草鱼出血病病毒基因组的cDNA合成、克隆及部分序列分析

采用差速离心的方法纯化感染草鱼出血病病毒的细胞悬液。提纯的病毒粒子经蛋白酶Ｋ处理和酚氯仿抽提,在１％琼脂糖凝胶电泳条件下分离得到１１条ｄｓＲＮＡ,利用柱离心式胶回收试剂盒纯化各基因片段。纯化的ｄｓＲＮＡ溶于９０％的ＤＭＳＯ中,７０℃变性１５ｍｉｎ,然后采用随机引物法反转录合成各基因片段的ｃＤＮＡ,并平头连接于ｐＺＥｒＯ２．０载体的ＥｃｏＲＶ位点,电转化ＴＯＰ１０感受态细胞。重组质粒经酶切、ＰＣＲ扩增得到大小不等的插入片段,ｃＤＮＡＲＮＡ斑点杂交的结果进一步证实其插入为目的基因片段。采用循环ＰＣＲ测序的方法,对其插入片段进行了序列测定,对其中第１１片段的部分序列作了报道。     

真菌一氧化氮还原酶细胞色素P—450nor 2 cDNA序列的测定

真菌一氧化氮还原酶细胞色素Ｐ４５０ｎｏｒ２ｃＤＮＡ序列的测定刘德立（华中师范大学生命科学学院武汉４３００７０）ＨｉｒｏｆｕｍｉＳＨＯＵＮ（筑波大学应用生物化学系日本）在真菌的反硝化作用中,一种细胞色素Ｐ４５０起着一氧化氮还原酶的作用,被称为细胞色...     

人乳铁蛋白cDNA的克隆及序列分析

从北京正常人乳腺组织中提取总RNA,用RT-PCR的方法扩增人乳铁蛋白(hLF)的cDNA,将其克隆到pGEM-T载体上并进行DNA序列测定。结果表明,所克隆的hLF cDNA序列全长为2136bp,其DNA序列与GenBank中另外5个hLF cDNA序列相比,有2个碱基与这5个序列不同：1740位这5个序列是G,本文序列是C;1756位这5个序列是T,本文序列是C。其中1740位碱基的变化导致了第580位氨基酸由Glu变为Asp。     

Identification of Sequence Motifs Causing Band Compressions on Human cDNA Sequencing

In order to characterize DNA sequences leadingto band compressionsin an automated dideoxy-DNA sequencing system which uses fluorescentdye primers, we compiled DNA sequences at compression sitesfrom accumulated sequence data of human cDNAs (about 205 kbin total length). The results clearly showed that almost allthe 3'-end regions at the compression sites (> 98%) carriedtwo types of common sequence motifs. The predominant one (about68%) contained a sequence of 5'-Y'GN_1–2AR'-3' (Y' andR': pyrimidine and purine residues capable of base pairing).The remainder (about 32%) carried a hairpin motif with a relativelystable GC-rich stem ( 3 bp) connected by a loop consistingof3 or 4 nucleotides. The occurrence of compressions at thesemotif sites was further confirmed by using synthetic DNAs withrandom sequences (about 58 kb in total length). Since DNA sequencesat compression sites analyzed so far shared either of the typeof motifs in the sequencing system employed here, it was possibleto predict the nucleotide residue to be located at a compressionsite by carefully checking the sequence preceding the site.     

Partial cDNA Cloning and Nucleotide Sequence of Rice Dwarf Virus Genome

Rice dwarf virus (RDV) was isolated and purified from infected rice leaves with chloro form extraction, PEG precipitation and sucrose gradient centrifugation. Total RDV RNA ge nome was separated in the agarose gel and segments of RDV RNA genome were purified. The cDNAs of several segments were synthesized with oligo dT as primer. Through cDNA mapping, subcloning and sequencing, we have obtained partial DNA sequence of those segments. Here we report the cloning and partial DNA sequence of segment 8 from RDV RNA genome.     

识别序列为非回纹对称结构限制核酸酶的正确切割位点

限制性内切核酸酶的切割识别序列分为回纹对称和非回纹对称结构两类,由于DNA是互补双链,所以对于识别序列为回纹对称结构的限制酶,其识别序列在DNA的两条链上是一致的,可以写为一个,但对于识别序列为非回纹对称结构的限制酶来说,其识别序列应为两个,而一些工具书、参考书中仅写为一个。本通过一个酶切实验证明其识别序列为两个。同时希望通过本,敦促一些工具书、参考书更正其错误。     

Brain metastasis in lung cancer: Building a molecular and systems-level understanding to improve outcomes

Lung cancer is a clinically difficult disease with rising disease burden around the world. Unfortunately, most lung cancers present at a clinically advanced stage. Of these cancers, many also present with brain metastasis which complicates the clinical picture. This review summarizes current knowledge on the molecular basis of lung cancer brain metastases. We start from the clinical perspective, aiming to provide a clinical context for a significant problem that requires much deeper scientific investigation. We review new research governing the metastatic process, including tumor cell signaling, establishment of a receptive tumor niches in the brain and evaluate potential new therapeutic options that take advantage of these new scientific advances.Lung cancer remains the largest single cause of cancer mortality in the United States (Siegel et al., 2015). This continues to be the clinical picture despite significant advances in therapy, including the advent of targeted molecular therapies and newly adopted immunotherapies for certain subtypes of lung cancer. In the vast majority of cases, lung cancer presents as advanced disease; in many instances, this advanced disease state is intimately associated with micro and macrometastatic disease (Goldberg et al., 2015). For both non-small cell lung cancer and small cell lung cancer patients, the predominant metastatic site is the brain, with up to 68% of patients with mediastinal lymph node metastasis eventually demonstrating brain metastasis (Wang et al., 2009).The frequency (incidence) of brain metastasis is highest in lung cancers, relative to other common epithelial malignancies (Schouten et al., 2002). Other studies have attempted to predict the risk of brain metastasis in the setting of previously non-metastatic disease. One of the largest studies to do this, analyzing historical data from 1973 to 2011 using the SEER database revealed a 9% risk of patients with previously non-metastatic NSCLC developing brain metastasis over the course of their disease, while 18% of small cell lung cancer patients without previous metastasis went on to develop brain metastasis as their disease progressed (Goncalves et al., 2016).The reasons underlying this predilection for the central nervous system, as well as the recent increase in the frequency of brain metastasis identified in patients remain important questions for both clinicians and basic scientists. More than ever, the question of how brain metastasis develop and how they can be treated and managed requires the involvement of interdisciplinary teams—and more importantly—scientists who are capable of thinking like clinicians and clinicians who are capable of thinking like scientists. This review aims to present a translational perspective on brain metastasis. We will investigate the scope of the problem of brain metastasis and the current management of the metastatic disease process in lung cancer. From this clinical starting point, we will investigate the literature surrounding the molecular underpinnings of lung tumor metastasis and seek to understand the process from a biological perspective to generate new hypotheses.     

The substance P/neurokinin-1 receptor system in lung cancer: Focus on the antitumor action of neurokinin-1 receptor antagonists

The last decades have seen no significant progress in extending the survival of lung cancer patients and there is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectively induce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of this system in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out in depth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23 (NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpress the tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5) NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines in a concentration-dependent manner; (6) the specific antitumor action of these antagonists against such cells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstrate the presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicate that the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists could be new candidate antitumor drugs in the treatment of SCLC and NSCLC.     

The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G₁/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.     

New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships

Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.     

DADS对人小细胞肺癌NCI-H446 细胞系增殖抑制的研究*

摘要目的：研究二烯丙基二硫（diallyl disulfide,DADS）对人小细胞肺癌NCI-H446细胞增殖的抑制作用,并探讨其作用机制。方法：体外培养NCI-H446 细胞,采用MTT、细胞计数实验方法检测DADS 抑制NCI-H446 细胞增殖;通过HE 染色和AO-EB 荧光染色方法,观察DADS 处理后NCI-H446 细胞的形态学改变。结果：MTT 结果显示：DADS作用于NCI-H446 细胞48 h后,代谢MTT 的能力明显降低,显示出较强的细胞毒性反应,IC50值介于20～40 滋g/ml之间。细胞计数结果表明：DADS 作用于NCI-H446细胞后,随DADS 浓度增加NCI-H446 细胞倍增时间延长。HE染色显示：NCI-H446 细胞经DADS处理24 h后,与对照组相比,细胞体积变小,胞浆丰富,细胞核变小,染色变淡。AO-EB 荧光染色显示：NCI-H446 细胞经DADS处理24 h后,与对照组相比,细胞皱缩、呈圆形,胞质黄色或橘红色,细胞核或细胞质内可见致密浓染的黄绿色或橘红色荧光,并可见橘红色碎片且随DADS 浓度增加,随DADS浓度增加细胞密度逐渐减少。结论：DADS 能抑制体外培养的NCI-H446 细胞增殖,作用效果与药物浓度及作用时间相关。     

DADS对人小细胞肺癌NCI-H446细胞系增殖抑制的研究

目的：研究二烯丙基二硫（diallyldisulfide,DADS）对人小细胞肺癌NCI．H446细胞增殖的抑制作用,并探讨其作用机制。方法：体外培养NCI-H446细胞,采用MTT、细胞计数实验方法检测DADS抑制NCI—H446细胞增殖;通过HE染色和AO—EB荧光染色方法,观察DADS处理后NCI—H446细胞的形态学改变。结果：MTT结果显示：DADS作用于NCI—H446细胞48h后,代谢MTT的能力明显降低,显示出较强的细胞毒性反应,IC50值介于20-40μg／ml之间。细胞计数结果表明：DADS作用于NCI—H446细胞后,随DADS浓度增加NCI—H446细胞倍增时间延长。HE染色显示：NCI—H446细胞经DADS处理24h后,与对照组相比,细胞体积变小,胞浆丰富,细胞核变小,染色变淡。AO-EB荧光染色显示：NCI-H446细胞经DADS处理24h后,与对照组相比,细胞皱缩、呈圆形,胞质黄色或橘红色,细胞核或细胞质内可见致密浓染的黄绿色或橘红色荧光,并可见橘红色碎片且随DADS浓度增加,随DADS浓度增加细胞密度逐渐减少。结论：DADS能抑制体外培养的NCI—H446细胞增殖,作用效果与药物浓度及作用时间相关。     

肺癌相关肿瘤抗原基因的分离及鉴定

通过分离肺癌肿瘤抗原 ,为肺癌早期诊断提供血清学标志物 ,并为研制肺癌疫苗提供候选抗原 ,成功构建了库容为 0 .8× 10 6个重组子的肺癌原发病灶组织的cDNA表达文库。采用SEREX技术对该cDNA表达文库进行筛选 ,获得了 33个阳性克隆 ,包括黑色素瘤抗原基因 (MAGE)、白斑相关蛋白基因、纤连蛋白基因、钠钾ATP酶基因等已知基因或EST ,另外有 18个基因或EST在GenBank数据库中没有同源性 ,可能是新的基因或EST。选取其中 3个克隆进行肺癌患者及正常人群血清的检测 ,结果显示肺癌患者的阳性率明显高于正常人群     

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC₅₀ = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC₅₀ = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC₅₀ = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC₅₀ = 1.53 ± 0.24 μm) than for hGDH1 (IC₅₀ = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.     

牛肝TrnaIle的序列分析和二级结构

用随机降解法和Donis-Keller酶分析法,测定了牛肝tRNA^Ile序列.牛肝tRNA^Ile长77个碱基;G5·G69不配对为其显著特征.依据tRNA螺旋区和环区自由能大小及Holley模型,确定了tRNA^Ile的二级结构.     

TN-C在小细胞肺癌中的表达及STAT3对TN-C表达的影响

目的：探讨小细胞肺癌（SCLC）组织和小细胞肺癌细胞（H446）中肌糖蛋白-C（TN-C）的表达及STAT3对TN-C表达的影响。方法：应用免疫组化法检测58例小细胞肺癌和17例癌旁正常组织中TN-C的表达水平,应用RT-PCR和Western blotting法检测STAT-siRNA和STAT3过表达的H446细胞中TN-C的表达水平。结果：（1）小细胞肺癌组织中TN-C的表达水平显著高于癌旁正常组织（P〈0.05）;（2）在H446细胞中,TN-C和STAT3均呈现高表达;（3）STAT3-siRNA处理的H446细胞中STAT3和TN-C的表达均显著降低（P〈0.05）,而STAT3过表达的H446细胞中STAT3和TN-C的表达均显著上调（P〈0.05）。结论：TN-C在小细胞肺癌中的表达上调,可能受到STAT3的调控。     

TN-C 在小细胞肺癌中的表达及STAT3 对TN-C 表达的影响

目的：探讨小细胞肺癌(SCLC)组织和小细胞肺癌细胞(H446)中肌糖蛋白-C(TN-C)的表达及STAT3 对TN-C表达的影响。 方法：应用免疫组化法检测58 例小细胞肺癌和17 例癌旁正常组织中TN-C 的表达水平,应用RT-PCR和Western blotting 法检测 STAT-siRNA和STAT3 过表达的H446 细胞中TN-C 的表达水平。结果：(1)小细胞肺癌组织中TN-C 的表达水平显著高于癌旁正 常组织(P<0.05);(2)在H446细胞中,TN-C 和STAT3 均呈现高表达;(3)STAT3-siRNA 处理的H446 细胞中STAT3 和TN-C 的表 达均显著降低(P<0.05),而STAT3 过表达的H446 细胞中STAT3 和TN-C 的表达均显著上调(P<0.05)。结论：TN-C 在小细胞肺癌 中的表达上调,可能受到STAT3 的调控。     

小细胞肺癌患者外周血淋巴细胞亚群分析

目的 探究化疗对小细胞肺癌(small cell lung cancer,SCLC)患者免疫功能的影响。 方法 选择2013年1月到2018年12月我院收治的95例小细胞肺癌患者为研究对象。患者第一周期、第二周期化疗前采用流式细胞术检测患者外周血淋巴细胞亚群水平,分别按照不同疗效及不同化疗方案对患者外周血淋巴细胞亚群进行比较。 结果 (1)化疗后,95例患者CD3+、CD4+、CD8+细胞平均值增加,CD19+、γδT细胞平均值减少,差异均有统计学意义(均P+、CD8+细胞平均值增加,CD19+细胞减少,差异有统计学意义(均P0.05)。(3)依托泊苷联合顺铂(EP)方案组化疗后患者CD3+、CD8+细胞平均值增多,CD19+细胞减少,差异均有统计学意义(均P0.05)。 结论 化疗可以调节小细胞肺癌患者的免疫功能,增强细胞免疫,降低体液免疫,其中EC方案对患者细胞免疫的增强作用较为显著。