸形患者的病历资料,以加强临床医师对该疾病的认识与防范。方法:对本例患者的病历资料进行回顾性阐述,结合该患者的临床特点揭示中耳术后并发耳廓软骨膜炎的发生发展过程,并通过文献回顾阐述该疾病的发病原因、病原学特点及治疗方法。结果:耳廓软骨膜炎是耳科手术术后较为棘手但相对少见的并发症,最为常见铜绿假单胞杆菌感染所致。治疗方法视患者病情而定,包括抗菌药物应用、脓肿切开引流、病变软骨切除术。对于合并糖尿病的老年患者中耳术后更易引发耳廓软骨膜炎,且疗程更长、预后更差。结论:中耳术后并发耳廓软骨膜炎临床需警惕,对合并糖尿病的老年患者应多加防范;早诊断,早治疗,避免增加患者痛苦、加重其经济负担,并与患者做好充分沟通,避免医疗纠纷的发生。     

Overexpression of integrin beta1 inhibits proliferation of hepatocellular carcinoma cell SMMC-7721 through preventing Skp2-dependent degradation of p27 via PI3K pathway

Integrins may play important roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We showed previously that overexpression of integrin beta1 inhibits cell proliferation in SMMC-7721 cells. Here we reported that one of the cyclin-dependent kinase (CDK) inhibitors, p27(Kip1) was involved in proliferation-inhibition induced by overexpression of integrin beta1. Overexpression of integrin beta1 upregulated p27(Kip1) at the protein level, but not mRNA level. The knock-down of p27(Kip1) expression restored cell growth in integrin beta1-overexpressing cells. Cycloheximide (Chx) treatment and pulse-chase experiments revealed that overexpression of integrin beta1 prolonged the half-life of p27(Kip1) by inhibiting its degradation. Proteasome inhibitor (MG132) treatment of the cells indicated that proteasome mediated degradation of p27, and Skp2-dependent degradation might be prevented. Overexpression of integrin beta1 decreased Skp2 at mRNA level, which was regulated by cell adhesion and the subsequent adhesion-dependent signaling. Overexpression of integrin beta1 reduced cell adhesion, accordingly, inactivated the phosphoinositide 3-kinase (PI3K) signaling. PI3K inhibitor LY294002 upregulated p27(Kip1) at post-translational level and downregulate Skp2 at mRNA level, which could mimic the effects of integrin beta1 overexpression on p27(Kip1) and Skp2. Together, these results suggested that overexpression of integrin beta1 inhibited cell proliferation by preventing the Skp2-dependent degradation of p27(Kip1) via PI3K pathway.     

GFP Stable Transfection Facilitated the Characterization of Lung Cancer Stem Cells

Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549–GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549–GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.     

胆囊收缩素及受体基因多态性与精神分裂症的相关性研究

目的：探讨胆囊收缩素（cholecystokinin,CCK）基因、胆囊收缩素A受体（cholecystokininAreceptor。CCKAR）基因和胆囊收缩素B受体（cholecystokinin A recepmr,CCKBR）基因多态性与精神分裂症之间的相关性。方法：采用聚合酶链式反应．限制性片段长度多态性方法,对420例精神分裂症患者（病例组）和455例健康个体（对照组）三个基因的6个单核苷酸多态性（single nucleotide polymorphism,SNPs）位点（rs11571842、rs13069836、rs1800908、rs1800857、rs1042047、rs4758092）的多态性进行检测。并比较两组人群中基因型和等位基因频率分布的差异。结果：对照组6个SNPs位点的基因型频率分布均符合Hardy-Weinbere平衡（P〉0．05）;CCKAR基因rs1800857位点基因型频率分布在精神分裂症组与正常对照组间存在显著性差异（P〈0．000）,病例组T等位基因频率显著高于对照组（P〈0．01）。结论：CCKAR基因多态性与精神分裂症相关,携带T等位基因的个体可能更容易患精神分裂症。