Complex terminal structure of a linear mitochondrial plasmid from Physarum polycephalum: three terminal inverted repeats and an ORF encoding DNA polymerase

The mitochondria of Physarum polycephalum have a linear plasmid (mF) which promotes mitochondrial fusion. To determine the terminal structure of the mF plasmid, restriction fragments derived from its ends were cloned and sequenced. The sequences showed that the mF plasmid has three kinds of terminal inverted repeats (TIRs). The most characteristic feature is a 144-bp repeating unit which exists between a 205-bp TIR at the extreme ends of the plasmid and another 591-bp TIR. All of the clones showed at least one of these 144-bp repeating units. The GC content of the 205-bp TIR (49%) was higher than those of the other TIRs and of another sequenced region (23%). This TIR can form three thermodynamically-stable hairpin structures based on complex internal palindromic components. Moreover, in the right terminal region of the mF plasmid, there is an open reading frame (ORF) which covers the entire 591-bp TIR and most of one of the 144-bp repeating units. This ORF encodes a 547-amino-acid polypeptide, ORF-547, and shows extensive homology with the polymerization domain of the putative DNA polymerases of linear mitochondrial plasmids from other sources.     

Trait anxiety and autonomic indicators of the processing of threatening information: a cued S1-S2 paradigm

The aim of this study was to use autonomic parameters in a cued S1-S2 task to examine associations between the processing of threatening information and trait anxiety in normal individuals. Forty-six student volunteers were designated high- or low-anxious due to pre-defined cutoff scores on the STAI. A cued S1-S2 task was presented in which the type of warning signal (S1) was consistently related to either threatening or non-threatening pictures (S2). Ten threat and 10 non-threat pictures were randomly presented. Heart rate and electrodermal activity were recorded in the time interval between S1 and S2. Results indicated deeper heart rate decelerations on threatening trials in high-anxious as compared to low-anxious individuals. For non-threatening trials, the opposite pattern was found. Moreover, high-anxious participants exhibited higher electrodermal responses to the S1, irrespective of the trial's valence as well as stronger responses to the threatening S2. Autonomic responses can, thus, be regarded as sensitive markers of information processing differences in trait anxiety.     

Oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling

Oleanolic acid has significant pharmacological activities, such as anti-tumor, regulating blood sugar level and liver protection, which are more effective compared with free aglyconeoleanolic acid. However, it is still unknown if oleanolic acid affects the proliferation of human bladder cancer. We utilized T24 cells to study the effect of oleanolic acid on the proliferation and apoptosis of human bladder cancer. In this study, we found that the anti-cancer effect of oleanolic acid significantly suppressed cell proliferation and increased apoptosis and caspase-3 activity of T24 cells. Furthermore, Akt, mTOR and S6K protein expression was greatly inhibited in T24 cells under oleanolic acid treatment. Meanwhile, ERK1/2 of phosphorylation protein expression was significantly promoted by oleanolic acid treatment. Taken together, we provided evidences that oleanolic acid was Akt/mTOR/S6K and ERK1/2 signaling-targeting anti-tumor agent. These findings represent new evidences that oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, and oleanolic acid may be used to prevent human bladder cancer.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.     

慢阻肺引发肺动脉高压预后评价与ET-1、H2S、NO的相关性研究

目的探讨慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)引发肺动脉高压(pulmonary arterial hypertension,PAH)预后评价与内皮素(ET-1)、内源性硫化氢(H_2S)和一氧化氮(NO)的相关性研究。方法选取2016年1月至2017年12月本院确诊的慢阻肺并发肺动脉高压患者120例,按肺动脉压水平分为并发轻度PAH组(45例)、中度PAH组(42例)、重度PAH组(33例),选取COPD不并发肺动脉高压的患者为对照组(40例)。分别检测各组受试者外周静脉血中ET-1、H_2S和NO表达水平。结果与对照组相比,慢阻肺并发PAH患者血浆内ET-1水平显著升高,H_2S和NO水平显著降低(P <0. 05);随着疾病程度的加重,患者血清内各指标变化趋势更为显著(P <0.05)。与治疗前相比,各组患者血清内ET-1水平显著降低,H_2S和NO水平显著升高(P<0.05)。与对照组相比,慢阻肺并发PAH患者FEV_1/FVC(%)及FEV_1%预计值显著降低(P<0.05)。随着疾病程度的加重,患者FEV_1/FVC(%)及FEV_1%预计值降低更为明显(P<0.05)。与治疗前相比,患者FEV_1/FVC(%)及FEV,%预计值显著升高(P<0.05)。血浆H_2S、NO水平与FEV_1/FVC(%)、FEV_1%预计值呈显著正相关(P<0.05),ET-1水平则与其呈明显负相关(P<0.05)。血浆H_2S、NO水平与患者肺动脉压力呈显著负相关(P<0.05),ET-1水平则与其呈明显正相关(P <0.05)。血浆H_2S水平和NO水平与呼吸系统评分、循环系统评分、躯体功能评分、认知功能评分、社会功能评分及生活质量评分呈显著正相关,ET-1水平与呼吸系统评分、循环系统评分、躯体功能评分、认知功能评分、社会功能评分及生活质量评分呈负相关。结论慢阻肺并发肺动脉高压患者体内ET-1、H_2S和NO表达水平与肺功能及病情变化密切相关,可作为监测病情严重程度的良好指标。     

Expression of the hMLH1 and hMSH2 proteins in normal tissues: relationship to cancer predisposition in hereditary non-polyposis colon cancer

The majority of tumours in patients with hereditary non-polyposis colon cancer (HNPCC) occur in large intestine and endometrium; also, other tissues are at increased risk. We studied expression of hMLH1 and hMSH2 proteins in 148 normal samples of various tissues from non-HNPCC patients and in 14 normal colon tissues from HNPCC patients. Immunohistochemical technique was used. Intensity of nuclear staining, percentage of stained cells and H-scores were calculated. Tissues were divided into groups. Groups A, B and C included tissues with increased risk of cancer in HNPCC A) stomach, small and large bowel; (B) endometrium; (C) ovary, ureter, urinary bladder, kidney and liver. Group D tissues were without increased risk. Expression of the proteins was significantly higher in groups A, B and C compared with group D (P<0.0001, P=0.0004 for hMSH2 in C versus D). The expression was highest in testis. In colons of HNPCC patients, expression of the mutated gene product was significantly lower than in non-HNPCC patients. In conclusion, hMLH1/hMSH2 protein expression is constitutively higher in certain cell types of certain tissues, including the majority of tissues that are at increased risk of cancer in HNPCC. However, association of strong hMLH1/hMSH2 expression with cancer risk is not strictly valid.     

Maturation of human monocyte-derived dendritic cells (MoDCs) in the presence of prostaglandin E2 optimizes CD4 and CD8 T cell-mediated responses to protein antigens: role of PGE2 in chemokine and cytokine expression by MoDCs

Prostaglandin E2 (PGE2) acts in synergy with other inflammatory stimuli such as tumor necrosis factor (TNF) to induce the maturation of migratory-type monocyte-derived dendritic cells (MoDCs). However, PGE2 has been reported to inhibit IL-12p70 production by MoDCs and to promote the generation of Th2 T cell responses. We demonstrate here that the addition of PGE2 to TNF for the maturation of MoDCs enhanced CD4 and CD8 T cell proliferative responses to neoantigen and recall antigen, and enhanced Th1-type responses. The increased stimulatory capacity of MoDCs matured with PGE2 was associated with a fully mature, migratory-type MoDC phenotype and more rapid down-regulation of the expression of inflammatory chemokines, with up-regulated expression of the constitutive chemokines TARC and MDC. In addition, although MoDCs matured with TNF and PGE2 selectively produced the inhibitory IL-12p40 subunit at steady state, they were able to produce the bioactive IL-12p70 heterodimer after stimulation with CD40 ligand and/or IFN-gamma. Despite increased IL-6 mRNA expression, MoDCs matured with PGE2 did not overcome the suppressive effects of CD4+ CD25+ T cells in allogeneic mixed lymphocyte reactions. In conclusion, MoDCs matured in the presence of PGE2 display characteristics of more efficient antigen-presenting cells that might be optimal for use in cancer vaccine-based clinical trials.     

Differential regulation of prohormone convertase 1/3, prohormone convertase 2 and phosphorylated cyclic-AMP-response element binding protein by short-term and long-term morphine treatment: implications for understanding the "switch" to opiate addiction

Drug addiction is a state of altered brain reward and self-regulation mediated by both neurotransmitter and hormonal systems. Although an organism's internal system attempts to maintain homeostasis when challenged by exogenous opiates and other drugs of abuse, it eventually fails, resulting in the transition from drug use to drug abuse. We propose that the attempted maintenance of hormonal homeostasis is achieved, in part, through alterations in levels of processing enzymes that control the ratio of active hormone to pro-hormone. Two pro-hormone convertases, PC1/3 and PC2 are believed to be responsible for the activation of many neurohormones and expression of these enzymes is dependent on the presence of a cyclic-AMP response element (CRE) in their promoters. Therefore, we studied the effects of short-term (24-h) and long-term (7-day) morphine treatment on the expression of hypothalamic PC1/3 and PC2 and levels of phosphorylated cyclic-AMP-response element binding protein (P-CREB). While short-term morphine exposure down-regulated, long-term morphine exposure up-regulated P-CREB, PC1/3 and PC2 protein levels in the rat hypothalamus as determined by Western blot analysis. Quantitative immunofluorescence studies confirmed these regulatory actions of morphine in the paraventricular and dorsomedial nucleus of the hypothalamus. Specific radioimmunoassays demonstrated that the increase in PC1/3 and PC2 levels following long-term morphine led to increased TRH biosynthesis as evidence by increased TRH/5.4 kDa C-terminal proTRH-derived peptide ratios in the median eminence. Promoter activity experiments in rat somatomammotrope GH3 cells containing the mu-opioid receptor demonstrated that the CRE(s) in the promoter of PC1/3 and PC2 is required for morphine-induced regulation of PC1/3 and PC2. Our data suggest that the regulation of the prohormone processing system by morphine may lead to alterations in the levels of multiple bioactive hormones and may be a compensatory mechanism whereby the organism tries to restore its homeostatic hormonal milieu. The down-regulation of PC1/3, PC2 and P-CREB by short-term morphine and up-regulation by long-term morphine treatment may be a signal mediating the switch from drug use to drug abuse.