PML/RARalpha fusion protein mediates the unique sensitivity to arsenic cytotoxicity in acute promyelocytic leukemia cells: Mechanisms involve the impairment of cAMP signaling and the aberrant regulation of NADPH oxidase

Acute promyelocytic leukemia (APL) cells are characterized by PML/RARalpha fusion protein, high responsiveness to arsenic trioxide (ATO)-induced cytotoxicity and an abundant generation of reactive oxygen species (ROS). In this study we investigated the association among these three features in APL-derived NB4 cells. We found that NADPH oxidase-derived ROS generation was more abundant in NB4 cells compared with monocytic leukemia U937 cells. By using PR9, a sub-line of U937 stably transduced with the inducible PML/RARalpha expression vectors, we attributed disparities on ROS generation and ATO sensitivity to the occurrence of PML/RARalpha fusion protein, since PML/RARalpha-expressing cells appeared higher NADPH oxidase activity, higher ROS level and higher sensitivity to ATO. On the other hand, the basal intensity of cAMP signaling pathway was compared between NB4 and U937 as well as between PR9 cells with or without PML/RARalpha, demonstrating that PML/RARalpha-expressing cells had an impaired cAMP signaling pathway which relieved its inhibitory effect on NADPH oxidase derived ROS generation. In summary, the present study demonstrated the correlation of PML/RARalpha with cAMP signaling pathway, NADPH oxidase and ROS generation in APL cells. PML/RARalpha that bestows NB4 cells various pathological features, paradoxically also endows these cells with the basis for susceptibility to ATO-induced cytotoxcity.     

Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell–cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.     

Structural Basis for the Biological Effects of Pr(III) Ions: Alteration of Cell Membrane Permeability

The biological effects of rare-earth ions on the organism have been studied using Pr³⁺ as a probe ion and Escherichia coli cell as a target. Atomic force microscopy (AFM) observation of the surface of E. coli cells shows that the presence of Pr³⁺ substantially changes the structure of the outer membrane. By induced coupled plasma-mass spectrometry (ICP-MS), more Cu²⁺ was found in the cells grown in the presence of Pr³⁺, indicating changes of cell permeability. Using energy dispersive X-ray spectroscopy (EDX), Ca²⁺ is found on the outer surface of the original cell. It is proposed that Pr³⁺ can replace Ca²⁺ from the binding sites because of their close ionic radii and similar ligand speciality.     

猪肥胖基因5'调控区与内含子1的测序分析及其两个新微卫星的发现

根据猪肥胖基因(Obese gene,Ob)部分序列设计引物,并通过Southern杂交和杂交阳性片段的克隆进行测序,首次得到猪Ob基因内含子1和5'调控区16.4 kb序列,将外显子1定位在起始密码子上游11.1 kb处,同时在内含子1中发现了两个新的微卫星,命名为SW200和SW160.调控区潜在的调控元件分析显示,-1～ -300 bp区间包含了C/EBP和两个Sp1,可能更直接高效的调节其转录.为研究SW200和SW160与一些重要经济性状的关系,分析了等位基因和基因型频率,用SAS8.2软件分析显示,两个微卫星多态仅对二花脸猪的头胎总产仔数有显著的效应.     

植物防御反应中活性氧的产生和作用

活性氧在植物抗病性中起着重要的作用。本文将对其在防御反应中的产生和作用进行简要的论述。     

人乳头状瘤病毒协同60钴照射促进食管上皮细胞恶性转化

为探明人乳头状瘤病毒(HPV)和促癌剂对食管上皮致癌作用,人胚食管上皮细胞转染HPV协同60钴(60Co)放射观察其恶性转化.用HPV18E6E7AAV转染的人胚食管上皮(SHEE),培养至13代,分为4组,实验组分别用60Co2、4、8Gy照射,每周1次共4周;SHEE未经照射为对照组.细胞形态用相差显微镜观察;细胞DNA合成和定量用3H-TdR掺入和用流式细胞仪分析;染色体众数用常规方法分析;致瘤性用软琼脂培养和裸小鼠接种;HPVDNA用PCR检测.经60Co照射后细胞呈凋亡和坏死(危象期).8周后SHEE 4Gy组细胞增殖,增殖指数(34%)和3H-TdR摄入增高,软琼脂培养和裸鼠接种出现致瘤性.对照组SHEE组细胞增殖指数24%,伴有少数3H-TdR掺入,裸鼠未成瘤.染色体众数:对照组,58～62;4Gy组,63～65;两组HPV18E6E7 PCR呈阳性条带.此结果表明,用HPV18E6E7协同60Coγ射线可以使人胚食管上皮恶性转化,60Co γ射线有加速食管上皮细胞恶性转化作用.     

Impact of emotion on consciousness: positive stimuli enhance conscious reportability

Emotion and reward have been proposed to be closely linked to conscious experience, but empirical data are lacking. The anterior cingulate cortex (ACC) plays a central role in the hedonic dimension of conscious experience; thus potentially a key region in interactions between emotion and consciousness. Here we tested the impact of emotion on conscious experience, and directly investigated the role of the ACC. We used a masked paradigm that measures conscious reportability in terms of subjective confidence and objective accuracy in identifying the briefly presented stimulus in a forced-choice test. By manipulating the emotional valence (positive, neutral, negative) and the presentation time (16 ms, 32 ms, 80 ms) we measured the impact of these variables on conscious and subliminal (i.e. below threshold) processing. First, we tested normal participants using face and word stimuli. Results showed that participants were more confident and accurate when consciously seeing happy versus sad/neutral faces and words. When stimuli were presented subliminally, we found no effect of emotion. To investigate the neural basis of this impact of emotion, we recorded local field potentials (LFPs) directly in the ACC in a chronic pain patient. Behavioural findings were replicated: the patient was more confident and accurate when (consciously) seeing happy versus sad faces, while no effect was seen in subliminal trials. Mirroring behavioural findings, we found significant differences in the LFPs after around 500 ms (lasting 30 ms) in conscious trials between happy and sad faces, while no effect was found in subliminal trials. We thus demonstrate a striking impact of emotion on conscious experience, with positive emotional stimuli enhancing conscious reportability. In line with previous studies, the data indicate a key role of the ACC, but goes beyond earlier work by providing the first direct evidence of interaction between emotion and conscious experience in the human ACC.     

Antifungal constituents from the Chinese moss Homalia trichomanoides

Bioautographic assay on TLC plates was adopted to guide the fractionation of the Et2O extract of Homalia trichomanoides (Hedw.) B. S. G., which led to the isolation of the novel p-terphenyl derivative trichomanin (= 4,4'-dihydroxy-1,1':4',1'-terphenyl-2',3',5',6'-tetrayl tetrakis(phenylacetate); 1), together with five known compounds: 3alpha-methoxyserrat-14-en-21beta-ol (2), 3beta-methoxyserrat-14-en-21beta-ol (3), 3beta-methoxyserrat-14-en-21-one (4), atranorin (5), and methyl 2,4-dihydroxy-3,6-dimethylbenzoate (6). Their structures were determined on the basis of spectral data (1D- and 2D-NMR, MS), X-ray crystallographic analysis, and chemical transformation. Compounds 3, 5, and 6 exhibited antifungal activity against Candida albicans, with minimum inhibitory doses (MID) of 2.0, 2.0, and 0.6 microg, respectively.     

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.