Validation of reference genes for real-time quantitative PCR normalization in soybean developmental and germinating seeds.

PubMed

Li, Qing; Fan, Cheng-Ming; Zhang, Xiao-Mei; Fu, Yong-Fu

2012-10-01

Most of traditional reference genes chosen for real-time quantitative PCR normalization were assumed to be ubiquitously and constitutively expressed in vegetative tissues. However, seeds show distinct transcriptomes compared with the vegetative tissues. Therefore, there is a need for re-validation of reference genes in samples of seed development and germination, especially for soybean seeds. In this study, we aimed at identifying reference genes suitable for the quantification of gene expression level in soybean seeds. In order to identify the best reference genes for soybean seeds, 18 putative reference genes were tested with various methods in different seed samples. We combined the outputs of both geNorm and NormFinder to assess the expression stability of these genes. The reference genes identified as optimums for seed development were TUA5 and UKN2, whereas for seed germination they were novel reference genes Glyma05g37470 and Glyma08g28550. Furthermore, for total seed samples it was necessary to combine four genes of Glyma05g37470, Glyma08g28550, Glyma18g04130 and UKN2 [corrected] for normalization. Key message We identified several reference genes that stably expressed in soybean seed developmental and germinating processes.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

PubMed

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

PubMed

Zhang, Shutao; Chen, Chun; Xie, Tingna; Ye, Sudan

2017-01-01

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

PubMed Central

2017-01-01

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies. PMID:28591185

Reference genes for reverse transcription quantitative PCR in canine brain tissue.

PubMed

Stassen, Quirine E M; Riemers, Frank M; Reijmerink, Hannah; Leegwater, Peter A J; Penning, Louis C

2015-12-09

In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis. Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area. Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.

Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages

PubMed Central

De Spiegelaere, Ward; Dern-Wieloch, Jutta; Weigel, Roswitha; Schumacher, Valérie; Schorle, Hubert; Nettersheim, Daniel; Bergmann, Martin; Brehm, Ralph; Kliesch, Sabine; Vandekerckhove, Linos; Fink, Cornelia

2015-01-01

Background An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. Results 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. Conclusions This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use. PMID:25825906

Reference gene validation for RT-qPCR, a note on different available software packages.

PubMed

De Spiegelaere, Ward; Dern-Wieloch, Jutta; Weigel, Roswitha; Schumacher, Valérie; Schorle, Hubert; Nettersheim, Daniel; Bergmann, Martin; Brehm, Ralph; Kliesch, Sabine; Vandekerckhove, Linos; Fink, Cornelia

2015-01-01

An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

PubMed

Płachetka-Bożek, Anna; Augustyniak, Maria

2017-08-21

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

NASA Astrophysics Data System (ADS)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

PubMed

Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

2014-01-01

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Validation of reference genes for quantitative gene expression analysis in experimental epilepsy.

PubMed

Sadangi, Chinmaya; Rosenow, Felix; Norwood, Braxton A

2017-12-01

To grasp the molecular mechanisms and pathophysiology underlying epilepsy development (epileptogenesis) and epilepsy itself, it is important to understand the gene expression changes that occur during these phases. Quantitative real-time polymerase chain reaction (qPCR) is a technique that rapidly and accurately determines gene expression changes. It is crucial, however, that stable reference genes are selected for each experimental condition to ensure that accurate values are obtained for genes of interest. If reference genes are unstably expressed, this can lead to inaccurate data and erroneous conclusions. To date, epilepsy studies have used mostly single, nonvalidated reference genes. This is the first study to systematically evaluate reference genes in male Sprague-Dawley rat models of epilepsy. We assessed 15 potential reference genes in hippocampal tissue obtained from 2 different models during epileptogenesis, 1 model during chronic epilepsy, and a model of noninjurious seizures. Reference gene ranking varied between models and also differed between epileptogenesis and chronic epilepsy time points. There was also some variance between the four mathematical models used to rank reference genes. Notably, we found novel reference genes to be more stably expressed than those most often used in experimental epilepsy studies. The consequence of these findings is that reference genes suitable for one epilepsy model may not be appropriate for others and that reference genes can change over time. It is, therefore, critically important to validate potential reference genes before using them as normalizing factors in expression analysis in order to ensure accurate, valid results. © 2017 Wiley Periodicals, Inc.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

PubMed

Zhou, Zhe; Cong, Peihua; Tian, Yi; Zhu, Yanmin

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

PubMed Central

Zhou, Zhe; Cong, Peihua; Tian, Yi

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization. PMID:28934340

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves.

PubMed

Fiallos-Jurado, Jennifer; Pollier, Jacob; Moses, Tessa; Arendt, Philipp; Barriga-Medina, Noelia; Morillo, Eduardo; Arahana, Venancio; de Lourdes Torres, Maria; Goossens, Alain; Leon-Reyes, Antonio

2016-09-01

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

PubMed

Wan, Qiao; Chen, Shuilian; Shan, Zhihui; Yang, Zhonglu; Chen, Limiao; Zhang, Chanjuan; Yuan, Songli; Hao, Qinnan; Zhang, Xiaojuan; Qiu, Dezhen; Chen, Haifeng; Zhou, Xinan

2017-01-01

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

PubMed

Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

PubMed Central

Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

2014-01-01

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

PubMed

Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

2016-10-13

In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various experimental conditions.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

PubMed Central

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults. PMID:26998411

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

PubMed

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been; Yang, Wei Cheng

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data

PubMed Central

Demidenko, Natalia V.; Logacheva, Maria D.; Penin, Aleksey A.

2011-01-01

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications – geNorm, NormFinder and BestKeeper - were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species. PMID:21589908

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

PubMed

Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

2016-07-15

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

PubMed

Lim, Q E; Zhou, L; Ho, Y K; Wan, G; Too, H P

2011-12-29

Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii.

PubMed

Pathan, Ejaj K; Ghormade, Vandana; Deshpande, Mukund V

2017-01-01

Benjaminiella poitrasii, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes viz. ACT, 18S rRNA, eEF1α, eEF-Tu,eIF-1A, Tub-α, Tub-b, Ubc, GAPDH, Try, WS-21, NADGDH and NADPGDH were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of B. poitrasii. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as Ubc, 18S rRNA and WS-21 were expressed at constant levels in each given subset of RNA samples from all the morphological phases of B. poitrasii. Therefore, these reference genes can be used to elucidate the role of morpho-genes in B. poitrasii. Further, use of the two most stably expressed genes (Ubc and WS-21) to normalize the expression of the ornithine decarboxylase gene (Bpodc) in different morphological forms of B. poitrasii, generated more reliable results, indicating that our selection of reference genes was appropriate.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

PubMed

Saraiva, Kátia D C; Fernandes de Melo, Dirce; Morais, Vanessa D; Vasconcelos, Ilka M; Costa, José H

2014-09-01

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Selection of reference genes for expression studies with fish myogenic cell cultures.

PubMed

Bower, Neil I; Johnston, Ian A

2009-08-10

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

PubMed

Durrenberger, Pascal F; Fernando, Francisca S; Magliozzi, Roberta; Kashefi, Samira N; Bonnert, Timothy P; Ferrer, Isidro; Seilhean, Danielle; Nait-Oumesmar, Brahim; Schmitt, Andrea; Gebicke-Haerter, Peter J; Falkai, Peter; Grünblatt, Edna; Palkovits, Miklos; Parchi, Piero; Capellari, Sabina; Arzberger, Thomas; Kretzschmar, Hans; Roncaroli, Federico; Dexter, David T; Reynolds, Richard

2012-12-01

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2016-08-01

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

PubMed Central

Mafra, Valéria; Kubo, Karen S.; Alves-Ferreira, Marcio; Ribeiro-Alves, Marcelo; Stuart, Rodrigo M.; Boava, Leonardo P.; Rodrigues, Carolina M.; Machado, Marcos A.

2012-01-01

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress. PMID:22347455

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes

PubMed Central

Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. PMID:29293626

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes.

PubMed

Xiang, Quanju; Li, Jin; Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

PubMed Central

Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

PubMed

Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

PubMed

Liu, Jing; Wang, Qun; Sun, Minying; Zhu, Linlin; Yang, Michael; Zhao, Yu

2014-01-01

Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

PubMed

Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

2015-10-01

Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

PubMed

Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

2014-08-01

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. Copyright © 2014 the American Physiological Society.

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.

PubMed

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels; Sørensen, Flemming Brandt; Jakobsen, Anders; Hansen, Torben Frøstrup

2016-01-01

MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer. We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

PubMed

Ma, Yue-Jiao; Sun, Xiao-Hong; Xu, Xiao-Yan; Zhao, Yong; Pan, Ying-Jie; Hwang, Cheng-An; Wu, Vivian C H

2015-01-01

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

Identification of stable reference genes in differentiating human pluripotent stem cells.

PubMed

Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

2015-06-01

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

EPA Science Inventory

Generation of Two Novel Cell Lines that Stably Express hAR and Firefly Luciferase Genes for Endocrine Screening
K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Divi...

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

PubMed

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

PubMed

Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

2014-07-01

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

PubMed Central

2011-01-01

Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

PubMed

Thiel, Cora S; Huge, Andreas; Hauschild, Swantje; Tauber, Svantje; Lauber, Beatrice A; Polzer, Jennifer; Paulsen, Katrin; Lier, Hartwin; Engelmann, Frank; Schmitz, Burkhard; Schütte, Andreas; Layer, Liliana E; Ullrich, Oliver

2017-01-01

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5 , GAPDH , HPRT1 , PLA2G4A , and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10 -4 and 9 g), 20-40% remained unchanged in microgravity (between 10 -4 and 10 -2  g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

PubMed

Almeida-Oliveira, Fernanda; Leandro, João G B; Ausina, Priscila; Sola-Penna, Mauro; Majerowicz, David

2017-04-01

Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

PubMed Central

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. PMID:25014071

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

PubMed

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

PubMed

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

PubMed Central

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. PMID:25774904

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

PubMed

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

PubMed

Die, Jose V; Baldwin, Ransom L; Rowland, Lisa J; Li, Robert; Oh, Sunghee; Li, Congjun; Connor, Erin E; Ranilla, Maria-Jose

2017-01-01

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).

PubMed

Johansen, Ilona; Andreassen, Rune

2014-12-23

MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature sequence in many aquaculture species. Therefore, they may also be suitable as reference genes in other teleosts. Finally, the systematic approach used in our study successfully identified suitable reference genes, suggesting that this may be a useful strategy to apply in similar validation studies in other aquaculture species.

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

PubMed

Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

2012-01-01

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

PubMed Central

Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

2016-01-01

Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

PubMed

Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A

2014-02-01

Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

PubMed Central

2014-01-01

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual. PMID:24884716

Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR.

PubMed

Xu, H; Li, C; Zeng, Q; Agrawal, I; Zhu, X; Gong, Z

2016-06-01

In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0·2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions. © 2016 The Fisheries Society of the British Isles.

Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

PubMed

Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

2015-10-01

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

PubMed Central

Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

2016-01-01

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

NASA Astrophysics Data System (ADS)

Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

PubMed Central

Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects. PMID:24466124

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

PubMed

Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

PubMed

Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Reliable Reference Genes for Quantification of MicroRNAs in Serum Samples of Sulfur Mustard-Exposed Veterans.

PubMed

Gharbi, Sedigheh; Shamsara, Mehdi; Khateri, Shahriar; Soroush, Mohammad Reza; Ghorbanmehr, Nassim; Tavallaei, Mahmood; Nourani, Mohammad Reza; Mowla, Seyed Javad

2015-01-01

In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims. In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene. miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that. We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers.

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

PubMed

Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

2011-05-01

To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

PubMed

Yin, Xiaotao; Li, Fanglong; Jin, Yipeng; Yin, Zhaoyang; Qi, Siyong; Wu, Shuai; Wang, Zicheng; Wang, Lin; Yu, Jiyun; Gao, Jiangping

2017-03-01

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

PubMed

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang

2009-05-15

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newlymore » developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.« less

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

PubMed Central

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-01-01

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies. PMID:27853238

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases.

PubMed

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-11-17

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer's disease (AD), Parkinson's disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.

Gaze behavior predicts memory bias for angry facial expressions in stable dysphoria.

PubMed

Wells, Tony T; Beevers, Christopher G; Robison, Adrienne E; Ellis, Alissa J

2010-12-01

Interpersonal theories suggest that depressed individuals are sensitive to signs of interpersonal rejection, such as angry facial expressions. The present study examined memory bias for happy, sad, angry, and neutral facial expressions in stably dysphoric and stably nondysphoric young adults. Participants' gaze behavior (i.e., fixation duration, number of fixations, and distance between fixations) while viewing these facial expressions was also assessed. Using signal detection analyses, the dysphoric group had better accuracy on a surprise recognition task for angry faces than the nondysphoric group. Further, mediation analyses indicated that greater breadth of attentional focus (i.e., distance between fixations) accounted for enhanced recall of angry faces among the dysphoric group. There were no differences between dysphoria groups in gaze behavior or memory for sad, happy, or neutral facial expressions. Findings from this study identify a specific cognitive mechanism (i.e., breadth of attentional focus) that accounts for biased recall of angry facial expressions in dysphoria. This work also highlights the potential for integrating cognitive and interpersonal theories of depression.

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).

PubMed

Huis, Rudy; Hawkins, Simon; Neutelings, Godfrey

2010-04-19

Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes. The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

Enhanced functional recombinant factor VII production by HEK 293 cells stably transfected with VKORC1 where the gamma-carboxylase inhibitor calumenin is stably suppressed by shRNA transfection.

PubMed

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo gamma-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational gamma-carboxylation, the recovery of fully gamma-carboxylated and functional proteins is low. In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K gamma-carboxylase cofactor and in addition stably silenced the gamma-carboxylase inhibitory protein calumenin. Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C.

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

PubMed

Kilby, N J; Davies, G J; Snaith, M R

1995-11-01

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age.

PubMed

Miyamoto, Yutaka; Kanzaki, Hiroyuki; Wada, Satoshi; Tsuruoka, Sari; Itohiya, Kanako; Kumagai, Kenichi; Hamada, Yoshiki; Nakamura, Yoshiki

2017-12-01

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Reference gene selection for molecular studies of dormancy in wild oat (Avena fatua L.) caryopses by RT-qPCR method.

PubMed

Ruduś, Izabela; Kępczyński, Jan

2018-01-01

Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α), AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development.

PubMed

Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2017-01-01

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development

PubMed Central

Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J.; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2017-01-01

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species. PMID:28900431

Establishment of Sf9 transformants constitutively expressing PBAN receptor variants: application to functional evaluation

USDA-ARS?s Scientific Manuscript database

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines stably expressing a number of fluorescent Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants incl...

Enhanced Functional Recombinant Factor VII Production by HEK 293 Cells Stably Transfected with VKORC1 where the Gamma-Carboxylase Inhibitor Calumenin is Stably Suppressed by shRNA Transfection

PubMed Central

Wajih, Nadeem; Owen, John; Wallin, Reidar

2008-01-01

Introduction Recombinant members of the vitamin K-dependent protein family (factors IX and VII and protein C) have become important pharmaceuticals in treatment of bleeding disorders and sepsis. However, because the in vivo γ-carboxylation system in stable cell lines used for transfection has a limited capacity of post translational γ carboxylation, the recovery of fully γ-carboxylated and functional proteins is low. Materials and Methods In this work we have engineered recombinant factor VII producing HEK 293 cells to stably overexpress VKORC1, the reduced vitamin K γ-carboxylase cofactor and in addition stably silenced the γ-carboxylase inhibitory protein calumenin. Results and Conclusions Stable cell lines transfected with only a factor VII cDNA had a 9% production of functional recombinant factor VII. On the other hand, these recombinant factor VII producing cells when engineered to overexpress VKORC1 and having calumenin stably suppressed more than 80% by shRNA expression, produced 68% functional factor VII. The technology presented should be applicable to all vertebrae members of the vitamin K-dependent protein family and should lower the production cost of the clinically used factors VII, IX and protein C. PMID:18177690

Highly stable maintenance of a mouse artificial chromosome in human cells and mice.

PubMed

Kazuki, Kanako; Takehara, Shoko; Uno, Narumi; Imaoka, Natsuko; Abe, Satoshi; Takiguchi, Masato; Hiramatsu, Kei; Oshimura, Mitsuo; Kazuki, Yasuhiro

2013-12-06

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: Potential as stable genetic markers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, S.T.; Stoker, A.W.; Bissell, M.J.

1991-12-01

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, the authors further show that blastoderms can be infected in culture and inmore » ovo. In ovo, lacZ expression is seen within 24 hours of virus inoculation, and by 96 hours stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.« less

Epigenomics: dissecting hybridization and polyploidization.

PubMed

Jackson, Scott A

2017-06-19

Epigenetic profiling in diploid, allopolyploid, and domesticated cotton shows that despite most DNA methylation being conserved and stably inherited, alterations likely due to hybridization and domestication affect gene expression.

Stable long-term indigo production by overexpression of dioxygenase genes using a chromosomal integrated cascade expression circuit.

PubMed

Royo, Jose Luis; Moreno-Ruiz, Emilia; Cebolla, Angel; Santero, Eduardo

2005-03-16

In our laboratory we have analyzed different factors to maximize the yield in heterologous protein expression for long-term cultivation, by combination of an efficient cascade expression system and stable integration in the bacterial chromosome. In this work, we have explored this system for the production of indigo dye as a model for biotechnological production, by expressing in Escherichia coli the thnA1A2A3A4 genes from Sphingomonas macrogolitabida strain TFA, which encode the components of a tetralin dioxygenase activity. We compared Ptac, and the Pm-based cascade expression circuit in a multicopy plasmid and stably integrated into the bacterial chromosome. Plasmid-based expression systems resulted in instability of indigo production when serially diluted batch experiments were performed without a selective pressure. This problem was solved by integrating the expression module in the chromosome. Despite the gene dosage reduction, the synergic effect of the cascade expression system produced comparable expression to the dioxygenase activity in the plasmid configuration but could be stably maintained for at least 5 days. Here, we show that the cascade amplification circuit integrated in the chromosome could be an excellent system for tight control and stable production of recombinant products.

Synchronization, TIGoRS, and Information Flow in Complex Systems: Dispositional Cellular Automata.

PubMed

Sulis, William H

2016-04-01

Synchronization has a long history in physics where it refers to the phase matching of two identical oscillators. This notion has been extensively studied in physics as well as in biology, where it has been applied to such widely varying phenomena as the flashing of fireflies and firing of neurons in the brain. Human behavior, however, may be recurrent but it is not oscillatory even though many physiological systems do exhibit oscillatory tendencies. Moreover, much of human behaviour is collaborative and cooperative, where the individual behaviours may be distinct yet contemporaneous (if not simultaneous) and taken collectively express some functionality. In the context of behaviour, the important aspect is the repeated co-occurrence in time of behaviours that facilitate the propagation of information or of functionality, regardless of whether or not these behaviours are similar or identical. An example of this weaker notion of synchronization is transient induced global response synchronization (TIGoRS). Previous work has shown that TIGoRS is a ubiquitous phenomenon among complex systems, enabling them to stably parse environmental transients into salient units to which they stably respond. This leads to the notion of Sulis machines, which emergently generate a primitive linguistic structure through their dynamics. This article reviews the notion of TIGoRS and its expression in several complex systems models including tempered neural networks, driven cellular automata and cocktail party automata. The emergent linguistics of Sulis machines are discussed. A new class of complex systems model, the dispositional cellular automaton is introduced. A new metric for TIGoRS, the excess synchronization, is introduced and applied to the study of TIGoRS in dispositional cellular automata. It is shown that these automata exhibit a nonlinear synchronization response to certain perturbing transients.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

PubMed

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

2015-08-03

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

PubMed Central

Rinnerthaler, Gabriel; Hackl, Hubert; Gampenrieder, Simon Peter; Hamacher, Frank; Hufnagl, Clemens; Hauser-Kronberger, Cornelia; Zehentmayr, Franz; Fastner, Gerd; Sedlmayer, Felix; Mlineritsch, Brigitte; Greil, Richard

2016-01-01

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)® microarrays from Agilent® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort. PMID:26821018

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

PubMed

Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

2012-12-04

The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites.

PubMed

Rinnerthaler, Gabriel; Hackl, Hubert; Gampenrieder, Simon Peter; Hamacher, Frank; Hufnagl, Clemens; Hauser-Kronberger, Cornelia; Zehentmayr, Franz; Fastner, Gerd; Sedlmayer, Felix; Mlineritsch, Brigitte; Greil, Richard

2016-01-26

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan(®) Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)(®) microarrays from Agilent(®) was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.

DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS

EPA Science Inventory

Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competi...

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family*

PubMed Central

Ogunbayo, Oluseye A.; Zhu, Yingmin; Shen, Bing; Agbani, Ejaife; Li, Jie; Ma, Jianjie; Zhu, Michael X.; Evans, A. Mark

2015-01-01

The organellar targeting of two-pore channels (TPCs) and their capacity to associate as homo- and heterodimers may be critical to endolysosomal signaling. A more detailed understanding of the functional association of vertebrate TPC1–3 is therefore necessary. We report here that when stably expressed in HEK293 cells, human (h) TPC1 and chicken (c) TPC3 were specifically targeted to different subpopulations of endosomes, hTPC2 was specifically targeted to lysosomes, and rabbit (r) TPC3 was specifically targeted to both endosomes and lysosomes. Intracellular dialysis of NAADP evoked a Ca2+ transient in HEK293 cells that stably overexpressed hTPC1, hTPC2, and rTPC3, but not in cells that stably expressed cTPC3. The Ca2+ transients induced in cells that overexpressed endosome-targeted hTPC1 were abolished upon depletion of acidic Ca2+ stores by bafilomycin A1, but remained unaffected following depletion of endoplasmic reticulum stores by thapsigargin. In contrast, Ca2+ transients induced via lysosome-targeted hTPC2 and endolysosome-targeted rTPC3 were abolished by bafilomycin A1 and markedly attenuated by thapsigargin. NAADP induced marked Ca2+ transients in HEK293 cells that stably coexpressed hTPC2 with hTPC1 or cTPC3, but failed to evoke any such response in cells that coexpressed interacting hTPC2 and rTPC3 subunits. We therefore conclude that 1) all three TPC subtypes may support Ca2+ signaling from their designate acidic stores, and 2) lysosome-targeted (but not endosome-targeted) TPCs support coupling to the endoplasmic reticulum. PMID:25451935

Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

PubMed

Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

2000-07-03

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level

PubMed Central

Yu, Shi; Pilot, Guillaume

2014-01-01

Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants. PMID:25477887

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling

PubMed Central

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I.

2015-01-01

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na+/Ca2+ exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies. PMID:26234466

Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells.

PubMed

Schweigmann, H; Sánchez-Guijo, A; Ugele, B; Hartmann, K; Hartmann, M F; Bergmann, M; Pfarrer, C; Döring, B; Wudy, S A; Petzinger, E; Geyer, J; Grosser, G

2014-09-01

16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 μM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 μM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 μM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta. Copyright © 2014 Elsevier Ltd. All rights reserved.

Semantic word impressions expressed by hue.

PubMed

Shinomori, Keizo; Komatsu, Honami

2018-04-01

We investigated the possibility of whether impressions of semantic words showing complex concepts could be stably expressed by hues. Using a paired comparison method, we asked ten subjects to select from a pair of hues the one that more suitably matched a word impression. We employed nine Japanese semantic words and used twelve hues from vivid tones in the practical color coordinate system. As examples of the results, for the word "vigorous" the most frequently selected color was yellow and the least selected was blue to purple; for "tranquil" the most selected was yellow to green and the least selected was red. Principal component analysis of the selection data indicated that the cumulative contribution rate of the first two components was 94.6%, and in the two-dimensional space of the components, all hues were distributed as a hue-circle shape. In addition, comparison with additional data of color impressions measured by a semantic differential method suggested that most semantic word impressions can be stably expressed by hue, but the impression of some words, such as "magnificent" cannot. These results suggest that semantic word impression can be expressed reasonably well by color, and that hues are treated as impressions from the hue circle, not from color categories.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

PubMed

Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

2013-11-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

PubMed

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

PubMed

Arazi, T; Slutsky, S G; Shiboleth, Y M; Wang, Y; Rubinstein, M; Barak, S; Yang, J; Gal-On, A

2001-04-27

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

[Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro].

PubMed

Lu, Y; Li, H; Fu, J

2000-04-01

To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells.

Imaging Prostate Cancer with Positron Emission Tomography

DTIC Science & Technology

2014-07-01

critical role in tumor development. The purpose of this proposal is to utilize fibroblast activation protein alpha ( FAP ) expression on TAFs within...based cell lines, which stably express eGFP and FAP . Ongoing experiments are focused on the in vitro and in vivo evaluation of each radiopharmaceutical...and on understanding the growth characteristics of each transfected cell line in vivo. 15. SUBJECT TERMS PET, Prostate Cancer, FAP , molecular

Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat.

PubMed

Schuhmacher, Laura-Nadine; Smith, Ewan St John

2016-12-13

Acid-sensing ion channels (ASICs) are a family of ion channels comprised of six subunits encoded by four genes and they are expressed throughout the peripheral and central nervous systems. ASICs have been implicated in a wide range of physiological and pathophysiological processes: pain, breathing, synaptic plasticity and excitotoxicity. Unlike mice and humans, naked mole-rats do not perceive acid as a noxious stimulus, even though their sensory neurons express functional ASICs, likely an adaptation to living in a hypercapnic subterranean environment. Previous studies of ASIC expression in the mammalian nervous system have often not examined all subunits, or have failed to adequately quantify expression between tissues; to date there has been no attempt to determine ASIC expression in the central nervous system of the naked mole-rat. Here we perform a geNorm study to identify reliable housekeeping genes in both mouse and naked mole-rat and then use quantitative real-time PCR to estimate the relative amounts of ASIC transcripts in different tissues of both species. We identify RPL13A (ribosomal protein L13A) and CANX (calnexin), and β-ACTIN and EIF4A (eukaryotic initiation factor 4a) as being the most stably expressed housekeeping genes in mouse and naked mole-rat, respectively. In both species, ASIC3 was most highly expressed in dorsal root ganglia (DRG), and ASIC1a, ASIC2b and ASIC3 were more highly expressed across all brain regions compared to the other subunits. We also show that ASIC4, a proton-insensitive subunit of relatively unknown function, was highly expressed in all mouse tissues apart from DRG and hippocampus, but was by contrast the lowliest expressed ASIC in all naked mole-rat tissues.

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light-dark cycle.

PubMed

Cleal, Jane K; Shepherd, James N; Shearer, Jasmine L; Bruce, Kimberley D; Cagampang, Felino R

2014-08-05

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Endomorphin-2: A Biased Agonist at the μ-Opioid Receptor

PubMed Central

Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J.; McArdle, Craig A.; Rosethorne, Elizabeth M.; Charlton, Steven J.; Krasel, Cornelius; Bailey, Christopher P.; Henderson, Graeme

2012-01-01

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K+ current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K+ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins. PMID:22553358

Endomorphin-2: a biased agonist at the μ-opioid receptor.

PubMed

Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J; McArdle, Craig A; Rosethorne, Elizabeth M; Charlton, Steven J; Krasel, Cornelius; Bailey, Christopher P; Henderson, Graeme; Kelly, Eamonn

2012-08-01

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding Yun; Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37208; Zhang Li

2008-02-01

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, therebymore » suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.« less

The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells.

PubMed

Nagaya, Shingo; Kawamura, Kazue; Shinmyo, Atsuhiko; Kato, Ko

2010-02-01

To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hajra, A.; Liu, P.; Collins, E.S.

1994-09-01

A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

Molecular Medicine II: Hormone Dependent Cancers

DTIC Science & Technology

2005-04-01

multimode, live-cell videomicroscopy - we are analyzing the cycles of centrosome amplification in living S-phase arrested cells. Finally, we will directly...living cells by time-lapse multi-mode videomicroscopy . The key construct for our work is the generation of a mammalian somatic cell line stably expressing

Antitumor Activity of a Mesenchymal Stem Cell Line Stably Secreting a Tumor-Targeted TNF-Related Apoptosis-Inducing Ligand Fusion Protein

PubMed Central

Marini, Irene; Siegemund, Martin; Hutt, Meike; Kontermann, Roland E.; Pfizenmaier, Klaus

2017-01-01

Mesenchymal stem cells (MSCs) are currently exploited as gene delivery systems for transient in situ expression of cancer therapeutics. As an alternative to the prevailing viral expression, we here describe a murine MSC line stably expressing a therapeutic protein for up to 42 passages, yet fully maintaining MSC features. Because of superior antitumoral activity of hexavalent TNF-related apoptosis-inducing ligand (TRAIL) formats and the advantage of a tumor-targeted action, we choose expression of a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) as a model. The bioactivity of Db-scTRAIL produced from an isolated clone (MSC.TRAIL) was revealed from cell death induction in Colo205 cells treated with either culture supernatants from or cocultured with MSC.TRAIL. In vivo, therapeutic activity of MSC.TRAIL was shown upon peritumoral injection in a Colo205 xenograft tumor model. Best antitumor activity in vitro and in vivo was observed upon combined treatment of MSC.TRAIL with bortezomib. Importantly, in vivo combination treatment did not cause apparent hepatotoxicity, weight loss, or behavioral changes. The development of well characterized stocks of stable drug-producing human MSC lines has the potential to establish standardized protocols of cell-based therapy broadly applicable in cancer treatment. PMID:28553285

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

PubMed Central

Fiedler, Nicola; Quant, Ellen; Fink, Ludger; Sun, Jianguang; Schuster, Ralph; Gerlich, Wolfram H; Schaefer, Stephan

2006-01-01

AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation. RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines. CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis. PMID:16937438

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

PubMed

Bahaji, Abdellatif; Ovecka, Miroslav; Bárány, Ivett; Risueño, María Carmen; Muñoz, Francisco José; Baroja-Fernández, Edurne; Montero, Manuel; Li, Jun; Hidalgo, Maite; Sesma, María Teresa; Ezquer, Ignacio; Testillano, Pilar S; Pozueta-Romero, Javier

2011-04-01

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

Novel Cadmium Responsive MicroRNAs in Daphnia pulex.

PubMed

Chen, Shuai; McKinney, Garrett J; Nichols, Krista M; Colbourne, John K; Sepúlveda, Maria S

2015-12-15

Daphnia pulex is a widely used toxicological model and is known for its sensitivity to cadmium (Cd). Recent research suggests that microRNAs (miRNAs) play a critical role in animal responses to heavy metals. To investigate the functions of D. pulex miRNAs under Cd exposure, we analyzed the miRNA profiles of D. pulex after 48 h using miRNA microarrays and validated our findings by q-PCR. miRNA dpu-let-7 was identified as a stably expressed gene and used as a reference. We identified 22 and 21 differentially expressed miRNAs under low (20 μg/L CdCl2) and high-exposure (40 μg/L CdCl2) concentrations compared to controls, respectively. Cellular functions of predicted miRNA target Cd-responsive genes included oxidative stress, ion transport, mitochondrial damage, and DNA repair. An insulin-related network was also identified in relation to several Cd-responsive miRNAs. The expression of three predicted target genes for miR-71 and miR-210 were evaluated, and expression of two of them (SCN2A and SLC31A1) was negatively correlated with the expression of their regulator miRNAs. We show miR-210 is hypoxia-responsive in D. pulex and propose Cd and hypoxia induce miR-210 via a same HIF1α modulated pathway. Collectively, this research advances our understanding on the role of miRNAs in response to heavy-metal exposure.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

PubMed

Tao, Qi-Fei; Yuan, Sheng-Xian; Yang, Fu; Yang, Sen; Yang, Yuan; Yuan, Ji-Hang; Wang, Zhen-Guang; Xu, Qing-Guo; Lin, Kong-Ying; Cai, Jie; Yu, Jian; Huang, Wei-Long; Teng, Xiao-Lei; Zhou, Chuan-Chuan; Wang, Fang; Sun, Shu-Han; Zhou, Wei-Ping

2015-09-17

Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

Resistance Emergence Mechanism and Mechanism of Resistance Suppression by Tobramycin for Cefepime for Pseudomonas aeruginosa

PubMed Central

Bonomo, Robert A.; Bahniuk, Nadzeya; Bulitta, Juergen B.; VanScoy, Brian; DeFiglio, Holland; Fikes, Steven; Brown, David; Drawz, Sarah M.; Kulawy, Robert; Louie, Arnold

2012-01-01

The panoply of resistance mechanisms in Pseudomonas aeruginosa makes resistance suppression difficult. Defining optimal regimens is critical. Cefepime is a cephalosporin whose 3′ side chain provides some stability against AmpC β-lactamases. We examined the activity of cefepime against P. aeruginosa wild-type strain PAO1 and its isogenic AmpC stably derepressed mutant in our hollow-fiber infection model. Dose-ranging studies demonstrated complete failure with resistance emergence (both isolates). Inoculum range studies demonstrated ultimate failure for all inocula. Lower inocula failed last (10 days to 2 weeks). Addition of a β-lactamase inhibitor suppressed resistance even with the stably derepressed isolate. Tobramycin combination studies demonstrated resistance suppression in both the wild-type and the stably derepressed isolates. Quantitating the RNA message by quantitative PCR demonstrated that tobramycin decreased the message relative to that in cefepime-alone experiments. Western blotting with AmpC-specific antibody for P. aeruginosa demonstrated decreased expression. We concluded that suppression of β-lactamase expression by tobramycin (a protein synthesis inhibitor) was at least part of the mechanism behind resistance suppression. Monte Carlo simulation demonstrated that a regimen of 2 g of cefepime every 8 h plus 7 mg/kg of body weight of tobramycin daily would provide robust resistance suppression for Pseudomonas isolates with cefepime MIC values up to 8 mg/liter and tobramycin MIC values up to 1 mg/liter. For P. aeruginosa resistance suppression, combination therapy is critical. PMID:22005996

Rhizosphere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant Bacteria

PubMed Central

Shim, Hojae; Chauhan, Sadhana; Ryoo, Doohyun; Bowers, Kally; Thomas, Stuart M.; Canada, Keith A.; Burken, Joel G.; Wood, Thomas K.

2000-01-01

Indigenous bacteria from poplar tree (Populus canadensis var. eugenei ‘Imperial Carolina’) and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 × 105 to 23 × 105 CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% ± 12% of all rhizosphere bacteria after 28 days (0.2 × 105 to 31 × 105 CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% ± 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively. PMID:11055909

The influence of monovalent cations on trimeric G protein G(i)1α activity in HEK293 cells stably expressing DOR-G(i)1α (Cys(351)-Ile(351)) fusion protein.

PubMed

Vošahlíková, M; Svoboda, P

2011-01-01

The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved.

Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

USDA-ARS?s Scientific Manuscript database

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...

The Role of GGAP2 in Prostate Cancer

DTIC Science & Technology

2009-03-01

show that GGAP2 protein expression is increased in HPC in both HPC cell lines and clinical patient samples. Biochemical studies indicate that GGAP2...GTPase domain of GGAP2 and enhance its effects on cancer growth. PC3 cells stably expressing wild-type GGAP2 form larger volume tumors in nude mice...reducing HPC incidence and slow down cancer development. 15. SUBJECT TERMS GGAP2, human PC, mutation 16. SECURITY CLASSIFICATION OF: 17

Androgen receptor agonism promotes an osteogenic gene program in preadipocytes

PubMed Central

Hartig, Sean M.; Feng, Qin; Ochsner, Scott A.; Xiao, Rui; McKenna, Neil J.; McGuire, Sean E.; He, Bin

2013-01-01

Androgens regulate body composition by interacting with the androgen receptor (AR) to control gene expression in a tissue-specific manner. To identify novel regulatory roles for AR in preadipocytes, we created a 3T3-L1 cell line stably expressing human AR. We found AR expression is required for androgen-mediated inhibition of 3T3-L1 adipogenesis. This inhibition is characterized by decreased lipid accumulation, reduced expression of adipogenic genes, and induction of genes associated with osteoblast differentiation. Collectively, our results suggest androgens promote an osteogenic gene program at the expense of adipocyte differentiation. PMID:23567971

Assessment of reference gene stability influenced by extremely divergent disease symptoms in Solanum lycopersicum L.

PubMed

Wieczorek, Przemysław; Wrzesińska, Barbara; Obrępalska-Stęplowska, Aleksandra

2013-12-01

Tomato (Solanum lycopersicum L.) is one of the most important vegetables of great worldwide economic value. The scientific importance of the vegetable results from the fact that the genome of S. lycopersicum has been sequenced. This allows researchers to study fundamental mechanisms playing an essential role during tomato development and response to environmental factors contributing significantly to cell metabolism alterations. Parallel with the development of contemporary genetics and the constant increase in sequencing data, progress has to be aligned with improvement of experimental methods used for studying genes functions and gene expression levels, of which the quantitative polymerase chain reaction (qPCR) is still the most reliable. As well as with other nucleic acid-based methods used for comparison of the abundance of specific RNAs, the RT-qPCR data have to be normalised to the levels of RNAs represented stably in a cell. To achieve the goal, the so-called housekeeping genes (i.e., RNAs encoding, for instance, proteins playing an important role in the cell metabolism or structure maintenance), are used for normalisation of the target gene expression data. However, a number of studies have indicated the transcriptional instability of commonly used reference genes analysed in different situations or conditions; for instance, the origin of cells, tissue types, or environmental or other experimental conditions. The expression of ten common housekeeping genes of S. lycopersicum, namely EF1α, TUB, CAC, EXP, RPL8, GAPDH, TBP, ACT, SAND and 18S rRNA were examined during viral infections of tomato. Changes in the expression levels of the genes were estimated by comparison of the non-inoculated tomato plants with those infected with commonly known tomato viral pathogens, Tomato torrado virus, Cucumber mosaic virus, Tobacco mosaic virus and Pepino mosaic virus, inducing a diverse range of disease symptoms on the common host, ranging from mild leaves chlorosis to very severe stem necrosis. It is emphasised that despite the wide range of diverse disease symptoms it is concluded that ACT, CAC and EF1α could be used as the most suitable reference genes in studies of host-virus interactions in tomato. Copyright © 2013 Elsevier B.V. All rights reserved.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis.

PubMed

Vestergaard, Anna L; Knudsen, Ulla B; Munk, Torben; Rosbach, Hanne; Martensen, Pia M

2011-04-01

Endometriosis is a painful chronic female disease defined by the presence of endometrial tissue implants in ectopic (Ec) locations. The pathogenesis is much debated, and type-I interferons (IFNs) could be involved. The expression of genes of the type-I IFN response were profiled by a specific PCR array of RNA obtained from Ec and eutopic (Eu) endometrium collected from nine endometriosis patients and nine healthy control women. Transcriptional expression levels of selected IFN-regulated and housekeeping genes (HKGs) were investigated by real-time quantitative reverse transcriptase PCR (qRT-PCR). Stably expressed HKGs for valid normalization of transcriptional studies of endometrium and endometriosis have not yet been published. Here, seven HKGs were evaluated for stability using the GeNorm and NormFinder software. A normalization factor based on HMBS, TBP and YWHAZ expression was suitable for normalization of qRT-PCR studies of Eu versus Ec endometrium. In the endometrial cell lines HEC1A, HEC1B, Ishikawa and RL95-2, HMBS and HPRT1 were the most stably expressed. The IFN-specific PCR array indicated significantly different expression of the genes BST2, COL16A1, HOXB2 and ISG20 between the endometrial tissue types. However, by correctly normalized qRT-PCR, levels of BST2, COL16A1 and the highly type-I IFN-stimulated genes ISG12A and 6-16 displayed insignificant variations. Conversely, HOXB2 and ISG20 transcriptions were significantly reduced in endometriosis lesions compared with endometrium from endometriosis patients and healthy controls. In conclusion, appropriate HKGs for normalization of qRT-PCR studies of endometrium and endometriosis have been identified here. Abolished expression of ISG20 and HOX genes could be important in endometriosis.

Comparison of Cell Expression Formats for the Characterization of GABAA Channels Using a Microfluidic Patch Clamp System

PubMed Central

Chen, Qin; Yim, Peter D.; Yuan, Nina; Johnson, Juliette; Cook, James M.; Smith, Steve; Ionescu-Zanetti, Cristian; Wang, Zhi-Jian; Arnold, Leggy A.

2012-01-01

Abstract Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABAA channels. A variety of cell types and methods of GABAA channel expression were successfully studied (defined as IGABA>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α1β3γ2 GABAA channels, frozen ready-to-assay (RTA) HEK cells expressing α1β3γ2 or α3β3γ2 GABAA channels, transiently transfected HEK293T cells expressing α1β3γ2 GABAA channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABAA channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABAA channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABAA channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABAA channels in a wide variety of cell formats can be performed using this automated patch clamp system. PMID:22574655

Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Li, Yonghe; Bu, Guojun

2005-04-15

The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.

Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidhauser, C. Bissell, M.J.; Myers, C.A.; Casperson, G.F.

1990-12-01

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with amore » series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.« less

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.

PubMed

Karlgren, Maria; Simoff, Ivailo; Backlund, Maria; Wegler, Christine; Keiser, Markus; Handin, Niklas; Müller, Janett; Lundquist, Patrik; Jareborg, Anne-Christine; Oswald, Stefan; Artursson, Per

2017-09-01

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

PubMed

Slusser-Nore, Andrea; Larson-Casey, Jennifer L; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R

2016-01-01

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

PubMed Central

Slusser-Nore, Andrea; Larson-Casey, Jennifer L.; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2016-01-01

Background This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Methods Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. Results It was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Conclusions Tumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA. PMID:26783756

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity.

PubMed

Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I

2002-06-15

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity.

PubMed Central

Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I

2002-01-01

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional. PMID:12049641

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

PubMed

Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

2015-05-01

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a novel herpes simplex virus type 1 transcript and protein (AL3) expressed during latency.

PubMed

Jaber, Tareq; Henderson, Gail; Li, Sumin; Perng, Guey-Chuen; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

2009-10-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected sensory neurons. In small animal models of infection, expression of the first 1.5 kb of LAT coding sequences is necessary and sufficient for wild-type reactivation from latency. The ability of LAT to inhibit apoptosis is important for reactivation from latency. Within the first 1.5 kb of LAT coding sequences and LAT promoter sequences, additional transcripts have been identified. For example, the anti-sense to LAT transcript (AL) is expressed in the opposite direction to LAT from the 5' end of LAT and LAT promoter sequences. In addition, the upstream of LAT (UOL) transcript is expressed in the LAT direction from sequences in the LAT promoter. Further examination of the first 1.5 kb of LAT coding sequences revealed two small ORFs that are anti-sense with respect to LAT (AL2 and AL3). A transcript spanning AL3 was detected in productively infected cells, mouse neuroblastoma cells stably expressing LAT and trigeminal ganglia (TG) of latently infected mice. Peptide-specific IgG directed against AL3 specifically recognized a protein migrating near 15 kDa in cells stably transfected with LAT, mouse neuroblastoma cells transfected with a plasmid containing the AL3 ORF and TG of latently infected mice. The inability to detect the AL3 protein during productive infection may have been because the 5' terminus of the AL3 transcript was downstream of the first in-frame methionine of the AL3 ORF during productive infection.

Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

NASA Astrophysics Data System (ADS)

Wang, Xia; Feng, Jianhua; Huang, Aiyou; He, Linwen; Niu, Jianfeng; Wang, Guangce

2017-11-01

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

PubMed

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

PubMed

Yamaji, Hideki

2014-03-01

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

In vitro mapping of Myotonic Dystrophy (DM) gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Storbeck, C.J.; Sabourin, L.; Baird, S.

1994-09-01

The Myotonic Dystrophy Kinase (DMK) gene has been cloned and shared homology to serine/threonine protein kinases. Overexpression of this gene in stably transfected mouse myoblasts has been shown to inhibit fusion into myotubes while myoblasts stably transfected with an antisense construct show increased fusion potential. These experiments, along with data showing that the DM gene is highly expressed in muscle have highlighted the possibility of DMK being involved in myogenesis. The promoter region of the DM gene lacks a consensus TATA box and CAAT box, but harbours numerous transcription binding sites. Clones containing extended 5{prime} upstream sequences (UPS) of DMKmore » only weakly drive the reporter gene chloramphenicol acetyl transferase (CAT) when transfected into C2C12 mouse myoblasts. However, four E-boxes are present in the first intron of the DM gene and transient assays show increased expression of the CAT gene when the first intron is present downstream of these 5{prime} UPS in an orientation dependent manner. Comparison between mouse and human sequence reveals that the regions in the first intron where the E-boxes are located are highly conserved. The mapping of the promoter and the importance of the first intron in the control of DMK expression will be presented.« less

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

2008-11-15

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

Comprehensive Characterization of Reference Standard Lots of HIV-1 Subtype C Gp120 Proteins for Clinical Trials in Southern African Regions

PubMed Central

Wang, Zihao; Lorin, Clarisse; Koutsoukos, Marguerite; Franco, David; Bayat, Babak; Zhang, Ying; Carfi, Andrea; Barnett, Susan W.; Porter, Frederick

2016-01-01

Two HIV-1 subtype C gp120 protein candidates were the selected antigens for several experimental vaccine regimens now under evaluation in HVTN 100 Phase I/II clinical trial aiming to support the start of the HVTN 702 Phase IIb/III trial in southern Africa, which is designed to confirm and extend the partial protection seen against HIV-1 infection in the RV144 Thai trial. Here, we report the comprehensive physicochemical characterization of the gp120 reference materials that are representative of the clinical trial materials. Gp120 proteins were stably expressed in Chinese Hamster Ovary (CHO) cells and subsequently purified and formulated. A panel of analytical techniques was used to characterize the physicochemical properties of the two protein molecules. When formulated in the AS01 Adjuvant System, the bivalent subtype C gp120 antigens elicited 1086.C- and TV1.C-specific binding antibody and CD4+ T cell responses in mice. All the characteristics were highly representative of the Clinical Trial Materials (CTM). Data from this report demonstrate the immunogenicity of the gp120 antigens, provide comprehensive characterization of the molecules, set the benchmark for assessment of current and future CTM lots, and lay the physicochemical groundwork for interpretation of future clinical trial data. PMID:27187483

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul

2008-10-10

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing eithermore » 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.« less

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2014-04-01

prostate cancer cells. In the third budget year, we achieved the following: a. Generation of retrovirus and lentivirus vectors expressing WT RUNX2 and S301A... retrovirus vectors will be developed that express β-galactosidase (negative control), wild type Runx2, S301A/S319A (non-phosphorylated) or S301E/S310E...constitutively active) Runx2 mutants. As described last year, retrovirus and lentivirus vectors were constructed to stably introduce wild type and mutant

Animal feed compositions containing phytase derived from transgenic alfalfa and methods of use thereof

DOEpatents

Austin-Phillips, Sandra; Koegel, Richard G.; Straub, Richard J.; Cook, Mark

1999-01-01

A value-added composition of matter containing plant matter from transgenic alfalfa which expresses exogenous phytase activity is disclosed. The phytase activity is a gene product of an exogenous gene encoding for phytase which has been stably incorporated into the genome of alfalfa plants. The transgenic alfalfa expresses phytase activity in nutritionally-significant amounts, thereby enabling its use in animal feeds to eliminate the need for phosphorous supplementation of livestock, poultry, and fish feed rations.

Animal feed compositions containing phytase derived from transgenic alfalfa and methods of use thereof

DOEpatents

Austin-Phillips, Sandra; Koegel, Richard G.; Straub, Richard J.; Cook, Mark

2001-01-01

A value-added composition of matter containing plant matter from transgenic alfalfa which expresses exogenous phytase activity is disclosed. The phytase activity is a gene product of an exogenous gene encoding for phytase which has been stably incorporated into the genome of alfalfa plants. The transgenic alfalfa expresses phytase activity in nutritionally-significant amounts, thereby enabling its use in animal feeds to eliminate the need for phosphorous supplementation of livestock, poultry, and fish feed rations.

Gravity Waves in the Martian Atmosphere detected by the Radio Science Experiment MaRS on Mars Express

NASA Astrophysics Data System (ADS)

Tellmann, S.; Pätzold, M.; Häusler, B.; Tyler, G. L.; Hinson, D. P.

2013-09-01

Gravity waves are an ubiquitous feature in all stably stratified planetary atmospheres. They are known to play a significant role in the energy and momentum budget of the Earth, and they are assumed to be of importance for the redistribution of energy and momentum throughout the Martian atmosphere.

Molecular characterization and localization of the first tyramine receptor of the American cockroach (Periplaneta americana).

PubMed

Rotte, C; Krach, C; Balfanz, S; Baumann, A; Walz, B; Blenau, W

2009-09-15

The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as "trace amines." Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC(50) approximately 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

PubMed

Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0. 05). A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

NASA Astrophysics Data System (ADS)

Lau, Yun-Fai; Kan, Yuet Wai

1983-09-01

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR.

PubMed

Theis, Torsten; Skurray, Ronald A; Brown, Melissa H

2007-08-01

Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus.

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

PubMed Central

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-01-01

AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.

PubMed

Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

2003-03-01

To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

PubMed

Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

2016-01-01

Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

A NOVEL CELL LINE, MDA-KB2, THAT STABLY EXPRESSES AN ANDROGEN AND GLUCOCORTICOID RESPONSIVE REPORTER FOR THE DETECTION OF HORMONE RECEPTOR AGONISTS AND ANTAGONISTS

EPA Science Inventory

The U.S. Environmental Protection Agency has proposed that in vitro assays for estrogen receptor (ER) and androgen receptor (AR) mediated actions be included in a Tier I screening battery to detect hormonally active chemicals. Herein we describe the development of a novel stab...

SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

EPA Science Inventory

A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

PubMed Central

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

In Situ Proximity Ligation Assays Indicate That Hemochromatosis Proteins Hfe and Transferrin Receptor 2 (Tfr2) Do Not Interact

PubMed Central

Rishi, Gautam; Crampton, Emily M.; Wallace, Daniel F.; Subramaniam, V. Nathan

2013-01-01

The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2) have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin. PMID:24155934

Type I human T cell leukemia virus tax protein transforms rat fibroblasts through the cyclic adenosine monophosphate response element binding protein/activating transcription factor pathway.

PubMed Central

Smith, M R; Greene, W C

1991-01-01

The Tax oncoprotein of the type I human T cell leukemia virus (HTLV-I) activates transcription of cellular and viral genes through at least two different transcription factor pathways. Tax activates transcription of the c-fos proto-oncogene by a mechanism that appears to involve members of the cAMP response element binding protein (CREB) and activating transcription factor (ATF) family of DNA-binding proteins. Tax also induces the nuclear expression of the NF-kappa B family of rel oncogene-related enhancer-binding proteins. We have investigated the potential role of these CREB/ATF and NF-kappa B/Rel transcription factors in Tax-mediated transformation by analyzing the oncogenic potential of Tax mutants that functionally segregate these two pathways of transactivation. Rat fibroblasts (Rat2) stably expressing either the wild-type Tax protein or a Tax mutant selectively deficient in the ability to induce NF-kappa B/Rel demonstrated marked changes in morphology and growth characteristics including the ability to form tumors in athymic mice. In contrast, Rat2 cells stably expressing a Tax mutant selectively deficient in the ability to activate transcription through CREB/ATF demonstrated no detectable changes in morphology or growth characteristics. These results suggest that transcriptional activation through the CREB/ATF pathway may play an important role in Tax-mediated cellular transformation. Images PMID:1832173

[Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

PubMed

Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

2009-09-01

To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum.

PubMed

El-Halawany, Medhat S; Ohkouchi, Susumu; Shibata, Hideki; Hitomi, Kiyotaka; Maki, Masatoshi

2004-06-01

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.

Nictaba Homologs from Arabidopsis thaliana Are Involved in Plant Stress Responses

PubMed Central

Eggermont, Lore; Stefanowicz, Karolina; Van Damme, Els J. M.

2018-01-01

Plants are constantly exposed to a wide range of environmental stresses, but evolved complicated adaptive and defense mechanisms which allow them to survive in unfavorable conditions. These mechanisms protect and defend plants by using different immune receptors located either at the cell surface or in the cytoplasmic compartment. Lectins or carbohydrate-binding proteins are widespread in the plant kingdom and constitute an important part of these immune receptors. In the past years, lectin research has focused on the stress-inducible lectins. The Nicotiana tabacum agglutinin, abbreviated as Nictaba, served as a model for one family of stress-related lectins. Here we focus on three non-chimeric Nictaba homologs from Arabidopsis thaliana, referred to as AN3, AN4, and AN5. Confocal microscopy of ArathNictaba enhanced green fluorescent protein (EGFP) fusion constructs transiently expressed in N. benthamiana or stably expressed in A. thaliana yielded fluorescence for AN4 and AN5 in the nucleus and the cytoplasm of the plant cell, while fluorescence for AN3 was only detected in the cytoplasm. RT-qPCR analysis revealed low expression for all three ArathNictabas in different tissues throughout plant development. Stress application altered the expression levels, but all three ArathNictabas showed a different expression pattern. Pseudomonas syringae infection experiments with AN4 and AN5 overexpression lines demonstrated a significantly higher tolerance of several transgenic lines to P. syringae compared to wild type plants. Finally, AN4 was shown to interact with two enzymes involved in plant defense, namely TGG1 and BGLU23. Taken together, our data suggest that the ArathNictabas represent stress-regulated proteins with a possible role in plant stress responses. On the long term this research can contribute to the development of more stress-resistant plants. PMID:29375596

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells.

PubMed

Yen, Andrew; Varvayanis, Susi; Smith, James L; Lamkin, Thomas J

2006-02-01

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

PubMed

He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

2015-03-01

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

Creating single-copy genetic circuits

PubMed Central

Lee, Jeong Wook; Gyorgy, Andras; Cameron, D. Ewen; Pyenson, Nora; Choi, Kyeong Rok; Way, Jeffrey C.; Silver, Pamela A.; Del Vecchio, Domitilla; Collins, James J.

2017-01-01

SUMMARY Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use. PMID:27425413

Construction of a novel shuttle vector for use in Gluconobacter oxydans.

PubMed

Zhang, Lin; Lin, Jinping; Ma, Yushu; Wei, Dongzhi; Sun, Ming

2010-11-01

A shuttle vector pZL1 which can replicate both in Gluconobacter oxydans and Escherichia coli was constructed based on G. oxydans DSM2003 cryptic plasmid pGOX3, a homology of G. oxydans 621H pGOX3, and E. coli cloning vector pUC18. It was found to be stably maintained in G. oxydans during the serial subcultures in the absence of antibiotic pressure for 144 h. With pGOX3 as the reference sample, the relative copy number of pZL1 in G. oxydans is 13 determined by real-time fluorescence quantitative PCR (qPCR). The copy number of pZL1 is much higher than pBBR1MCS5 in E. coli. The vector pZL1 contains six commonly used restriction endonuclease sites, HindIII, SalI, XbaI, BamHI, SmaI, KpnI, and SacI, and is easy to manipulate in molecular biology experiments. The shuttle vector was used to express a reporter protein wasabi successfully in G. oxydans DSM2003 under the control of the tufB promoter.

MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p.

PubMed

Floris, Ilaria; Billard, Hélène; Boquien, Clair-Yves; Joram-Gauvard, Evelyne; Simon, Laure; Legrand, Arnaud; Boscher, Cécile; Rozé, Jean-Christophe; Bolaños-Jiménez, Francisco; Kaeffer, Bertrand

2015-01-01

Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48-72 hours.

Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas.

PubMed

Jaeschke, Holger; Mueller, Sandra; Eszlinger, Markus; Paschke, Ralf

2010-12-01

Constitutively activating mutations (CAMs) of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas. Mutations L677V and T620I identified in hot thyroid carcinomas were previously characterized in CHO and in 3T3-Vill cell lines, respectively, stably expressing the mutant without determination of TSHR expression. F666L identified in a patient with hot thyroid nodules, I691F in a family with nonautoimmune hyperthyroidism and F631I identified in a hot thyroid carcinoma were not characterized for their in vitro function. Therefore, we decided to (re)evaluate the in vitro function of these five TSHR variants by determination of cell surface expression, and intracellular cAMP and inositol phosphate levels and performed additionally linear regression analyses to determine basal activity independently from the mutant's cell surface expression in COS-7 and HEK(GT) cells. Only one (F631I) of the five investigated TSHR variants displayed constitutive activity for G(α) s signalling and showed correlation with the clinical phenotype. The previous false classification of T620I and L677V as CAMs is most likely related to the fact that both mutations were characterized in cell lines stably expressing the mutated receptor construct without assessing the respective receptor number per cell. Other molecular aetiologies for the nonautoimmune hyperthyroidism and/or hot thyroid carcinomas in these three patients and one family should be elucidated. © 2010 Blackwell Publishing Ltd.

Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

PubMed

Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

2013-05-01

Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

PubMed Central

Miklík, Dalibor; Šenigl, Filip; Hejnar, Jiří

2018-01-01

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features—especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species. PMID:29517993

Identification of herpes simplex virus type 1 proteins encoded within the first 1.5 kb of the latency-associated transcript.

PubMed

Henderson, Gail; Jaber, Tareq; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

2009-09-01

Expression of the first 1.5 kb of the latency-associated transcript (LAT) that is encoded by herpes simplex virus type 1 (HSV-1) is sufficient for wild-type (wt) levels of reactivation from latency in small animal models. Peptide-specific immunoglobulin G (IgG) was generated against open reading frames (ORFs) that are located within the first 1.5 kb of LAT coding sequences. Cells stably transfected with LAT or trigeminal ganglionic neurons of mice infected with a LAT expressing virus appeared to express the L2 or L8 ORF. Only L2 ORF expression was readily detected in trigeminal ganglionic neurons of latently infected mice.

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

Silencing of grapevine pectate lyase-like genes VvPLL2 and VvPLL3 confers resistance against Erysiphe necator and differentially modulates gene expression

USDA-ARS?s Scientific Manuscript database

Broad-spectrum resistance against powdery mildew (PM) has been reported by silencing susceptibility genes in the model plant Arabidopsis. Here we used artificial microRNA constructs in PM-susceptible Vitis vinifera cv. Chardonnay to stably silence two pectate lyase-like orthologs (VvPLL2 and VvPLL3)...

Effect of DJ-1 overexpression on the proliferation, apoptosis, invasion and migration of laryngeal squamous cell carcinoma SNU-46 cells through PI3K/AKT/mTOR.

PubMed

Wang, Bin; Qin, Hao; Wang, Yuejian; Chen, Weixiong; Luo, Jie; Zhu, Xiaolin; Wen, Weiping; Lei, Wenbin

2014-09-01

The aim of the present study was to explore the effect of DJ-1-mediated PI3K/AKT/mTOR pathway on the proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous cell SNU-46, through stable transfection and overexpression of the DJ-1 gene. Retrovirus carrying DJ-1 gene was used to stabilize transfected human laryngeal squamous carcinoma SNU-46 cell line, and monoclonal cell line of stably overexpressed DJ-1 protein was screened out by G418. DJ-1 protein expression was determined by western blotting, and changes of p-AKT, p-mTOR and PTEN protein content were detected, followed by the detection of changes in proliferation, apoptosis, invasion, migration and other tumor biological characteristics of laryngeal squamous carcinoma cell line with stably transfected DJ-1 protein overexpression by flow cytometry, CCK-8 method and Transwell. We successfully constructed a laryngeal squamous carcinoma cell line of stably overexpressed DJ-1 protein and termed it SNU-46-DJ-1. After overexpression of DJ-1 protein, the levels of PTEN expression in laryngeal squamous cell SNU-46 decreased and p-AKT and p-mTOR protein expression levels increased. Compared to the untreated SNU-46 cells, the proliferation rate of SNU-46-DJ-1 cells increased (0.834±0.336 vs. 0.676±0.112; p<0.001); invasiveness was enhanced (165.7±13.6 vs. 100.0±17.4; p=0.001), the migration ability was enhanced (207.3±13.1 vs. 175.3±13.3; p=0.036), and the apoptosis rate decreased (3.533±5.167 vs. 16.397±5.447%; p=0.019). The overexpression of DJ-1 protein in laryngeal squamous carcinoma SNU-46 cells can accelerate proliferation rate, increase the invasion and migration capacity, and reduce apoptosis, by activating the PI3K/AKT/mTOR pathway.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis.

PubMed

Deeg, Cornelia A; Hauck, Stefanie M; Amann, Barbara; Kremmer, Elisabeth; Stangassinger, Manfred; Ueffing, Marius

2007-07-01

Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites within retina is evident. In contrast to stable expression of autoantigens, rhodopsin, the major component of phototransduction in photoreceptors, disappeared from destructed retinas. These results explain persistent uveitic attacks even in severely damaged eyes and draw the attention to further investigations of biological pathways and regulations in autoimmune target tissues.

Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

PubMed

Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

2017-06-01

Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

PubMed

Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

2015-07-01

Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

DTIC Science & Technology

2007-11-01

cells ( gray fill), cells preincubated with PBS and infected with virus (solid line), and cells preincubated with recombinant knob protein and incubated...U118-hCAR-tailless cells (b). Gray line¼ cells alone, solid line¼ cells+Ad5Luc1-CK1, dashed line¼ cells+Ad5Luc1. Figure 5 Ad5Luc1-CK1CAR-independent...line, whereas U118MG-hCAR-tailless stably expresses the extracellular domain of human CAR. Cells were infected with Ad5Luc1 ( gray bar) and Ad5-r1 (black

Screening and verifying potential NTCP inhibitors from herbal medicinal ingredients using the LLC-PK1 cell model stably expressing human NTCP.

PubMed

Shen, Zhuo-Wei; Luo, Meng-Yue; Hu, Hai-Hong; Zhou, Hui; Jiang, Hui-Di; Yu, Lu-Shan; Zeng, Su

2016-07-01

NTCP is specifically expressed on the basolateral membrane of hepatocytes, participating in the enterohepatic circulation of bile salts, especially conjugated bile salts, to maintain bile salts homeostasis. In addition, recent studies have found that NTCP is a functional receptor of HBV and HDV. Therefore, it is important to study the interaction between drugs and NTCP and identify the inhibitors/substrates of NTCP. In the present study, a LLC-PK1 cell model stably expressing human NTCP was established, which was simple and suitable for high throughput screening, and utilized to screen and verify the potential inhibitors of NTCP from 102 herbal medicinal ingredients. The results showed that ginkgolic acid (GA) (13 : 0), GA (15 : 1), GA (17 : 1), erythrosine B, silibinin, and emodin have inhibitory effects on NTCP uptake of TCNa in a concentration-dependent manner. Among them, GA (13 : 0) and GA (15 : 1) exhibited the stronger inhibitory effects, with IC50 values being less than 8.3 and 13.5 μmol·L(-1), respectively, than the classical inhibitor, cyclosporin A (CsA) (IC50 = 20.33 μmol·L(-1)). Further research demonstrated that GA (13 : 0), GA (15 : 1), GA (17 : 1), silibinin, and emodin were not substrates of NTCP. These findings might contribute to a better understanding of the disposition of the herbal ingredients in vivo, especially in biliary excretion. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis*

PubMed Central

Ward, Richard J.; Pediani, John D.; Godin, Antoine G.; Milligan, Graeme

2015-01-01

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor. PMID:25825490

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Takahiro; Kanamori, Takao; Inouye, Satoshi

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- andmore » 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs. - Highlights: • An imaging method for protein secretion from 3D-cultured cells was established. • The fused protein of insulin to GLase, Insulin-GLase, was used as a reporter. • Synchronous insulin secretion was visualized in rat islets and spheroidal beta cells. • A rat beta cell line stably expressing Insulin-GLase, named iGL, was established. • Effect of an antidiabetic drug on insulin secretion was visualized in iGL cells.« less

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

PubMed

Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

2012-05-01

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Cereal transformation through particle bombardment

NASA Technical Reports Server (NTRS)

Casas, A. M.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.; Mitchell, C. A. (Principal Investigator)

1995-01-01

The review focuses on experiments that lead to stable transformation in cereals using microprojectile bombardment. The discussion of biological factors that affect transformation examines target tissues and vector systems for gene transfer. The vector systems include reporter genes, selectable markers, genes of agronomic interest, and vector constructions. Other topics include physical parameters that affect DNA delivery, selection of stably transformed cells and plant regeneration, and analysis of gene expression and transmission to the progeny.

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

PubMed Central

Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

2013-01-01

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

PubMed

Zhang, Min; Shi, Shuo; Guo, Rui; Miao, Yin; Li, Biao

2016-10-01

Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and in vivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. In vitro and in vivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS-based gene therapy and imaging using the hTERT promoter and 188Re may be possible.

Effect of Overproduction of Mitochondrial Uncoupling Protein 2 on Cos7 Cells: Induction of Senescent-like Morphology and Oncotic Cell Death.

PubMed

Nishio, Koji; Ma, Qian

2016-01-01

The maintenance of mitochondrial membrane potential is essential for cell growth and survival. Mitochondrial uncoupling protein 2 plays the most important roles in uncoupling oxidative phosphorylation and decreasing mitochondrial O2- production by regulating the mitochondrial membrane potential. We propose that mouse UCP2 has two glycine-rich motifs, motif 1: EGIRGLWKG (170-178) and a known Walker A-like motif 2: EGPRAFYKG (264-272). These motifs seem to be important for the function of UCP2. We investigated the biological effects of overproduced-UCP2 and its physiological consequence in Cos7 cells. We introduced several amino acid changes in the motif 1. The expression vectors of the green fluorescent protein (GFP)-fused UCP2 and mutant UCP2 were constructed and expressed in Cos7 cells. The UCP2-GFP-expressed cells significantly down-regulated the mitochondrial membrane potentials and induced the enlarged cell shapes. Next we generated the stably UCP2-GFP-expressed Cos7 cells by selection with the antibiotic Genecitin (G418). Within the first few weeks following G418-selection, the stably UCP2-GFP-expressed cells could not divide well and gradually manifested the irregular and enlarged senescent-like cell morphology. The UCP2/K177E- or UCP2/G174L-expressed cells did not induce the enlarged cell shapes. Hence, UCP2/K177E and UCP2/G174L produced the functional incompetence of the glycine-rich motif 1. The senescent-like cells significantly decreased the mitochondrial membrane potentials and finally died nearly one month. Overproduction of UCP2 irreversibly reduces the mitochondrial membrane potentials and induces the senescent-like morphology and finally oncotic cell death in Cos7 cells. These changes seem to occur from the irreversible metabolic changes following total loss of cellular ATP.

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Sox17 drives functional engraftment of endothelium converted from non-vascular cells.

PubMed

Schachterle, William; Badwe, Chaitanya R; Palikuqi, Brisa; Kunar, Balvir; Ginsberg, Michael; Lis, Raphael; Yokoyama, Masataka; Elemento, Olivier; Scandura, Joseph M; Rafii, Shahin

2017-01-16

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function.

Over-expression of Flt3 induces NF-kappaB pathway and increases the expression of IL-6.

PubMed

Takahashi, Shinichiro; Harigae, Hideo; Ishii, Keiko Kumura; Inomata, Mitsue; Fujiwara, Tohru; Yokoyama, Hisayuki; Ishizawa, Kenichi; Kameoka, Junichi; Licht, Jonathan D; Sasaki, Takeshi; Kaku, Mitsuo

2005-08-01

Activating mutations or over-expression of the Flt3 is prevalent in acute myeloblastic leukemia (AML), associated with activation of Ras/MAP kinase and other signaling pathways. In this study, we addressed the role of Flt3 in the activation of nuclear factor-kappa B (NF-kappaB), which is a target molecule of these kinase pathways. In BaF3 cells stably expressing Flt3, a NF-kappaB-responsive reporter was upregulated and its target gene, IL-6, was increased by the involvement of Flt3-ERK/MAPK-NF-kappaB pathway. Furthermore, we found a modest positive correlation (r=0.35, p=0.096) between Flt3 and IL-6 mRNA expression in 24 AML specimens. These results suggest a role of Flt3 over-expression in NF-kappaB pathway.

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.

PubMed

Archer, T K; Fryer, C J; Lee, H L; Zaniewski, E; Liang, T; Mymryk, J S

1995-06-01

The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin structure is dependent upon the cell type and receptor status of the recipient cell into which the MMTV promoter is stably introduced.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges

PubMed Central

Gardner, Matthew R.; Kattenhorn, Lisa M.; Kondur, Hema R.; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J.; Haworth, Kevin G.; Decker, Julie M.; Alpert, Michael D.; Bailey, Charles C.; Neale, Ernest S.; Fellinger, Christoph H.; Joshi, Vinita R.; Fuchs, Sebastian P.; Martinez-Navio, Jose M.; Quinlan, Brian D.; Yao, Annie Y.; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C.; Burton, Dennis R.; Kwong, Peter D.; Piatak, Michael; Lifson, Jeffrey D.; Gao, Guangping; Desrosiers, Ronald C.; Evans, David T.; Hahn, Beatrice H.; Ploss, Alexander; Cannon, Paula M.; Seaman, Michael S.; Farzan, Michael

2015-01-01

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling.

PubMed

Beitner-Johnson, D; Blakesley, V A; Shen-Orr, Z; Jimenez, M; Stannard, B; Wang, L M; Pierce, J; LeRoith, D

1996-04-19

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase

PubMed Central

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate its role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of the estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of the ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E2), whereas growth hormone plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E2, their proliferation rate was not different from controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E2 treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E2-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Conclusions: increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein. PMID:20974639

Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase.

PubMed

Mendoza, Rhone A; Moody, Emily E; Enriquez, Marlene I; Mejia, Sylvia M; Thordarson, Gudmundur

2011-01-01

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate p38 MAPK's role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E₂), whereas GH plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E₂, their proliferation rate was lower compared to controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E₂ treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E₂-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in the apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein.

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.

PubMed

Shin, Youngmi; Cho, Nam Jeong

2014-04-01

Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

PubMed

Furuya, Takahito; Takehara, Issey; Shimura, Asuka; Kishimoto, Hisanao; Yasujima, Tomoya; Ohta, Kinya; Shirasaka, Yoshiyuki; Yuasa, Hiroaki; Inoue, Katsuhisa

2018-01-15

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K m ) of 0.23 μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent K m of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. Copyright © 2017 Elsevier Inc. All rights reserved.

[Breeding of transgenic mice expressing human tau isoform with P301L mutation and identification of homozygous transgenic mice].

PubMed

Wang, Yan-yan; Chen, Ru-zhui; Zhu, Xiao-nani; Liu, Jing; Li, Zhi-hui; Liu, Xiu-juan; Li, Zhi-hui; Na, Xin; Liang, Shan-shan; Qiu, Guo-guang; Zhang, Wei; Wang, Hai; Wang, Xue-lan

2012-05-01

To establish homozygous transgenic mouse strain expressing human tau isoform with P301L mutation. Five transgenic mice expressing human tau isoform with P301L mutation were obtained by microinjection into male nuclei. Homozygote and hemizygote were identified by PCR and real-time fluorescent quantitative PCR. Ninety five homozygous transgenic mice were selected, and the results indicated that homozygous transgenic mice were superior to hemizygote in simulating the changes of biological characteristics. Exogenous gene tau is able to stably transmit to next generation and the combination of SYBR Green real-time fluorescent quantitative PCR with the traditional mating is a fast, reliable and economical way to screen homozygous and hemizygous transgenic mice.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

2014-01-01

Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530

Breakthrough in chloroplast genetic engineering of agronomically important crops

PubMed Central

Daniell, Henry; Kumar, Shashi; Dufourmantel, Nathalie

2012-01-01

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development. PMID:15866001

Characterization of IKBKE as a Breast Cancer Oncogene

DTIC Science & Technology

2011-10-01

HMLE -MEKDD cells stably expressing either pWZL or MF-IKKε. Immunoblot analysis by IKKε antibody. (D) IP with an IKK antibody from MCF-7 breast cancer ...summary is presented of research performed during three years of a project to further characterize the breast cancer oncogene IKKε. Two specific aims...constitutive IKKε transgenic mouse model to study the role of IKKε in breast cancer initiation and maintenance. The long term goals of this research

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

PubMed Central

Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

2001-01-01

The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process. PMID:11483711

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Identification, Expression Analysis, and Target Prediction of Flax Genotroph MicroRNAs Under Normal and Nutrient Stress Conditions

PubMed Central

Melnikova, Nataliya V.; Dmitriev, Alexey A.; Belenikin, Maxim S.; Koroban, Nadezhda V.; Speranskaya, Anna S.; Krinitsina, Anastasia A.; Krasnov, George S.; Lakunina, Valentina A.; Snezhkina, Anastasiya V.; Sadritdinova, Asiya F.; Kishlyan, Natalya V.; Rozhmina, Tatiana A.; Klimina, Kseniya M.; Amosova, Alexandra V.; Zelenin, Alexander V.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.

2016-01-01

Cultivated flax (Linum usitatissimum L.) is an important plant valuable for industry. Some flax lines can undergo heritable phenotypic and genotypic changes (LIS-1 insertion being the most common) in response to nutrient stress and are called plastic lines. Offspring of plastic lines, which stably inherit the changes, are called genotrophs. MicroRNAs (miRNAs) are involved in a crucial regulatory mechanism of gene expression. They have previously been assumed to take part in nutrient stress response and can, therefore, participate in genotroph formation. In the present study, we performed high-throughput sequencing of small RNAs (sRNAs) extracted from flax plants grown under normal, phosphate deficient and nutrient excess conditions to identify miRNAs and evaluate their expression. Our analysis revealed expression of 96 conserved miRNAs from 21 families in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of the identified miRNAs were transcribed from LIS-1. Expression of seven miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as most suitable for this purpose. Down-regulation of novel potential lus-miR-N1 and up-regulation of conserved miR399 were revealed under the phosphate deficient conditions. In addition, the negative correlation of expression of lus-miR-N1 and its predicted target, ubiquitin-activating enzyme E1 gene, as well as, miR399 and its predicted target, ubiquitin-conjugating enzyme E2 gene, was observed. Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide new insights into nutrient stress response regulation in plastic flax cultivars. PMID:27092149

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle.

PubMed

Ono, Takeshi; Tadakuma, Takushi; Rodriguez, Ana

2007-03-01

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM).

PubMed

Xiong, Kai; Zhou, Yan; Hyttel, Poul; Bolund, Lars; Freude, Kristine Karla; Luo, Yonglun

2016-11-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

Convergence of miR-143 overexpression, oxidative stress and cell death in HCT116 human colon cancer cells.

PubMed

Gomes, Sofia E; Pereira, Diane M; Roma-Rodrigues, Catarina; Fernandes, Alexandra R; Borralho, Pedro M; Rodrigues, Cecília M P

2018-01-01

MicroRNAs (miRNAs) regulate a wide variety of biological processes, including tumourigenesis. Altered miRNA expression is associated with deregulation of signalling pathways, which in turn cause abnormal cell growth and de-differentiation, contributing to cancer. miR-143 and miR-145 are anti-tumourigenic and influence the sensitivity of tumour cells to chemotherapy and targeted therapy. Comparative proteomic analysis was performed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145. Immunoblotting analysis validated the proteomic data in stable and transient miRNA overexpression conditions in human colon cancer cells. We show that approximately 100 proteins are differentially expressed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145 compared to Empty control cells. Further, Gene Ontology and pathway enrichment analysis indicated that proteins involved in specific cell signalling pathways such as cell death, response to oxidative stress, and protein folding might be modulated by these miRNAs. In particular, antioxidant enzyme superoxide dismutase 1 (SOD1) was downregulated by stable expression of either miR-143 or miR-145. Further, SOD1 gain-of-function experiments rescued cells from miR-143-induced oxidative stress. Moreover, miR-143 overexpression increased oxaliplatin-induced apoptosis associated with reactive oxygen species generation, which was abrogated by genetic and pharmacological inhibition of oxidative stress. Overall, miR-143 might circumvent resistance of colon cancer cells to oxaliplatin via increased oxidative stress in HCT116 human colon cancer cells.

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geller, A.I.; Keyomarsi, K.; Bryan, J.

1990-11-01

The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; tsmore » mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.« less

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

1998-10-13

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

1998-01-01

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2007-03-20

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2000-08-22

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Inhibition of apoptosis by knockdown of caspase-3 with siRNA in rat bone marrow mesenchymal stem cells.

PubMed

Hua, Ping; Liu, Li-Bao; Liu, Jia-Liang; Wang, Meng; Jiang, Hui-Qi; Zeng, Kuan; Yang, Yan-Qi; Yang, Song-Ran

2013-09-01

Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Genetic engineering of somatic cells to study and improve cardiac function.

PubMed

Kirkton, Robert D; Bursac, Nenad

2012-11-01

To demonstrate the utility of genetically engineered excitable cells for studies of basic electrophysiology and cardiac cell therapy. 'Zig-zag' networks of neonatal rat ventricular myocytes (NRVMs) were micropatterned onto thin elastomeric films to mimic the slow action potential (AP) conduction found in fibrotic myocardium. Addition of genetically engineered excitable human embryonic kidney cells (HEK-293 cells) ('Ex-293' cells stably expressing Kir2.1, Na(v)1.5, and Cx43 channels) increased both cardiac conduction velocity by 370% and twitch force amplitude by 64%. Furthermore, we stably expressed mutant Na(v)1.5 [A1924T (fast sodium channel mutant (substitution of alanine by threonine at amino acid 1924)] channels with hyperpolarized steady-state activation and showed that, despite a 71.6% reduction in peak I(Na), these cells propagated APs at the same velocity as the wild-type Na(v)1.5-expressing Ex-293 cells. Stable expression of Ca(v)3.3 (T-type voltage-gated calcium) channels in Ex-293 cells (to generate an 'ExCa-293' line) significantly increased their AP duration and reduced repolarization gradients in cocultures of these cells and NRVMs. Additional expression of an optogenetic construct [ChIEF (light-gated Channelrhodopsin mutant)]enabled light-based control of AP firing in ExCa-293 cells. We show that, despite being non-contractile, genetically engineered excitable cells can significantly improve both electrical and mechanical function of engineered cardiac tissues in vitro. We further demonstrate the utility of engineered cells for tissue-level studies of basic electrophysiology and cardiac channelopathies. In the future, this novel platform could be utilized in the high-throughput design of new genetically encoded indicators of cell electrical function, validation, and improvement of computer models of AP conduction, and development of novel engineered somatic cell therapies for the treatment of cardiac infarction and arrhythmias.

Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases

PubMed Central

Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

2008-01-01

Background Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time. Results Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus. Conclusion Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts. PMID:18416852

Identifying p53 Transactivation Domain 1-Specific Inhibitors to Alleviate the Side Effects of Prostate Cancer Therapy

DTIC Science & Technology

2014-10-01

harnessed the latest genome editing tools of the CRISPR /Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats) system to generate...immortalized MEF reporter lines that stably express reporters under the control of the endogenous genes. The CRISPR /Cas9 system, which was recently developed...cells carrying different retroviral reporter constructs. Thus, even though using the CRISPR /Cas9 system is inherently a longer process, the use of

Plastid transformation in lettuce (Lactuca sativa L.) by biolistic DNA delivery.

PubMed

Ruhlman, Tracey A

2014-01-01

The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa L.). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide an ideal platform for the production of biopharmaceuticals.

Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

DTIC Science & Technology

2001-08-01

Utilization of green fluorescent protein for the identification of metastasis in an in vivo breast cancer model system. In Preparation. REPRINTS OF ALL...phenotype. Utilizing the SUM-159PT cell line stably transfected with pEGFP-Ci (enhanced green fluorescent protein ) we have been able to successfully...accurately detected. To develop a model with enhanced resolution of micrometastases we created a stable cell line expressing green fluorescent protein

Prostate Cancer Evaluation: Design, Synthesis and Evaluation of Novel Enzyme-Activated Proton MRI Contrast Agents

DTIC Science & Technology

2009-10-01

NUMBER E -Mail: 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER...transcriptional activation, protein expression, and protein interaction, lacZ gene encoding E . coli β-gal has already been recognized as the most...1) Cell preparation (a) Stably transfected PC3 cell line: E . coli lacZ gene (from pSV-β-gal vector, Promega, Madison,WI) was inserted into high

Targeting CD81 to Prevent Metastases in Breast Cancer

DTIC Science & Technology

2015-10-01

in tumor cells would curb the formation of CTCs. Briefly, 4T1 cells either WT or cells in which CD81 has been knocked down stably using CRISPR -Cas9...expression in breast cancer cells impairs the number of circulating tumor cells . The experiments were performed using a protocol that we standardized for...detection of circulating tumor cells in an immunocompetent syngeneic mouse model of breast cancer using FASTcell™ system. 15. SUBJECT TERMS Breast

The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

PubMed Central

Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

2009-01-01

Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

PubMed Central

Rahman, Saifur; Quann, Kevin; Pandya, Devanshi; Singh, Shruti; Khan, Zafar K.; Jain, Pooja

2012-01-01

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type. PMID:22496815

Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B–ESAT6 fusion protein

PubMed Central

Hall, Lindsay J.; Clare, Simon; Pickard, Derek; Clark, Simon O.; Kelly, Dominic L.F.; Ghany, Moataz Abd El; Hale, Christine; Dietrich, Jes; Andersen, Peter; Marsh, Philip D.; Dougan, Gordon

2009-01-01

A recombinant Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain was constructed that stably expressed the Mycobacterium tuberculosis fusion antigen Ag85B–ESAT6 from the chromosome. Live oral vaccination of mice with the Salmonella/Ag85B–ESAT6 strain generated a potent anti-Ag85B–ESAT6 TH1 response with high antibody titres with a IgG2a-bias and significant IFN-γ production lasting over a 120-day period. When mice primed with the Salmonella/Ag85B–ESAT6 vaccine were mucosally boosted with the Ag85B–ESAT6 antigen and adjuvant the IFN-γ responses increased markedly. To determine the protective efficacy of this vaccine strain, guinea pigs were immunised and followed for a 30-week period after aerosol challenge with M. tuberculosis. The heterologous prime-boost strategy of live Salmonella vaccine followed by a systemic boost of antigen and adjuvant reduced the levels of M. tuberculosis bacteria in the lungs and spleen to the same extent as BCG. Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunisation with BCG. Thus, live oral priming with the recombinant Salmonella/Ag85B–ESAT6 and boosting with Ag85B–ESAT6 plus the adjuvant LTK63 represents an effective mucosal vaccination regimen. PMID:19755145

Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme

PubMed Central

Mokri, Poroshista; Lamp, Nora; Linnebacher, Michael; Classen, Carl Friedrich; Erbersdobler, Andreas; Schneider, Björn

2017-01-01

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies. PMID:28934307

Abnormal Epigenetic Regulation of Immune System during Aging.

PubMed

Jasiulionis, Miriam G

2018-01-01

Epigenetics refers to the study of mechanisms controlling the chromatin structure, which has fundamental role in the regulation of gene expression and genome stability. Epigenetic marks, such as DNA methylation and histone modifications, are established during embryonic development and epigenetic profiles are stably inherited during mitosis, ensuring cell differentiation and fate. Under the effect of intrinsic and extrinsic factors, such as metabolic profile, hormones, nutrition, drugs, smoke, and stress, epigenetic marks are actively modulated. In this sense, the lifestyle may affect significantly the epigenome, and as a result, the gene expression profile and cell function. Epigenetic alterations are a hallmark of aging and diseases, such as cancer. Among biological systems compromised with aging is the decline of immune response. Different regulators of immune response have their promoters and enhancers susceptible to the modulation by epigenetic marks, which is fundamental to the differentiation and function of immune cells. Consistent evidence has showed the regulation of innate immune cells, and T and B lymphocytes by epigenetic mechanisms. Therefore, age-dependent alterations in epigenetic marks may result in the decline of immune function and this might contribute to the increased incidence of diseases in old people. In order to maintain health, we need to better understand how to avoid epigenetic alterations related to immune aging. In this review, the contribution of epigenetic mechanisms to the loss of immune function during aging will be discussed, and the promise of new means of disease prevention and management will be pointed.

Expression and Characterization of Functional Recombinant Bet v 1.0101 in the Chloroplast of Chlamydomonas reinhardtii.

PubMed

Hirschl, Sonja; Ralser, Claudia; Asam, Claudia; Gangitano, Alessandro; Huber, Sara; Ebner, Christof; Bohle, Barbara; Wolf, Martin; Briza, Peter; Ferreira, Fatima; Griesbeck, Christoph; Wallner, Michael

2017-01-01

Allergen immunotherapy (AIT) still plays a minor role in the treatment of allergic diseases. To improve the acceptance of AIT by allergic patients, the treatment has to become more convenient and efficacious. One possibility is the oral application of allergens or derivatives thereof. Therefore, we sought to produce a recombinant allergen in the green alga Chlamydomonas reinhardtii as a novel production platform. The major birch pollen allergen Bet v 1 was selected as candidate molecule, and a codon-optimized gene was synthesized and stably integrated into the microalga C. reinhardtii FUD50. Positive transformants were identified by PCR, cultured, and thereafter cells were disrupted by sonication. Bet v 1 was purified from algal total soluble protein (TSP) by affinity chromatography and characterized physicochemically as well as immunologically. All transformants showed expression of the allergen with yields between 0.01 and 0.04% of TSP. Algal-derived Bet v 1 displayed similar secondary structure elements as the Escherichia coli-produced reference allergen. Moreover, Bet v 1 produced in C. reinhardtii showed binding comparable to human IgE as well as murine Bet v 1-specific IgG. We could successfully produce recombinant Bet v 1 in C. reinhardtii. As microalgae are classified as GRAS (generally recognized as safe), the pilot study supports the development of novel allergy treatment concepts such as the oral administration of allergen-containing algal extracts for therapy. © 2017 The Author(s) Published by S. Karger AG, Basel.

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

PubMed Central

Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

2012-01-01

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

Sox17 drives functional engraftment of endothelium converted from non-vascular cells

PubMed Central

Schachterle, William; Badwe, Chaitanya R.; Palikuqi, Brisa; Kunar, Balvir; Ginsberg, Michael; Lis, Raphael; Yokoyama, Masataka; Elemento, Olivier; Scandura, Joseph M.; Rafii, Shahin

2017-01-01

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function. PMID:28091527

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Development of gradient descent adaptive algorithms to remove common mode artifact for improvement of cardiovascular signal quality.

PubMed

Ciaccio, Edward J; Micheli-Tzanakou, Evangelia

2007-07-01

Common-mode noise degrades cardiovascular signal quality and diminishes measurement accuracy. Filtering to remove noise components in the frequency domain often distorts the signal. Two adaptive noise canceling (ANC) algorithms were tested to adjust weighted reference signals for optimal subtraction from a primary signal. Update of weight w was based upon the gradient term of the steepest descent equation: [see text], where the error epsilon is the difference between primary and weighted reference signals. nabla was estimated from Deltaepsilon(2) and Deltaw without using a variable Deltaw in the denominator which can cause instability. The Parallel Comparison (PC) algorithm computed Deltaepsilon(2) using fixed finite differences +/- Deltaw in parallel during each discrete time k. The ALOPEX algorithm computed Deltaepsilon(2)x Deltaw from time k to k + 1 to estimate nabla, with a random number added to account for Deltaepsilon(2) . Deltaw--> 0 near the optimal weighting. Using simulated data, both algorithms stably converged to the optimal weighting within 50-2000 discrete sample points k even with a SNR = 1:8 and weights which were initialized far from the optimal. Using a sharply pulsatile cardiac electrogram signal with added noise so that the SNR = 1:5, both algorithms exhibited stable convergence within 100 ms (100 sample points). Fourier spectral analysis revealed minimal distortion when comparing the signal without added noise to the ANC restored signal. ANC algorithms based upon difference calculations can rapidly and stably converge to the optimal weighting in simulated and real cardiovascular data. Signal quality is restored with minimal distortion, increasing the accuracy of biophysical measurement.

MiRNA Analysis by Quantitative PCR in Preterm Human Breast Milk Reveals Daily Fluctuations of hsa-miR-16-5p

PubMed Central

Floris, Ilaria; Billard, Hélène; Boquien, Clair-Yves; Joram-Gauvard, Evelyne; Simon, Laure; Legrand, Arnaud; Boscher, Cécile; Rozé, Jean-Christophe; Bolaños-Jiménez, Francisco; Kaeffer, Bertrand

2015-01-01

Background and Aims Human breast milk is an extremely dynamic fluid containing many biologically-active components which change throughout the feeding period and throughout the day. We designed a miRNA assay on minimized amounts of raw milk obtained from mothers of preterm infants. We investigated changes in miRNA expression within month 2 of lactation and then over the course of 24 hours. Materials and Methods Analyses were performed on pooled breast milk, made by combining samples collected at different clock times from the same mother donor, along with time series collected over 24 hours from four unsynchronized mothers. Whole milk, lipids or skim milk fractions were processed and analyzed by qPCR. We measured hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-146-5p, and hsa-let-7a, d and g (all -5p). Stability of miRNA endogenous controls was evaluated using RefFinder, a web tool integrating geNorm, Normfinder, BestKeeper and the comparative ΔΔCt method. Results MiR-21 and miR-16 were stably expressed in whole milk collected within month 2 of lactation from four mothers. Analysis of lipids and skim milk revealed that miR-146b and let-7d were better references in both fractions. Time series (5H-23H) allowed the identification of a set of three endogenous reference genes (hsa-let-7d, hsa-let-7g and miR-146b) to normalize raw quantification cycle (Cq) data. We identified a daily oscillation of miR-16-5p. Perspectives Our assay allows exploring miRNA levels of breast milk from mother with preterm baby collected in time series over 48–72 hours. PMID:26474056

Suppression of the lipopolysaccharide-induced expression of MARCKS-related protein (MRP) affects transmigration in activated RAW264.7 cells.

PubMed

Chun, Kwang-Rok; Bae, Eun Mi; Kim, Jae-Kwan; Suk, Kyoungho; Lee, Won-Ha

2009-01-01

The molecular action mechanism of MRP, one of the protein kinase C (PKC) substrates, has been under intense investigation, but reports on its role in macrophage function remain controversial. The treatment of macrophage cell lines with bacterial lipopolysaccharide (LPS) induced a high level of MRP expression suggesting that MRP plays a role in the function of activated macrophages. In order to investigate the role of MRP in activated RAW264.7 cells, we stably transfected MRP-specific shRNA expression constructs and tested for alterations in macrophage-related functions. The down-regulation of MRP expression resulted in a marked reduction in chemotaxis toward MCP-1 or extracellular matrix proteins. Furthermore, pharmacological inhibitors of PKC significantly inhibited the chemotaxis in RAW264.7 cells. These data reveals the pivotal role of MRP in the transmigration of activated RAW264.7 cells.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Understanding the Relative Contributions of and Critical Enzymes for the Three Pathways for Intracrine Metabolism of Testicular Androgens in Advanced Prostate Cancer

DTIC Science & Technology

2017-10-01

of the project as stated in the approved SOW. If the application listed milestones/target dates for important activities or phases of the project...These fluorescent AND, 5α-dione and DIOL derivatives retained the needed level of biological activity in biological assays (vida infra). In...stably expressed AR fused with green fluorescent protein (GFP) was used to determine cellular uptake of fluorescent- androgens, AR activation and

Global circular RNA expression profile of human gastric cancer and its clinical significance.

PubMed

Shao, Yongfu; Li, Jinyun; Lu, Rongdan; Li, Tianwen; Yang, Yunben; Xiao, Bingxiu; Guo, Junming

2017-06-01

Circular RNAs (circRNAs) are a new class of noncoding RNAs. However, the expression profile and clinical significance of circRNAs in human gastric cancer is unclear. The global circRNA expression profile in human gastric cancer was measured by circRNA microarray. Hsa_circ_0014717, one of the most downregulated circRNAs in microarray, was selected as a targeted circRNA to explore its levels in gastric tissues and gastric juice. Freeze-thaw experiment and incubation experiment confirmed the stability of gastric juice circRNAs. A total of 308 circRNAs, including 107 (34.74%) upregulated and 201 (65.26%) downregulated circRNAs, were found significantly aberrantly expressed in gastric cancer tissues. The top ten upregulated in gastric cancer tissues were hsa_circ_0035445, hsa_circ_0003789, hsa_circ_0063809, hsa_circ_0074362, hsa_circ_0006282, hsa_circ_0011107, hsa_circ_0084606, hsa_circ_0005556, hsa_circ_0050547, and hsa_circ_0006470, while the top ten downregulated ones were hsa_circ_0007099, hsa_circ_0001897, hsa_circ_0007707, hsa_circ_0008832, hsa_circ_0001546, hsa_circ_0002089, hsa_circ_0004680, hsa_circ_0000154, hsa_circ_0004458, and hsa_circ_0008394. The hot-point chromosomes were chr1, chr2, chr3, chr9, and chr17. Hsa_circ_0014717 was significantly downregulated in 77.2% (74/96) gastric cancer tissues. Its levels in gastric cancer tissues were related to tumor stage (P = 0.037), distal metastasis (P = 0.048), tissue carcinoembryonic antigen (P = 0.001), and carbohydrate antigen 19-9 expression (P = 0.021). More importantly, hsa_circ_0014717 can stably exist in human gastric juice; and its nature meets the requirements of clinical detection. Our study uncovered the circRNA expression profile in human gastric cancer. Moreover, some circRNAs can stably exist in human body fluid, and has the potential to be used as novel biomarkers for the screening of high-risk gastric cancer patients. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Structure-Activity Correlations for β-Phenethylamines at Human Trace Amine Receptor 1

PubMed Central

Lewin, Anita H.; Navarro, Hernán A.; Mascarella, S. Wayne

2008-01-01

A cell line in which RD-HGA16 cells were stably transfected with the hTAAR 1 receptor was created and utilized to carry out a systematic evaluation of a series of β-phenethylamines. Fair agreement was observed with data obtained for aryl and ethylene chain substituted analogs in an AV12-664 cell line in which hemagglutinin-tagged hTAAR 1 was stably co-expressed with rat Gαs. Analogs with multiple substituents as well as analogs with bulky groups were found to be partial agonists. Analogs in which the primary amino group was converted to a secondary or a tertiary amino group by N-methylation were also partial agonists. Comparative Molecular Field Analysis (CoMFA) using the potency data yielded a regression coefficient r2 of 0.824. The steric field contribution to the model was 61% with the balance (39%) contributed by the electrostatic field. The collective results suggest that increasing steric bulk at both the amino nitrogen, particularly by N-dimethylation, and at the 4-position of the aromatic ring, leads to low efficacy ligands. PMID:18602830

In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors

PubMed Central

Richter, Maximilian; Saydaminova, Kamola; Yumul, Roma; Krishnan, Rohini; Liu, Jing; Nagy, Eniko-Eva; Singh, Manvendra; Izsvák, Zsuzsanna; Cattaneo, Roberto; Uckert, Wolfgang; Palmer, Donna; Ng, Philip; Haworth, Kevin G.; Kiem, Hans-Peter; Ehrhardt, Anja; Papayannopoulou, Thalia

2016-01-01

Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking in BM HSPCs (Lin−Sca1+Kit− cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. PMID:27554082

Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

PubMed

Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

2014-01-01

Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

PubMed

Zhang, Jialin; Liu, Wenxing; Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao

2016-01-01

From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

Evaluation of microRNA stability in feces from healthy dogs.

PubMed

Cirera, Susanna; Willumsen, Line M; Johansen, Thea T; Nielsen, Lise N

2018-03-01

Gastrointestinal cancer accounts for approximately 8% of all canine malignancies. Early detection of cancer may have a tremendous impact on both treatment options and prognosis. MicroRNAs (miRNAs), a class of noncoding RNAs that can be found stably expressed in body fluids and feces, have been suggested as valuable human cancer biomarkers. The purpose of the study was to investigate the feasibility of detecting miRNAs in canine feces and to determine the miRNA stability in fecal samples stored at different temperatures for different duration. The levels of 4 Canine familiaris (cfa) miRNAs (cfa-miR-16, cfa-miR-20a, cfa-miR-21, and cfa-miR-92a) were investigated by quantitative real-time PCR(qPCR) in fecal samples from 10 healthy dogs. Fecal samples were collected at 3 different time points and samples from the first time point were stored at different temperatures and for a different duration. A statistically significant difference was found in miRNA levels from samples stored at room temperature compared with samples stored at -20°C for cfa-miR-16 and cfa-miR-21. No significant difference was found in the level of the investigated miRNAs over time. Overall, miRNAs are present in dog feces at measurable levels. Some miRNAs seem to be subject to a higher degree of degradation in samples stored at room temperature for 24 hours compared with samples frozen after collection at -20°C. The investigated miRNAs were stably expressed over time. This study provides the basis for further research on miRNA expression profiles as biomarkers for gastrointestinal cancer in dogs. © 2018 American Society for Veterinary Clinical Pathology.

Compact reflection holographic recording system with high angle multiplexing

NASA Astrophysics Data System (ADS)

Kanayasu, Mayumi; Yamada, Takehumi; Takekawa, Shunsuke; Akieda, Kensuke; Goto, Akiyo; Yamamoto, Manabu

2011-02-01

Holographic memory systems have been widely researched since 1963. However, the size of the drives required and the deterioration of reconstructed data resulting from shrinkage of the medium have made practical use of a hologram memory difficult. In light of this, we propose a novel holographic recording/reconstructing system: a dual-reference beam reflection system that is smaller than conventional systems such as the off-axis or co-axis types, and which is expected to increase the number of multiplexing in angle multiplexed recording. In this multiplex recording system, two laser beams are used as reference beams, and the recorded data are reconstructed stably, even if there is shrinkage of the recording medium. In this paper, a reflection holographic memory system is explained in detail. In addition, the change in angle selectivity resulting from shrinkage of the medium is analyzed using the laminated film three-dimensional simulation method. As a result, we demonstrate that a dual-reference beam multiplex recording system is effective in reducing the influence of medium shrinkage.

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

PubMed Central

Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

1998-01-01

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

PubMed Central

Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

2013-01-01

Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector

PubMed Central

Werner, Stefan; Breus, Oksana; Symonenko, Yuri; Marillonnet, Sylvestre; Gleba, Yuri

2011-01-01

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 104-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing. PMID:21825158

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

PubMed

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

Heat shock protein 90{beta}: A novel mediator of vitamin D action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angelo, Giana; Mineral Bioavailability Laboratory, 711 Washington Street, Boston, MA 02111; Lamon-Fava, Stefania

2008-03-14

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90{beta} expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90{beta} by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%,more » respectively, in Hsp90{beta}-deficient cells. Nuclear protein for VDR and RXR{alpha}, its heterodimer partner, were not reduced in Hsp90{beta}-deficient cells. These findings indicate that Hsp90{beta} is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90{beta} in VDR signaling.« less

Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

PubMed Central

Leurs, R.; Smit, M. J.; Menge, W. M.; Timmerman, H.

1994-01-01

1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7921611

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

PubMed Central

Turowski, Vanessa; Sperling, Beatrice; Hanczaruk, Matthias A.; Göbel, Thomas W.; Viertlboeck, Birgit C.

2016-01-01

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50–70%. PMID:26967520

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

PubMed

Mahakapuge, T A N; Scheerlinck, J-P Y; Rojas, C A Alvarez; Every, A L; Hagen, J

2016-03-01

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep. Copyright © 2016. Published by Elsevier B.V.

Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein.

PubMed

Gauson, Elaine J; Windle, Brad; Donaldson, Mary M; Caffarel, Maria M; Dornan, Edward S; Coleman, Nicholas; Herzyk, Pawel; Henderson, Scott C; Wang, Xu; Morgan, Iain M

2014-11-01

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome. Copyright © 2014 Elsevier Inc. All rights reserved.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria.

PubMed

Bron, Peter A; Benchimol, Marcos G; Lambert, Jolanda; Palumbo, Emmanuelle; Deghorain, Marie; Delcour, Jean; De Vos, Willem M; Kleerebezem, Michiel; Hols, Pascal

2002-11-01

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.

[Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding].

PubMed

Zhang, P; Wang, J G; Wan, J G; Liu, W Q

2010-01-01

The frequent disease outbreaks caused by avian influenza virus not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis in combination with RNAi to specifically inhibit avian enza virus gene expression has been proposed to make chickens resistant to the infection. For the transgenic breeding, screening in vitro efficient siRNAs as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing siRNAs targeted towards different mRNA regions of H5N1 avian influenza virus. Five rational siRNAs were chosen by this method, five U6 promoter-driven shRNA expression plasmids containing the siRNA genes were constructed and used for producing stably transfected MDCK cells. The data obtained by virus titration, IFA, PI-stained flow cytometry, real-time quantitative RT-PCR, and DAS-ELISA analyses showed that all five stably transfected cell lines we re resistant to virusreplication when exposed to 100 CCID50 of avian influenza virus H5N1. Finally, most effective plasmids (pSi-604i and pSi-1597i) as the candidates for making the transgenic chickens were chosen. These findings provide baseline information on use of RNAi technique for breeding transgenic chickens resistant to avian influenza virus.

Rapid resensitization of purinergic receptor function in human platelets.

PubMed

Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

2008-08-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides1[W][OPEN

PubMed Central

Muthukumar, Balasubramaniam; Joyce, Blake L.; Elless, Mark P.; Stewart, C. Neal

2013-01-01

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns. PMID:23933990

Limiting an Insect Infestation of Nitrogen-Fixing Root Nodules of the Pigeon Pea (Cajanus cajan) by Engineering the Expression of an Entomocidal Gene in Its Root Nodules

PubMed Central

Nambiar, P. T. C.; Ma, S.-W.; Iyer, V. N.

1990-01-01

A region of DNA which determined the production of the insecticidal toxin of Bacillus thuringiensis subsp. israelensis was cloned into a derivative of a broad-host-range group IncQ plasmid vector of gram-negative bacteria. The plasmid which we constructed was transferred by conjugative mobilization into a Bradyrhizobium species that nodulates pigeon peas. In this species the construction was maintained stably in the absence of selection and expressed the gene that was installed. Experiments in a greenhouse with the strain which we constructed indicated that this organism provides protection against root nodule damage by the larvae of the insect Rivellia angulata (Diptera). Images PMID:16348294

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

PubMed Central

Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

2012-01-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

2012-07-01

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

Expression of human oxoguanine glycosylase 1 or formamidopyrimidine glycosylase in human embryonic kidney 293 cells exacerbates methylmercury toxicity in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ondovcik, Stephanie L.; Preston, Thomas J.; McCallum, Gordon P.

Exposure to methylmercury (MeHg) acutely at high levels, or via chronic low-level dietary exposure from daily fish consumption, can lead to adverse neurological effects in both the adult and developing conceptus. To determine the impact of variable DNA repair capacity, and the role of reactive oxygen species (ROS) and oxidatively damaged DNA in the mechanism of toxicity, transgenic human embryonic kidney (HEK) 293 cells that stably express either human oxoguanine glycosylase 1 (hOgg1) or its bacterial homolog, formamidopyrimidine glycosylase (Fpg), which primarily repair the oxidative lesion 8-oxo-2′-deoxyguanosine (8-oxodG), were used to assess the in vitro effects of MeHg. Western blottingmore » confirmed the expression of hOgg1 or Fpg in both the nuclear and mitochondrial compartments of their respective cell lines. Following acute (1–2 h) incubations with 0–10 μM MeHg, concentration-dependent decreases in clonogenic survival and cell growth accompanied concentration-dependent increases in lactate dehydrogenase (LDH) release, ROS formation, 8-oxodG levels and apurinic/apyrimidinic (AP) sites, consistent with the onset of cytotoxicity. Paradoxically, hOgg1- and Fpg-expressing HEK 293 cells were more sensitive than wild-type cells stably transfected with the empty vector control to MeHg across all cellular and biochemical parameters, exhibiting reduced clonogenic survival and cell growth, and increased LDH release and DNA damage. Accordingly, upregulation of specific components of the base excision repair (BER) pathway may prove deleterious potentially due to the absence of compensatory enhancement of downstream processes to repair toxic intermediary abasic sites. Thus, interindividual variability in DNA repair activity may constitute an important risk factor for environmentally-initiated, oxidatively damaged DNA and its pathological consequences. - Highlights: • hOgg1 and Fpg repair oxidatively damaged DNA. • hOgg1- and Fpg-expressing cells are more sensitive to MeHg toxicity. • Enhanced sensitivity is likely due to an accumulation of toxic repair intermediates. • Interindividual variability in DNA repair activity may modulate toxicological risk.« less

Relativistic electrons and whistlers in Jupiter's magnetosphere

NASA Technical Reports Server (NTRS)

Barbosa, D. D.; Coroniti, F. V.

1976-01-01

The paper examines some of the consequences of relativistic electrons in stably trapped equilibrium with parallel propagating whistlers in the inner magnetosphere of Jupiter. Approximate scaling laws for the stably trapped electron flux and equilibrium wave intensity are derived, and the equatorial growth rate for whistlers is determined. It is shown that fluxes are near the stably trapped limit, which suggests that whistler intensities may be high enough to cause significant diffusion of electrons, accounting for the observed reduction of phase space densities.

E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

PubMed

Carvell, Melanie J; Marsh, Phil J; Persaud, Shanta J; Jones, Peter M

2007-01-01

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation. Copyright (c) 2007 S. Karger AG, Basel.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Ping; Jiang Xiaohong; Arcasoy, Murat O.

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutivelymore » active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.« less

Podoplanin increases migration and angiogenesis in malignant glioma

PubMed Central

Grau, Stefan J; Trillsch, Fabian; Tonn, Joerg-Christian; Goldbrunner, Roland H; Noessner, Elfriede; Nelson, Peter J; von Luettichau, Irene

2015-01-01

Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373Phigh/U87Phigh). Transfection did not influence the production of pro-angiogenic factors including VEGF, VEGF-C and D. Also, expression of VEGF receptors (VEGFR) remained unchanged except for U87Phigh, where a VEGFR3 expression was induced. U373Phigh showed significantly reduced proliferation as compared to mock transfected group. By contrast, podoplanin significantly increased migration and invasion into collagen matrix. Furthermore, conditioned media from Phigh glioma cells strongly induced tube formation on matrigel. In conclusion, podoplanin increased migration of tumor cells and enhanced tube formation activity in endothelial cells independent from VEGF. Thus, podoplanin expression may be an important step in tumor progression. PMID:26339454

Activating Cell Death Ligand Signaling Through Proteasome Inhibition

DTIC Science & Technology

2009-05-01

cell system would work and, if not, identify a new system. First, wild-type caspase-8 was stably expressed in the SH -SY-5Y line . SH -SY-5Y caspase-8...little success with the SH -SY-5Y cells we sought to use the I9.2 caspase-8 deficient Jurkat cell line . Our initial data confirmed that the caspase-8...shown that TRAIL can induce apop- tosis in a variety of human tumor cell lines .3,12,13 However, a large number of prostate cancer cells exhibit a

A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.

PubMed Central

Piechaczek, C; Fetzer, C; Baiker, A; Bode, J; Lipps, H J

1999-01-01

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure. PMID:9862961

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

PubMed

Kirkton, Robert D; Bursac, Nenad

2011-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression

PubMed Central

Balda, Maria S.; Matter, Karl

2000-01-01

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation. PMID:10790369

Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.

PubMed

Zhang, Jiang; Khan, Sher Afzal; Hasse, Claudia; Ruf, Stephanie; Heckel, David G; Bock, Ralph

2015-02-27

Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the β-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides. Copyright © 2015, American Association for the Advancement of Science.

Human fatigue expression recognition through image-based dynamic multi-information and bimodal deep learning

NASA Astrophysics Data System (ADS)

Zhao, Lei; Wang, Zengcai; Wang, Xiaojin; Qi, Yazhou; Liu, Qing; Zhang, Guoxin

2016-09-01

Human fatigue is an important cause of traffic accidents. To improve the safety of transportation, we propose, in this paper, a framework for fatigue expression recognition using image-based facial dynamic multi-information and a bimodal deep neural network. First, the landmark of face region and the texture of eye region, which complement each other in fatigue expression recognition, are extracted from facial image sequences captured by a single camera. Then, two stacked autoencoder neural networks are trained for landmark and texture, respectively. Finally, the two trained neural networks are combined by learning a joint layer on top of them to construct a bimodal deep neural network. The model can be used to extract a unified representation that fuses landmark and texture modalities together and classify fatigue expressions accurately. The proposed system is tested on a human fatigue dataset obtained from an actual driving environment. The experimental results demonstrate that the proposed method performs stably and robustly, and that the average accuracy achieves 96.2%.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

PubMed

Schaumberg, Katherine A; Antunes, Mauricio S; Kassaw, Tessema K; Xu, Wenlong; Zalewski, Christopher S; Medford, June I; Prasad, Ashok

2016-01-01

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to mitochondrial dysfunction.

PubMed

Nakashima-Kamimura, Naomi; Asoh, Sadamitsu; Ishibashi, Yoshitomo; Mukai, Yuri; Shidara, Yujiro; Oda, Hideaki; Munakata, Kae; Goto, Yu-Ichi; Ohta, Shigeo

2005-11-15

To investigate the regulatory system in mitochondrial biogenesis involving crosstalk between the mitochondria and nucleus, we found a factor named MIDAS (mitochondrial DNA absence sensitive factor) whose expression was enhanced by the absence of mitochondrial DNA (mtDNA). In patients with mitochondrial diseases, MIDAS expression was increased only in dysfunctional muscle fibers. A majority of MIDAS localized to mitochondria with a small fraction in the Golgi apparatus in HeLa cells. To investigate the function of MIDAS, we stably transfected HeLa cells with an expression vector carrying MIDAS cDNA or siRNA. Cells expressing the MIDAS protein and the siRNA constitutively showed an increase and decrease in the total mass of mitochondria, respectively, accompanying the regulation of a mitochondria-specific phospholipid, cardiolipin. In contrast, amounts of the mitochondrial DNA, RNA and proteins did not depend upon MIDAS. Thus, MIDAS is involved in the regulation of mitochondrial lipids, leading to increases of total mitochondrial mass in response to mitochondrial dysfunction.

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

PubMed

Le Maout, S; Sewing, S; Coudrier, E; Elalouf, J M; Pongs, O; Merot, J

1996-01-01

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

Optimal bipedal interactions with dynamic terrain: synthesis and analysis via nonlinear programming

NASA Astrophysics Data System (ADS)

Hubicki, Christian; Goldman, Daniel; Ames, Aaron

In terrestrial locomotion, gait dynamics and motor control behaviors are tuned to interact efficiently and stably with the dynamics of the terrain (i.e. terradynamics). This controlled interaction must be particularly thoughtful in bipeds, as their reduced contact points render them highly susceptible to falls. While bipedalism under rigid terrain assumptions is well-studied, insights for two-legged locomotion on soft terrain, such as sand and dirt, are comparatively sparse. We seek an understanding of how biological bipeds stably and economically negotiate granular media, with an eye toward imbuing those abilities in bipedal robots. We present a trajectory optimization method for controlled systems subject to granular intrusion. By formulating a large-scale nonlinear program (NLP) with reduced-order resistive force theory (RFT) models and jamming cone dynamics, the optimized motions are informed and shaped by the dynamics of the terrain. Using a variant of direct collocation methods, we can express all optimization objectives and constraints in closed-form, resulting in rapid solving by standard NLP solvers, such as IPOPT. We employ this tool to analyze emergent features of bipedal locomotion in granular media, with an eye toward robotic implementation.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malinowsky, Katharina; Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg; Luksza, Julia

2008-06-20

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tailmore » required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of virus-cell fusion confirming the correlation between post-translational modification of tubulin and virus-cell fusion. These results thus identify tubulin and its post-translational modification as a new cellular target for interference with HIV-cell fusion.« less

[Effect of Ech1 overexpression on biological behavior of mouse hepatocarcinoma Hca-P cells in vitro].

PubMed

Wang, Mei; Song, Bo; Wang, Bo; Zhang, Jun; Tang, Jian-wu

2013-05-01

To investigate the effect of enoyl coenzyme A hydratase-1 (Ech1) on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro. Recombinant pcDNA3.1(+)-Ech1 gene and pcDNA3.1(+) were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEch1 cells that stably expressing Ech1 and clone of control Pvector cells were screened by G418. The Ech1 expression was identified subsequently by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test. The cell line Hca-P cells stably expressing Ech1 gene was constructed. The relative expression of Ech1 mRNA in the PEch1 group was 3.21 ± 0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups (P = 0.029). The results of ELISA revealed that the expression of Ech1 protein was 0.140 ± 0.005 in the PEch1 group, 0.088 ± 0.003 in the Pvector group, and 0.078 ± 0.006 in the Hca-P group, showing a significant difference between the PEch1 group and the Pvector and Hca-P groups (P < 0.05). Transwell migration test showed that the number of penetrated cells in the PEch1 group was 143.00 ± 7.25 cells, significantly higher than that of the Pvector group (95.73 ± 3.88 cells) and un-treated Hca-1 group (106.67 ± 3.54 cells, both P < 0.05). The Transwell invasion assay showed that the number of penetrated cells was 77.20 ± 5.46 cells in the PEch1 group, significantly higher than 46.34 ± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-1 group (both P < 0.05). The results showed that overexpressed Ech1 in Hca-P cells may significantly increase the cell proliferation in a time-dependent manner. The up-regulation of Ech1 may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Ech1 expression may become a therapeutic target in the treatment of hepatocarcinoma.

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

PubMed

Bastide, C; Maroc, N; Bladou, F; Hassoun, J; Maitland, N; Mannoni, P; Bagnis, C

2003-01-01

In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

PubMed

Li, Zhili; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Sun, Jiazeng; Yi, Bao; Hou, Qiang; Mao, Yaping; Liu, Weiquan

2015-06-01

Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals.

The fluorescent photobleaching properties of GFP expressed in human lung cancer cells

NASA Astrophysics Data System (ADS)

Jin, Ying; Xing, Da

2003-12-01

The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the dicistronic expression vector (pEGFP-C1) was used to transfected into human lung cancer cell line (ASTC-a-1) and a positive clone which stably expressed GFP in high level was obtained. After more than three months' passengers, the cells were also remained the strong fluorescence under fluorescent microscope. The results showed that the green fluorescent protein expressed in tumor cells was also photobleached under intense irradiation (approximately 488 nm) and the degree of photobleaching varied with the difference of the intensity of the excitation. Using different interdiction parcel (None, ND4, ND8, ND16), there were significant differences in photobleaching among the different excitation. The photobleaching was also affected by the time length of excitation, and the intensity of fluorescence was obviously decreased along with the increasing of excitation time, especially to stronger excitation.

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

PubMed

Ma, Bing-cun; Yang, Xin; Wang, Hong-ning; Cao, Hai-peng; Xu, Peng-wei; Ding, Meng-die; Liu, Hui

2016-01-01

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

A novel packaging system for the generation of helper-free oncolytic MVM vector stocks.

PubMed

Brandenburger, A; Russell, S

1996-10-01

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.

Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein

PubMed Central

Takenaka-Uema, Akiko; Murata, Yousuke; Gen, Fumihiro; Ishihara-Saeki, Yukari; Watanabe, Ken-ichi; Uchida, Kazuyuki; Kato, Kentaro; Murakami, Shin; Haga, Takeshi

2015-01-01

ABSTRACT We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases. PMID:26157127

S-phase arrest after vincristine treatment may promote hepatitis B virus replication

PubMed Central

Xu, Lei; Tu, Zeng; Xu, Ge; Hu, Jie-Li; Cai, Xue-Fei; Zhan, Xing-Xing; Wang, Yu-Wei; Huang, Yuan; Chen, Juan; Huang, Ai-Long

2015-01-01

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication. PMID:25663769

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

NASA Astrophysics Data System (ADS)

Chishti, Arif A.; Hellweg, Christine E.; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther

2015-01-01

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

The Prenylflavonoid Xanthohumol Reduces Alzheimer-Like Changes and Modulates Multiple Pathogenic Molecular Pathways in the Neuro2a/APPswe Cell Model of AD.

PubMed

Huang, Xianfeng; Wang, Jing; Chen, Xiao; Liu, Pan; Wang, Shujin; Song, Fangchen; Zhang, Zaijun; Zhu, Feiqi; Huang, Xinfeng; Liu, Jianjun; Song, Guoqiang; Spencer, Peter S; Yang, Xifei

2018-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that has proved refractory to drug treatment. Given evidence of neuroprotection in animal models of ischemic stroke, we assessed the prenylflavonoid xanthohumol from the Common Hop ( Humulus lupulus L.) for therapeutic potential in murine neuroblastoma N2a cells stably expressing human Swedish mutant amyloid precursor protein (N2a/APP), a well-characterized cellular model of AD. The ELISA and Western-blot analysis revealed that xanthohumol (Xn) inhibited Aβ accumulation and APP processing, and that Xn ameliorated tau hyperphosphorylation via PP2A, GSK3β pathways in N2a/APP cells. The amelioration of tau hyperphosphorylation by Xn was also validated on HEK293/Tau cells, another cell line with tau hyperphosphorylation. Proteomic analysis (2D-DIGE-coupled MS) revealed a total of 30 differentially expressed lysate proteins in N2a/APP vs. wild-type (WT) N2a cells (N2a/WT), and a total of 21 differentially expressed proteins in lysates of N2a/APP cells in the presence or absence of Xn. Generally, these 51 differential proteins could be classified into seven main categories according to their functions, including: endoplasmic reticulum (ER) stress-associated proteins; oxidative stress-associated proteins; proteasome-associated proteins; ATPase and metabolism-associated proteins; cytoskeleton-associated proteins; molecular chaperones-associated proteins, and others. We used Western-blot analysis to validate Xn-associated changes of some key proteins in several biological/pathogenic processes. Taken together, we show that Xn reduces AD-related changes in stably transfected N2a/APP cells. The underlying mechanisms involve modulation of multiple pathogenic pathways, including those involved in ER stress, oxidative stress, proteasome molecular systems, and the neuronal cytoskeleton. These results suggest Xn may have potential for the treatment of AD and/or neuropathologically related neurodegenerative diseases.

Partial nephrogenic diabetes insipidus caused by a novel AQP2 variation impairing trafficking of the aquaporin-2 water channel.

PubMed

Dollerup, Pia; Thomsen, Troels Møller; Nejsum, Lene N; Færch, Mia; Österbrand, Martin; Gregersen, Niels; Rittig, Søren; Christensen, Jane H; Corydon, Thomas J

2015-12-29

Autosomal dominant inheritance of congenital nephrogenic diabetes insipidus (CNDI) is rare and usually caused by variations in the AQP2 gene. We have investigated the genetic and molecular background underlying symptoms of diabetes insipidus (DI) in a Swedish family with autosomal dominant inheritance of the condition. The proband and her father were subjected to water deprivation testing and direct DNA sequencing of the coding regions of the AQP2 and AVP genes. Madin-Darby canine kidney (MDCK) cells stably expressing AQP2 variant proteins were generated by lentiviral gene delivery. Localization of AQP2 variant proteins in the cells under stimulated and unstimulated conditions was analyzed by means of immunostaining and confocal laser scanning microscopy. Intracellular trafficking of AQP2 variant proteins was studied using transient expression of mutant dynamin2-K44A-GFP protein and AQP2 variant protein phosphorylation levels were assessed by Western blotting analysis. Clinical and genetic data suggest that the proband and her father suffer from partial nephrogenic DI due to a variation (g.4807C > T) in the AQP2 gene. The variation results in substitution of arginine-254 to tryptophan (p.R254W) in AQP2. Analysis of MDCK cells stably expressing AQP2 variant proteins revealed disabled phosphorylation, impaired trafficking and intracellular accumulation of AQP2-R254W protein. Notably, blocking of the endocytic pathway demonstrated impairment of AQP2-R254W to reach the cell surface. Partial CNDI in the Swedish family is caused by an AQP2 variation that seems to disable the encoded AQP2-R254W protein to reach the subapical vesicle population as well as impairing its phosphorylation at S256. The AQP2-R254W protein is thus unable to reach the plasma membrane to facilitate AVP mediated urine concentration.

Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation.

PubMed

Almeida, Luis E F; Pereira, Edna F R; Camara, Adriana L; Maelicke, Alfred; Albuquerque, Edson X

2004-04-19

In HEK293 cells stably expressing alpha4beta2 nAChRs, naltrexone, but not naloxone, blocked alpha4beta2 nAChRs via an open-channel blocking mechanism. In primary hippocampal cultures, naltrexone inhibited alpha7 nAChRs up-regulated by nicotine, and in organotypic hippocampal cultures naltrexone caused a time-dependent up-regulation of functional alpha7 nAChRs that was detected after removal of the drug. These results indicate that naltrexone could be used as a smoking cessation aid.

Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

PubMed

Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

2017-10-13

Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

Differential homologous desensitization of the human histamine H3 receptors of 445 and 365 amino acids expressed in CHO-K1 cells.

PubMed

García-Gálvez, Ana-Maricela; Escamilla-Sánchez, Juan; Flores-Maldonado, Catalina; Contreras, Rubén-Gerardo; Arias, Juan-Manuel; Arias-Montaño, José-Antonio

2018-01-01

Histamine H 3 receptors (H 3 Rs) signal through Gα i/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H 3 R (hH 3 R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH 3 R 445 and hH 3 R 365 ) are widely expressed in the human brain. We previously showed that the hH 3 R 445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH 3 R 365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH 3 R 445 . In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH 3 R 445 and hH 3 R 365 , respectively), there were no differences in receptor affinity for selective H 3 R ligands or for agonist-induced [ 35 S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H 3 R agonist RAMH (1 μM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH 3 R 445 and hH 3 R 365 , respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH 3 R 365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methadone but not morphine inhibits lubiprostone-stimulated Cl- currents in T84 intestinal cells and recombinant human ClC-2, but not CFTR Cl- currents.

PubMed

Cuppoletti, John; Chakrabarti, Jayati; Tewari, Kirti; Malinowska, Danuta H

2013-05-01

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Functions of MiRNA-128 on the regulation of head and neck squamous cell carcinoma growth and apoptosis.

PubMed

Hauser, Belinda; Zhao, Yuan; Pang, Xiaowu; Ling, Zhiqiang; Myers, Ernest; Wang, Paul; Califano, Joseph; Gu, Xinbin

2015-01-01

Incidence of head and neck squamous cell carcinoma (HNSCC) has continuously increased in past years while its survival rate has not been significantly improved. There is a critical need to better understand the genetic regulation of HNSCC tumorigenesis and progression. In this study, we comprehensively analyzed the function of miRNA-128 (miR-128) in the regulation of HNSCC growth and its putative targets in vitro and in vivo systems. The function and targets of miR-128 were investigated in human HNSCC cell lines (JHU-13 and JHU-22), which were stably transfected with the miR-128 gene using a lentiviral delivery system. The expression levels of miR-128 and its targeted proteins were analyzed with qRT-PCR, Western blotting and flow cytometry. The binding capacity of miRNA-128 to its putative targets was determined using a luciferase report assay. MTT, colony formation, and a tumor xenograft model further evaluated the effects of miR-128 on HNSCC growth. We generated two miR-128 stably transfected human HNSCC cell lines (JHU-13miR-128 and JHU-22miR-128). Enforced expression of miR-128 was detected in both cultured JHU-13miR-128 and JHU-22miR-128 cell lines, approximately seventeen to twenty folds higher than in vector control cell lines. miRNA-128 was able to bind with the 3'-untranslated regions of BMI-1, BAG-2, BAX, H3f3b, and Paip2 mRNAs, resulting in significant reduction of the targeted protein levels. We found that upregulated miR-128 expression significantly inhibited both JHU-13miR-128 and JHU-22miR-128 cell viability approximately 20 to 40%, and the JHU-22miR-128 tumor xenograft growth compared to the vector control groups. miR-128 acted as a tumor suppressor inhibiting the HNSCC growth by directly mediating the expression of putative targets. Our results provide a better understanding of miRNA-128 function and its potential targets, which may be valuable for developing novel diagnostic markers and targeted therapy.

PLUME DISPERSION IN STABLY STRATIFIED FLOWS OVER COMPLEX TERRAIN, PHASE 2

EPA Science Inventory

Laboratory experiments were conducted in a stratified towing tank to investigate plume dispersion in stably stratified flows. First, plume dispersion over an idealized terrain model with a simulated elevated inversion in the atmosphere was investigated. These results were compare...

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

PubMed

Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

2002-07-15

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells

PubMed Central

S. CHAHAL, MANPREET; TERESA KU, H.; ZHANG, ZHIHONG; M. LEGASPI, CHRISTIAN; LUO, ANGELA; M. HOPKINS, MANDI; E. MEIER, KATHRYN

2016-01-01

Background: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Materials and Methods: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Results: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). Conclusion: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. PMID:27807066

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

The circadian gene Rev-erbα improves cellular bioenergetics and provides preconditioning for protection against oxidative stress.

PubMed

Sengupta, Shaon; Yang, Guang; O'Donnell, John C; Hinson, Maurice D; McCormack, Shana E; Falk, Marni J; La, Ping; Robinson, Michael B; Williams, Monica L; Yohannes, Mekdes T; Polyak, Erzsebet; Nakamaru-Ogiso, Eiko; Dennery, Phyllis A

2016-04-01

Diurnal oscillations in the expression of antioxidant genes imply that protection against oxidative stress is circadian-gated. We hypothesized that stabilization of the core circadian gene Rev-erbα (Nr1d1) improves cellular bioenergetics and protects against nutrient deprivation and oxidative stress. Compared to WT, mouse lung fibroblasts (MLG) stably transfected with a degradation resistant Rev-erbα (Ser(55/59) to Asp; hence referred to as SD) had 40% higher protein content, 1.5-fold higher mitochondrial area (confocal microscopy), doubled oxidative phosphorylation by high-resolution respirometry (Oroboros) and were resistant to glucose deprivation for 24h. This resulted from a 4-fold reduction in mitophagy (L3CB co-localized with MitoTracker Red) versus WT. Although PGC1α protein expression was comparable between SD and WT MLG cells, the role of mitochondrial biogenesis in explaining increased mitochondrial mass in SD cells was less clear. Embryonic fibroblasts (MEF) from C57Bl/6-SD transgenic mice, had a 9-fold induction of FoxO1 mRNA and increased mRNA of downstream antioxidant targets heme oxygenase-1 (HO-1), Mn superoxide dismutase and catalase (1.5, 2 fold and 2 fold respectively) versus WT. This allowed the SD cells to survive 1h incubation with 500 µM H2O2 as well as 24h of exposure to 95% O2 and remain attached whereas most WT cells did not. These observations establish a mechanistic link between the metabolic functions of Rev-erbα with mitochondrial homeostasis and protection against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.

PubMed

Lemaire, Géraldine; Mnif, Wissem; Pascussi, Jean-Marc; Pillon, Arnaud; Rabenoelina, Fanja; Fenet, Hélène; Gomez, Elena; Casellas, Claude; Nicolas, Jean-Claude; Cavaillès, Vincent; Duchesne, Marie-Josèphe; Balaguer, Patrick

2006-06-01

Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

PubMed

Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

2016-08-01

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Formation of temperature front in stably stratified turbulence

NASA Astrophysics Data System (ADS)

Kimura, Yoshifumi; Sullivan, Peter; Herring, Jackson

2016-11-01

An important feature of stably stratified turbulence is the significant influence of internal gravity waves which makes stably stratified turbulence unique compared to homogeneous isotropic turbulence. In this paper, we investigate the genesis of temperature fronts-a crucial subject both practically and fundamentally-in stably stratified turbulence using Direct Numerical Simulations (DNS) of the Navier-Stokes equation under the Boussinesq approximation with 10243 grid points. Vertical profiles of temperature fluctuations show almost vertically periodic sawtooth wavy structures with negative and positive layers stacked together with clear boundaries implying a sharp temperature fronts. The sawtooth waves consist of gradual decreasing temperature fluctuations with rapid recovery to a positive value as the frontal boundary is crossed vertically. This asymmetry of gradients comes from the structure that warm temperature region lies on top of cool temperature region, and can be verified in the skewed probability density function (PDF) of vertical temperature gradient. We try to extract the flow structures and mechanism for the formation and maintenance of the strong temperature front numerically.

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach

PubMed Central

Beger, Carmela; Pierce, Leigh N.; Krüger, Martin; Marcusson, Eric G.; Robbins, Joan M.; Welcsh, Piri; Welch, Peter J.; Welte, Karl; King, Mary-Claire; Barber, Jack R.; Wong-Staal, Flossie

2001-01-01

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an “inverse genomics” approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer. PMID:11136250

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

PubMed

Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

2007-02-01

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha1A1-adrenoceptors.

PubMed

Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork; Pickering, Darryl S; Kristensen, Torsten

2008-10-31

Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant which was relevant, we used quantitative real-time polymerase chain reaction (qPCR) to determine the prevalence in human subcutaneous small arteries of three of the five splice variants ADRA1A_v1-5, which encode functional protein: alpha(1A1)-, alpha(1A3)-, alpha(1A4)-adrenoceptors. Our statistical analysis showed higher transcription levels of alpha(1A1)- than of alpha(1A3)- and alpha(1A4)-adrenoceptors (1.6 and 5.8 times, respectively). We therefore chose to study the alpha(1A1)-adrenoceptor, and the cDNA encoding it was transfected into the Flp-In-293 (modified from HEK-293) cell line to produce a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors to yield pK(B) values of 8.40, 8.05, 8.26 and 7.38, respectively. In [7-methoxy-(3)H]-prazosin binding experiments, high expression was seen (B(max)=48.5+/-16.7 pmol/mg protein, +/-S.E.M.) along with high affinity binding to a single site (K(d)=0.210+/-0.034 nM). The pharmacological profiles of recombinant human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol.

Partial apamin sensitivity of human small conductance Ca2+-activated K+ channels stably expressed in Chinese hamster ovary cells.

PubMed

Dale, T J; Cryan, J E; Chen, M X; Trezise, D J

2002-11-01

The bee venom toxin apamin is an important drug tool for characterising small conductance Ca(2+)-activated K(+) channels (SK channels). In recombinant expression systems both rSK2 and rSK3 channels are potently blocked by apamin, whilst the sensitivity of SK1 channels is somewhat less clear. In the present study we have conducted a detailed analysis by patch clamp electrophysiology of the effects of apamin on human SK channels (SK1, SK2 and SK3) stably expressed in Chinese hamster ovary (CHO-K1) cells. CHO-K1 cell lines expressing either hSK1, 2 or 3 channels were first validated using specific antibodies and Western blotting. Specific protein bands of a size corresponding to the predicted channel tetramer (approximately 250-290 kDa) were detected. In each cell line, but not wild-type untransfected cells, large, time-independent inwardly rectifying Ca(2+)-dependent K(+) currents were observed under voltage-clamp. In CHO-hSK1, this current was markedly reduced by apamin (IC(50) value 8 nM), however, a significant fraction of the current remained unblocked (39+/-5%), even at saturating concentrations (1 microM apamin). The apamin-sensitive and -insensitive currents possess very similar biophysical and pharmacological properties. Each are Ca(2+)-dependent, inwardly rectify and have relative ionic permeabilities of K(+)>Cs(+)>Li(+)=Na(+). Both components were resistant to block by charybdotoxin and iberiotoxin, known IK and BK channel blockers, but were attenuated by the tricyclic antidepressant cyproheptadine (>95% block at 1 mM). The SK channel opener 1-EBIO could still produce channel activation in the presence of apamin. Importantly, hSK2 and hSK3 channels also exhibit partial apamin sensitivity in our experimental paradigm (IC(50) values of 0.14 nM and 1.1 nM, respectively, and maximal percentage inhibition values of 47+/-7% and 58+/-9%, respectively). Our data indicate that, at least in a recombinant expression system, all three SK channels can be partially apamin-sensitive. The explanation for this finding is presently unclear but may be due to regulatory subunits, phosphorylation or other types of post translational modification. Ascribing particular SK channels to physiological roles using apamin as a drug tool needs to be done cautiously in light of these findings.

Generation of a new Gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells.

PubMed

De Groote, Philippe; Grootjans, Sasker; Lippens, Saskia; Eichperger, Chantal; Leurs, Kirsten; Kahr, Irene; Tanghe, Giel; Bruggeman, Inge; De Schamphelaire, Wouter; Urwyler, Corinne; Vandenabeele, Peter; Haustraete, Jurgen; Declercq, Wim

2016-01-01

In contrast to most common gene delivery techniques, lentiviral vectors allow targeting of almost any mammalian cell type, even non-dividing cells, and they stably integrate in the genome. Therefore, these vectors are a very powerful tool for biomedical research. Here we report the generation of a versatile new set of 22 lentiviral vectors with broad applicability in multiple research areas. In contrast to previous systems, our platform provides a choice between constitutive and/or conditional expression and six different C-terminal fusions. Furthermore, two compatible selection markers enable the easy derivation of stable cell lines co-expressing differently tagged transgenes in a constitutive or inducible manner. We show that all of the vector features are functional and that they contribute to transgene overexpression in proof-of-principle experiments.

Reliable transformation system for Microbotryum lychnidis-dioicae informed by genome and transcriptome project.

PubMed

Toh, Su San; Treves, David S; Barati, Michelle T; Perlin, Michael H

2016-10-01

Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.

Parenting in On/Off Relationships: The Link Between Relationship Churning and Father Involvement.

PubMed

Turney, Kristin; Halpern-Meekin, Sarah

2017-06-01

Family systems theory points to the interconnected nature of dyadic relationships within the family unit, arguing for attention to how the parental relationship shapes their ties to and interactions with their children. Grounded in family systems theory, we consider how relationship churning-defined as being in an on-again/off-again relationship with the same partner-is associated with father involvement. We use data from the Fragile Families and Child Wellbeing Study to examine how father involvement among relationship churners compares with father involvement among those in three other relationship types (measured during the first five years of the focal child's life): stably together relationships, stably broken-up relationships, and repartnered relationships. First, we find that churning fathers remain more involved with their 9-year-old children than do parents who stably break up or repartner, but they are less involved than those who are stably together. Second, lower relationship quality among churners-and, to a lesser extent, repartnering and childbearing with a new partner-explains some of the differences in father involvement between churners and the stably together. Third, these differences are most apparent among parents not living together when father involvement is measured. Taken together, the focus on relationship churning extends prior research on the association between relationship transitions and father involvement by separating relationship instability from partner change.

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

PubMed Central

Stringer, J R; Kuhn, R M; Newman, J L; Meade, J C

1985-01-01

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene. Images PMID:3016511

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

PubMed

Bobek, Vladimir; Plachy, Jiri; Pinterova, Daniela; Kolostova, Katarina; Boubelik, Michael; Jiang, Ping; Yang, Meng; Hoffman, Robert M

2004-01-01

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

NASA Technical Reports Server (NTRS)

Ludwig, D. L.; Bruschi, C. V.

1991-01-01

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression.

PubMed

Koenigsberger, C; Chiappa, S; Brimijoin, S

1997-10-01

Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

PubMed Central

Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

2010-01-01

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

PubMed

Edmonds, Tara G; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S; Conway, Joan A; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T; Montefiori, David C; Kappes, John C; Ochsenbauer, Christina

2010-12-05

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. Copyright © 2010 Elsevier Inc. All rights reserved.

Expression of insulin-like growth factor-2 receptors on EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2007-01-01

In the present study, we report the upregulation of functional IGF-2Rs in cells overexpressing growth hormone (GH). EL4 lymphoma cells stably transfected with an rGH cDNA overexpression vector (GHo) exhibited an increase in the binding of (125)I-IGF-2 with no change in the binding affinity compared to vector alone controls. An increase in the expression of the insulin-like growth factor-2 receptor (IGF-2R) in cells overexpressing GH was confirmed by Western blot analysis and IGF-2R promoter luciferase assays. EL4 cells produce insulin-like growth factor-2 (IGF-2) as detected by the reverse transcription-polymerase chain reaction (RT-PCR); however, no IGF-2 protein was detected by Western analysis. The increase in the expression of the IGF-2R resulted in greater levels of IGF-2 uptake in GHo cells compared to vector alone controls. The data suggest that one of the consequences of the overexpression of GH is an increase in the expression of the IGF-2R.

Resetting the transcription factor network reverses terminal chronic hepatic failure

PubMed Central

Nishikawa, Taichiro; Bell, Aaron; Brooks, Jenna M.; Setoyama, Kentaro; Melis, Marta; Han, Bing; Fukumitsu, Ken; Handa, Kan; Tian, Jianmin; Kaestner, Klaus H.; Vodovotz, Yoram; Locker, Joseph; Soto-Gutierrez, Alejandro; Fox, Ira J.

2015-01-01

The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement. PMID:25774505

Transcription elongation factors are involved in programming hormone production in pituitary neuroendocrine GH4C1 cells.

PubMed

Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

2010-05-05

Transcription elongation of many eukaryotic genes is regulated. Two negative transcription elongation factors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) are known to stall collaboratively RNA polymerase II promoter proximally. We discovered that DSIF and NELF are linked to hormone expression in rat pituitary GH4C1 cells. When NELF-E, a subunit of NELF or Spt5, a subunit of DSIF was stably knocked-down, prolactin (PRL) expression was increased both at the mRNA and protein levels. In contrast, stable knock-down of only Spt5 abolished growth hormone (GH) expression. Transient NELF-E knock-down increased coincidentally PRL expression and enhanced transcription of a PRL-promoter reporter gene. However, no direct interaction of NELF with the PRL gene could be demonstrated by chromatin immuno-precipitation. Thus, NELF suppressed PRL promoter activity indirectly. In conclusion, transcription regulation by NELF and DSIF is continuously involved in the control of hormone production and may contribute to neuroendocrine cell differentiation. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Oocyte formation by mitotically-active germ cells purified from ovaries of reproductive age women

PubMed Central

White, Yvonne A. R.; Woods, Dori C.; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L.

2012-01-01

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. PMID:22366948

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.

PubMed

White, Yvonne A R; Woods, Dori C; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L

2012-02-26

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

PubMed

Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

2005-01-01

Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

2014-01-01

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

PubMed

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Green tea polyphenols as potent enhancers of glucocorticoid-induced mouse mammary tumor virus gene expression.

PubMed

Abe, I; Umehara, K; Morita, R; Nemoto, K; Degawa, M; Noguchi, H

2001-02-16

The effect of natural and synthetic galloyl esters on glucocorticoid-induced gene expression was evaluated by using rat fibroblast 3Y1 cells stably transfected with a luciferase reporter gene under the transcriptional regulation of the mouse mammary tumor virus promoter. The glucocorticoid-induced gene transcription was strongly suppressed by synthetic alkyl esters; n-dodecyl gallate showed the most potent inhibition (66% inhibition at 10 microM), which was far more potent than that of crude tannic acid. n-Octyl and n-cetyl gallate also showed good inhibition, while gallic acid itself was not so active, suggesting that the presence of hydrophobic side chain is important for the suppressive effect. On the other hand, surprisingly, green tea gallocatechins, (-)-epigallocatechin-3-O-gallate and theasinensin A, potently enhanced the promoter activity (182 and 247% activity at 1 microM, respectively). The regulation of the level of the glucocorticoid-induced gene expression by the antioxidative gallates is of great interest from a therapeutic point of view.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies

PubMed Central

Kirkton, Robert D.; Bursac, Nenad

2012-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. PMID:21556054

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

PubMed Central

Enuka, Yehoshua; Lauriola, Mattia; Feldman, Morris E.; Sas-Chen, Aldema; Ulitsky, Igor; Yarden, Yosef

2016-01-01

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. PMID:26657629

Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.

PubMed

Martínez, R; Estrada, M P; Berlanga, J; Guillén, I; Hernández, O; Cabrera, E; Pimentel, R; Morales, R; Herrera, F; Morales, A; Piña, J C; Abad, Z; Sánchez, V; Melamed, P; Lleonart, R; de la Fuente, J

1996-03-01

The generation of transgenic fish with the transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. The tilapia growth hormone (tiGH) cDNA was linked to the human cytomegalovirus (CMV) enhancer-promoter and used to generate transgenic tilapia by microinjection into one-cell embryos. Five transgenic tilapia were obtained from 40 injected embryos. A transgenic animal containing one copy of the transgene per cell was selected to establish a transgenic line. The transgene was stably transmitted to F1 and F2 generations in a Mendelian fashion. Ectopic, low-level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by immunohystochemical analysis of tissue sections. Nine-month-old transgenic F1 progeny were 82% larger than nontransgenic fish at p = .001. These results showed that low-level ectopic expression of tiGH resulted in a growth acceleration in transgenic tilapia. Tilapia GH gene transfer is an alternative for growth acceleration in tilapia.

[Antiapoptotic Effect of the Leukemia Associated Gene MLAA-34 in HeLa Cells].

PubMed

Zhang, Peng-Yu; Zhao, Xuan; Zhang, Wen-Juan; Zhang, Wang-Gang; Chen, Yin-Xia

2016-04-01

To study the antiapoptotic effect of leukemia-associated gene MLAA-34 in HeLa cells. The MLAA-34 recombinant lentiviral expression vector was constructed, and the stably transfected HeLa cell line with high expression of MLAA-34 was set up; As(2)O(3) was used to induce apoptosis; the MTT assay, colony formation test and flow cytometry were used to detect the ability of cell proliferation, colong formation, apoptosis and cell cycle changes respectively. After treatment with As(2)O(3), the survival rate of HeLa cells with MLAA-34 overexpression was significantly higher than that of the control cells, and the colony formation ability of MLAA-34 significantly increased, and the high expression of MLAA-34 gene significantly decreased the apoptosis rate of HeLa cells, and decreased the proportion of G(2)/M phase cells. The leukemia-associated gene MLAA-34 has been comfirmed to show antiapoptotic effect in HeLa cells which are induced by As(2)O(3).

EXPERIMENTS ON STABLY AND NEUTRALLY STRATIFIED FLOW OVER A MODEL THREE-DIMENSIONAL HILL

EPA Science Inventory

The flow structure over a bell shaped hill (reciprocal of a fourth order polynomial in cross section and height h) was studied in large and small stably stratified towing tanks (with uniform density gradients) and in an unstratified wind tunnel. Observations were made at Froude n...

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

PubMed

Glasser, Stephan W; Eszterhas, Susan K; Detmer, Emily A; Maxfield, Melissa D; Korfhagen, Thomas R

2005-04-01

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Bioelectric memory: modeling resting potential bistability in amphibian embryos and mammalian cells.

PubMed

Law, Robert; Levin, Michael

2015-10-15

Bioelectric gradients among all cells, not just within excitable nerve and muscle, play instructive roles in developmental and regenerative pattern formation. Plasma membrane resting potential gradients regulate cell behaviors by regulating downstream transcriptional and epigenetic events. Unlike neurons, which fire rapidly and typically return to the same polarized state, developmental bioelectric signaling involves many cell types stably maintaining various levels of resting potential during morphogenetic events. It is important to begin to quantitatively model the stability of bioelectric states in cells, to understand computation and pattern maintenance during regeneration and remodeling. To facilitate the analysis of endogenous bioelectric signaling and the exploitation of voltage-based cellular controls in synthetic bioengineering applications, we sought to understand the conditions under which somatic cells can stably maintain distinct resting potential values (a type of state memory). Using the Channelpedia ion channel database, we generated an array of amphibian oocyte and mammalian membrane models for voltage evolution. These models were analyzed and searched, by simulation, for a simple dynamical property, multistability, which forms a type of voltage memory. We find that typical mammalian models and amphibian oocyte models exhibit bistability when expressing different ion channel subsets, with either persistent sodium or inward-rectifying potassium, respectively, playing a facilitative role in bistable memory formation. We illustrate this difference using fast sodium channel dynamics for which a comprehensive theory exists, where the same model exhibits bistability under mammalian conditions but not amphibian conditions. In amphibians, potassium channels from the Kv1.x and Kv2.x families tend to disrupt this bistable memory formation. We also identify some common principles under which physiological memory emerges, which suggest specific strategies for implementing memories in bioengineering contexts. Our results reveal conditions under which cells can stably maintain one of several resting voltage potential values. These models suggest testable predictions for experiments in developmental bioelectricity, and illustrate how cells can be used as versatile physiological memory elements in synthetic biology, and unconventional computation contexts.

Shearing-induced asymmetry in entorhinal grid cells.

PubMed

Stensola, Tor; Stensola, Hanne; Moser, May-Britt; Moser, Edvard I

2015-02-12

Grid cells are neurons with periodic spatial receptive fields (grids) that tile two-dimensional space in a hexagonal pattern. To provide useful information about location, grids must be stably anchored to an external reference frame. The mechanisms underlying this anchoring process have remained elusive. Here we show in differently sized familiar square enclosures that the axes of the grids are offset from the walls by an angle that minimizes symmetry with the borders of the environment. This rotational offset is invariably accompanied by an elliptic distortion of the grid pattern. Reversing the ellipticity analytically by a shearing transformation removes the angular offset. This, together with the near-absence of rotation in novel environments, suggests that the rotation emerges through non-coaxial strain as a function of experience. The systematic relationship between rotation and distortion of the grid pattern points to shear forces arising from anchoring to specific geometric reference points as key elements of the mechanism for alignment of grid patterns to the external world.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoo, Masako; Fujita, Ryosuke; Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021

Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes locatedmore » between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.« less

G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid.

PubMed

Ylikomi, Timo; Vienonen, Annika; Ahola, Tytti M

2004-11-01

G protein-coupled receptor 30 (GPR30) has previously been described to be important in steroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) cells using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They expressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished the expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. When HME cells were transiently transfected with the glucocorticoid response element MMTV-luc reporter plasmid, stable expression of GPR30 resulted in the abolition of ligand-induced transactivation of the promoter. In COS cells, transient transfection of GPR30 with glucocorticoid receptor alpha resulted in an abrogation of the MMTV-luc and GRE-luc reporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling interferes with steroid signaling.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation

PubMed Central

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-01-01

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells. PMID:25110433

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

PubMed

Zhai, Hui-Hong; Meng, Juan; Wang, Jing-Bo; Liu, Zhen-Xiong; Li, Yuan-Fei; Feng, Shan-Shan

2014-08-07

To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

Thyroid cell lines in research on goitrogenesis.

PubMed

Gerber, H; Peter, H J; Asmis, L; Studer, H

1991-12-01

Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

PubMed Central

Rodriguez, Michelle D.; Paul, Zubin; Wood, Charles E.; Rice, Kelly C.; Triplett, Eric W.

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources. PMID:29312199

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

PubMed

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

PubMed Central

Venkatachalam, Veena; Kralj, Joel M.; Dib-Hajj, Sulayman D.; Waxman, Stephen G.; Cohen, Adam E.

2013-01-01

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators. PMID:24391999

Heat shock proteins HSP70 and MRJ cooperatively regulate cell adhesion and migration through urokinase receptor.

PubMed

Lin, Yuli; Peng, Nana; Zhuang, Hongqin; Zhang, Di; Wang, Yao; Hua, Zi-Chun

2014-08-30

The urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein. We performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay. HSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR. These findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.

Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

NASA Astrophysics Data System (ADS)

Paproski, Robert J.; Zemp, Roger J.

2012-02-01

We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells.

PubMed

Chahal, Manpreet S; Ku, H Teresa; Zhang, Zhihong; Legaspi, Christian M; Luo, Angela; Hopkins, Mandi M; Meier, Kathryn E

Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Paired box 7 inhibits differentiation in 3T3-L1 preadipocytes.

PubMed

Izumi, Wakana; Takuma, Yuko; Ebihara, Ryo; Mizunoya, Wataru; Tatsumi, Ryuichi; Nakamura, Mako

2018-06-13

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets. © 2018 Japanese Society of Animal Science.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

PubMed

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Li, Chuanfeng; Chen, Zongyan; Liu, Guangqing

2016-11-01

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. Copyright © 2016 Elsevier B.V. All rights reserved.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

PubMed

Pateraki, Irini; Andersen-Ranberg, Johan; Jensen, Niels Bjerg; Wubshet, Sileshi Gizachew; Heskes, Allison Maree; Forman, Victor; Hallström, Björn; Hamberger, Britta; Motawia, Mohammed Saddik; Olsen, Carl Erik; Staerk, Dan; Hansen, Jørgen; Møller, Birger Lindberg; Hamberger, Björn

2017-03-14

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii , in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13 R -manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13 R -manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana . The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

Contactin‑associated protein‑like 2 expression in SH‑SY5Y cells is upregulated by a FOXP2 mutant with a shortened poly‑glutamine tract.

PubMed

Zhao, Yunjing; Liu, Xiaoliang; Sun, Hongwei; Wang, Yueping; Yang, Wenzhu; Ma, Hongwei

2015-12-01

The forkhead box protein P2 (FOXP2) gene encodes an important transcription factor that contains a polyglutamine (poly‑Q) tract and a forkhead DNA binding domain. It has been observed that FOXP2 is associated with speech sound disorder (SSD), and mutations that decrease the length of the poly‑Q tract were identified in the FOXP2 gene of SSD patients. However, the exact role of poly‑Q reduction is not well understood. In the present study, constructs expressing wild‑type and poly‑Q reduction mutants of FOXP2 were generated by polymerase chain reaction (PCR) using lentiviral vectors and transfected into the SH‑SY5Y neuronal cell line. Quantitative reverse transcription (qRT)‑PCR and western blotting indicated that infected cells stably expressed high levels of FOXP2. Using this cell model, the impact of FOXP2 on the expression of contactin‑associated protein‑like 2 (CNTNAP2) were investigated, and CNTNAP2 mRNA expression levels were observed to be significantly higher in cells expressing poly‑Q‑reduced FOXP2. In addition, the expression level of CASPR2, a mammalian homolog of Drosophila Neurexin IV, was increased in cells expressing the FOXP2 mutant. Demonstration of regulation by FOXP2 indicates that CNTNAP2 may also be involved in SSD.

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed Central

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-01-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver. PMID:9343392

Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

PubMed

Chaya, D; Fougère-Deschatrette, C; Weiss, M C

1997-11-01

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

Dynamics of Wolbachia pipientis Gene Expression Across the Drosophila melanogaster Life Cycle

PubMed Central

Gutzwiller, Florence; Carmo, Catarina R.; Miller, Danny E.; Rice, Danny W.; Newton, Irene L. G.; Hawley, R. Scott; Teixeira, Luis; Bergman, Casey M.

2015-01-01

Symbiotic interactions between microbes and their multicellular hosts have manifold biological consequences. To better understand how bacteria maintain symbiotic associations with animal hosts, we analyzed genome-wide gene expression for the endosymbiotic α-proteobacteria Wolbachia pipientis across the entire life cycle of Drosophila melanogaster. We found that the majority of Wolbachia genes are expressed stably across the D. melanogaster life cycle, but that 7.8% of Wolbachia genes exhibit robust stage- or sex-specific expression differences when studied in the whole-organism context. Differentially-expressed Wolbachia genes are typically up-regulated after Drosophila embryogenesis and include many bacterial membrane, secretion system, and ankyrin repeat-containing proteins. Sex-biased genes are often organized as small operons of uncharacterized genes and are mainly up-regulated in adult Drosophila males in an age-dependent manner. We also systematically investigated expression levels of previously-reported candidate genes thought to be involved in host-microbe interaction, including those in the WO-A and WO-B prophages and in the Octomom region, which has been implicated in regulating bacterial titer and pathogenicity. Our work provides comprehensive insight into the developmental dynamics of gene expression for a widespread endosymbiont in its natural host context, and shows that public gene expression data harbor rich resources to probe the functional basis of the Wolbachia-Drosophila symbiosis and annotate the transcriptional outputs of the Wolbachia genome. PMID:26497146

Acoustic suspension system

NASA Technical Reports Server (NTRS)

Lee, M. C.; Wang, T. G. (Inventor)

1983-01-01

An acoustic levitation system is described, with single acoustic source and a small reflector to stably levitate a small object while the object is processed as by coating or heating it. The system includes a concave acoustic source which has locations on opposite sides of its axis that vibrate towards and away from a focal point to generate a converging acoustic field. A small reflector is located near the focal point, and preferably slightly beyond it, to create an intense acoustic field that stably supports a small object near the reflector. The reflector is located about one-half wavelength from the focal point and is concavely curved to a radius of curvature (L) of about one-half the wavelength, to stably support an object one-quarter wavelength (N) from the reflector.

Adolescents Exiting Homelessness Over Two Years: The Risk Amplification and Abatement Model

PubMed Central

Milburn, Norweeta G.; Rice, Eric; Rotheram-Borus, Mary Jane; Mallett, Shelley; Rosenthal, Doreen; Batterham, Phillip; May, Susanne J.; Witkin, Andrea; Duan, Naihua

2014-01-01

The Risk Amplification and Abatement Model (RAAM), demonstrates that negative contact with socializing agents amplify risk, while positive contact abates risk for homeless adolescents. To test this model, the likelihood of exiting homelessness and returning to familial housing at 2 years and stably exiting over time are examined with longitudinal data collected from 183 newly homeless adolescents followed over 2 years in Los Angeles, CA. In support of RAAM, unadjusted odds of exiting at 2 years and stably exiting over2 years revealed that engagement with pro-social peers, maternal social support, and continued school attendance all promoted exiting behaviors. Simultaneously, exposure to family violence and reliance on shelter services discouraged stably exiting behaviors. Implications for family-based interventions are proposed. PMID:25067896

Selection of housekeeping genes for use in quantitative reverse transcription PCR assays on the murine cornea.

PubMed

Ren, Shengwei; Zhang, Feng; Li, Changyou; Jia, Changkai; Li, Siyuan; Xi, Haijie; Zhang, Hongbo; Yang, Lingling; Wang, Yiqiang

2010-06-11

To evaluate the suitability of common housekeeping genes (HKGs) for use in quantitative reverse transcription PCR (qRT-PCR) assays of the cornea in various murine disease models. CORNEAL DISEASE MODELS STUDIED WERE: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin [ACTB], lactate dehydrogenase A [LDHA], ribosomal protein L5 [RPL5], ubiquitin C [UBC], peptidylprolyl isomerase A [PPIA], TATA-box binding protein [TBP1], and hypoxanthine guanine phosphoribosyl transferase [HPRT1]) in the cornea were measured at various time points by microarray hybridization or qRT-PCR and the data analyzed using geNorm and NormFinder. Microarray results showed that under the CorNV condition the expression stability of the 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. qRT-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA and HRPT1 and PPIA and RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice, respectively, while UBC and HPRT1 and UBC and LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice, respectively. When qRT-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.

Sensitivity of the Geomagnetic Octupole to a Stably Stratified Layer in the Earth's Core

NASA Astrophysics Data System (ADS)

Yan, C.; Stanley, S.

2017-12-01

The presence of a stably stratified layer at the top of the core has long been proposed for Earth, based on evidence from seismology and geomagnetic secular variation. Geodynamo modeling offers a unique window to inspect the properties and dynamics in Earth's core. For example, numerical simulations have shown that magnetic field morphology is sensitive to the presence of stably stratified layers in a planet's core. Here we use the mMoSST numerical dynamo model to investigate the effects of a thin stably stratified layer at the top of the fluid outer core in Earth on the resulting large-scale geomagnetic field morphology. We find that the existence of a stable layer has significant influence on the octupolar component of the magnetic field in our models, whereas the quadrupole doesn't show an obvious trend. This suggests that observations of the geomagnetic field can be applied to provide information of the properties of this plausible stable layer, such as how thick and how stable this layer could be. Furthermore, we have examined whether the dominant thermal signature from mantle tomography at the core-mantle boundary (CMB) (a degree & order 2 spherical harmonic) can influence our results. We found that this heat flux pattern at the CMB has no outstanding effects on the quadrupole and octupole magnetic field components. Our studies suggest that if there is a stably stratified layer at the top of the Earth's core, it must be limited in terms of stability and thickness, in order to be compatible with the observed paleomagnetic record.

From Near-Neutral to Strongly Stratified: Adequately Modelling the Clear-Sky Nocturnal Boundary Layer at Cabauw.

PubMed

Baas, P; van de Wiel, B J H; van der Linden, S J A; Bosveld, F C

2018-01-01

The performance of an atmospheric single-column model (SCM) is studied systematically for stably-stratified conditions. To this end, 11 years (2005-2015) of daily SCM simulations were compared to observations from the Cabauw observatory, The Netherlands. Each individual clear-sky night was classified in terms of the ambient geostrophic wind speed with a [Formula: see text] bin-width. Nights with overcast conditions were filtered out by selecting only those nights with an average net radiation of less than [Formula: see text]. A similar procedure was applied to the observational dataset. A comparison of observed and modelled ensemble-averaged profiles of wind speed and potential temperature and time series of turbulent fluxes showed that the model represents the dynamics of the nocturnal boundary layer (NBL) at Cabauw very well for a broad range of mechanical forcing conditions. No obvious difference in model performance was found between near-neutral and strongly-stratified conditions. Furthermore, observed NBL regime transitions are represented in a natural way. The reference model version performs much better than a model version that applies excessive vertical mixing as is done in several (global) operational models. Model sensitivity runs showed that for weak-wind conditions the inversion strength depends much more on details of the land-atmosphere coupling than on the turbulent mixing. The presented results indicate that in principle the physical parametrizations of large-scale atmospheric models are sufficiently equipped for modelling stably-stratified conditions for a wide range of forcing conditions.

Construction of trypanosome artificial mini-chromosomes.

PubMed Central

Lee, M G; E, Y; Axelrod, N

1995-01-01

We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucei PARP promoter driving a chloramphenicol acetyltransferase (CAT) gene. In the 10 kb construct the CAT gene is followed by one hygromycin phosphotransferase (Hph) gene, and in the 13 kb construct the CAT gene is followed by three tandemly linked Hph genes. At each end of these linear molecules are telomere repeats and subtelomeric sequences. Electroporation of these linear DNA constructs into the procyclic form of T.brucei generated hygromycin-B resistant cell lines. In these cell lines, the input DNA remained linear and bounded by the telomere ends, but it increased in size. In the cell lines generated by the 10 kb construct, the input DNA increased in size to 20-50 kb. In the cell lines generated by the 13 kb constructs, two sizes of linear DNAs containing the input plasmid were detected: one of 40-50 kb and the other of 150 kb. The increase in size was not the result of in vivo tandem repetitions of the input plasmid, but represented the addition of new sequences. These Hph containing linear DNA molecules were maintained stably in cell lines for at least 20 generations in the absence of drug selection and were subsequently referred to as trypanosome artificial mini-chromosomes, or TACs. Images PMID:8532534

From Near-Neutral to Strongly Stratified: Adequately Modelling the Clear-Sky Nocturnal Boundary Layer at Cabauw

NASA Astrophysics Data System (ADS)

Baas, P.; van de Wiel, B. J. H.; van der Linden, S. J. A.; Bosveld, F. C.

2018-02-01

The performance of an atmospheric single-column model (SCM) is studied systematically for stably-stratified conditions. To this end, 11 years (2005-2015) of daily SCM simulations were compared to observations from the Cabauw observatory, The Netherlands. Each individual clear-sky night was classified in terms of the ambient geostrophic wind speed with a 1 m s^{-1} bin-width. Nights with overcast conditions were filtered out by selecting only those nights with an average net radiation of less than - 30 W m^{-2}. A similar procedure was applied to the observational dataset. A comparison of observed and modelled ensemble-averaged profiles of wind speed and potential temperature and time series of turbulent fluxes showed that the model represents the dynamics of the nocturnal boundary layer (NBL) at Cabauw very well for a broad range of mechanical forcing conditions. No obvious difference in model performance was found between near-neutral and strongly-stratified conditions. Furthermore, observed NBL regime transitions are represented in a natural way. The reference model version performs much better than a model version that applies excessive vertical mixing as is done in several (global) operational models. Model sensitivity runs showed that for weak-wind conditions the inversion strength depends much more on details of the land-atmosphere coupling than on the turbulent mixing. The presented results indicate that in principle the physical parametrizations of large-scale atmospheric models are sufficiently equipped for modelling stably-stratified conditions for a wide range of forcing conditions.

Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

PubMed

Zhu, Wuzheng; Lin, Yaqiu; Liao, Honghai; Wang, Yong

2015-01-01

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

Application of Hanging Drop Technique for Kidney Tissue Culture.

PubMed

Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

2017-01-01

The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

F-prostanoid receptor regulation of fibroblast growth factor 2 signaling in endometrial adenocarcinoma cells.

PubMed

Sales, Kurt J; Boddy, Sheila C; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N

2007-08-01

Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.

Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

PubMed Central

Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

2014-01-01

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

PubMed

Charoensri, Nicha; Suphatrakul, Amporn; Sriburi, Rungtawan; Yasanga, Thippawan; Junjhon, Jiraphan; Keelapang, Poonsook; Utaipat, Utaiwan; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

2014-09-01

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Unprecedented Cell-Selection Using Ultra-Quick Freezing Combined with Aquaporin Expression

PubMed Central

Kato, Yasuhiro; Miyauchi, Takayuki; Abe, Youichiro; Kojić, Dušan; Tanaka, Manami; Chikazawa, Nana; Nakatake, Yuhki; Ko, Shigeru B. H.; Kobayashi, Daisuke; Hazama, Akihiro; Fujiwara, Shoko; Uchida, Tatsuya; Yasui, Masato

2014-01-01

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols. PMID:24558371

Low BRMS1 expression promotes nasopharyngeal carcinoma metastasis in vitro and in vivo and is associated with poor patient survival

PubMed Central

2012-01-01

Background Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene. This study aimed to investigate the impact of BRMS1 on metastasis in nasopharyngeal carcinoma (NPC) and to evaluate the prognostic significance of BRMS1 in NPC patients. Methods BRMS1 expression was examined in NPC cell lines using quantitative reverse transcription-polymerase chain reaction and Western blotting. NPC cells stably expressing BRMS1 were used to perform wound healing and invasion assays in vitro and a murine xenograft assay in vivo. Immunohistochemical staining was performed in 274 paraffin-embedded NPC specimens divided into a training set (n = 120) and a testing set (n = 154). Results BRMS1 expression was down-regulated in NPC cell lines. Overexpression of BRMS1 significantly reversed the metastatic phenotype of NPC cells in vitro and in vivo. Importantly, low BRMS1 expression was associated with poor distant metastasis-free survival (DMFS, P < 0.001) and poor overall survival (OS, P < 0.001) in the training set; these results were validated in the testing set and overall patient population. Cox regression analysis demonstrated that low BRMS1 expression was an independent prognostic factor for DMFS and OS in NPC. Conclusions Low expression of the metastasis suppressor BRMS1 may be an independent prognostic factor for poor prognosis in NPC patients. PMID:22931099

Overexpression of microRNA-21 strengthens stem cell-like characteristics in a hepatocellular carcinoma cell line.

PubMed

Jiang, Jinghang; Yang, Peipei; Guo, Zhe; Yang, Rirong; Yang, Haojie; Yang, Fuquan; Li, Lequn; Xiang, Bangde

2016-10-28

Liver cancer stem cells (LCSCs) have been shown to express higher levels of microRNA-21 (miR-21). Here, we examine the possible contributions of miR-21 to the phenotype of LCSCs in culture and in xenograft tumors in nude mice. The hepatocellular carcinoma cell line MHCC-97H was stably transformed with a retroviral vector to establish cells overexpressing miR-21, while a cell line transformed with empty vector served as a negative control. RT-PCR and Western blotting were used to evaluate the effects of miR-21 overexpression on the expression of various LCSC markers, a Transwell assay was used to assess the effects on cell migration and invasion, and a spheroid formation assay was used to examine the effects on clonogenesis. The effects of miR-21 overexpression were also examined in tumors in nude mice. An MHCC-97H cell line was constructed that stably overexpresses miR-21 at 7.78 ± 1.51-fold higher levels than the negative control cell line. Expression of the LCSC markers CD13, Ep-CAM, CD90, and OCT4 was significantly higher in the miR-21-overexpressing cell line than in the negative control at both mRNA and protein levels. The overexpressing cell line formed larger, tighter, and more numerous spheroids. Overexpression of miR-21 was associated with greater cell migration and invasion. Tumors of overexpressing cells in nude mice had a significantly larger mean volume after 34 days of growth (773.62 ± 163.46 mm 3 ) than tumors of negative control cells (502.79 ± 33.94 mm 3 , p = 0.048), as well as greater mean weight (0.422 ± 0.019 vs. 0.346 ± 0.006 g, p = 0.003). Overexpression of miR-21 strengthens the phenotype of LCSCs, facilitating invasion, migration, and tumorigenesis in hepatocellular carcinoma.

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

PubMed

Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

1993-07-15

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Differential sensitivity of porcine endogenous retrovirus to APOBEC3-mediated inhibition.

PubMed

Park, Sung-Han; Kim, Jin Ha; Jung, Yong-Tae

2015-08-01

Pigs are considered to be suitable xenotransplantation organ donors. However, the risk of pathogen transmission from pigs to humans is a major concern in the transplantation of porcine tissues. The porcine endogenous retroviruses (PERVs) PERV-A, PERV-A/C, and PERV-B can infect human cells, but PERV-C is an ecotropic virus infecting only pig cells. Thus, several strategies have been proposed to reduce PERV transmission in xenograft recipients. Human APOBEC3G (huA3G) is a single-strand DNA cytosine deaminase, which inactivates the coding capacity of the virus by deamination of cDNA cytosines to uracils. This reaction occurs within the (-) DNA strand during reverse transcription, resulting in a G-to-A mutation in the (+) strand. While recent data have shown that PERV-B is severely inhibited by huA3G and porcine A3Z2-Z3 (poA3F) in a pseudotype assay, little is known about PERV-C. Here, we compare the antiretroviral activities of huA3G, huA3F and poA3Z2-Z3 against PERV-C. Our data show that APOBEC3 was packaged into PERV-C particles and inhibited PERV-C replication in a dose-dependent manner. PERV-C infectivity was strongly inhibited by poA3Z2-Z3, but it did not markedly reduce PERV-B infectivity. This suggests that PERV-C Gag interacts efficiently with poA3Z2-Z3. In addition, we constructed stably huA3G- and poA3Z2-Z3-expressing 293-PERV-PK-CIRCE cells (human 293 cells infected with PK15-derived PERVs) to examine whether PERV is resistant to poA3Z2-Z3 in a virus-spreading assay. The stably expressed huA3G and poA3Z2-Z3 were more packaging-competent than transiently expressed APOBEC3 proteins. These results suggest that poA3Z2-Z3 can inhibit PERV replication in a pseudotype assay as well as in a virus-spreading assay.

Loss of HSulf-1 expression enhances tumorigenicity by inhibiting Bim expression in ovarian cancer.

PubMed

He, Xiaoping; Khurana, Ashwani; Roy, Debarshi; Kaufmann, Scott; Shridhar, Viji

2014-10-15

The expression of human Sulfatase1 (HSulf-1) is downregulated in the majority of primary ovarian cancer tumors, but the functional consequence of this downregulation remains unclear. Using two different shRNAs (Sh1 and Sh2), HSulf-1 expression was stably downregulated in ovarian cancer OV202 cells. We found that HSulf-1-deficient OV202 Sh1 and Sh2 cells formed colonies in soft agar. In contrast, nontargeting control (NTC) shRNA-transduced OV202 cells did not form any colonies. Moreover, subcutaneous injection of OV202 HSulf-1-deficient cells resulted in tumor formation in nude mice, whereas OV202 NTC cells did not. Also, ectopic expression of HSulf-1 in ovarian cancer SKOV3 cells significantly suppressed tumor growth in nude mice. Here, we show that HSulf-1-deficient OV202 cells have markedly decreased expression of proapoptotic Bim protein, which can be rescued by restoring HSulf-1 expression in OV202 Sh1 cells. Enhanced expression of HSulf-1 in HSulf-1-deficient SKOV3 cells resulted in increased Bim expression. Decreased Bim levels after loss of HSulf-1 were due to increased p-ERK, because inhibition of ERK activity with PD98059 resulted in increased Bim expression. However, treatment with a PI3 kinase/AKT inhibitor, LY294002, failed to show any change in Bim protein level. Importantly, rescuing Bim expression in HSulf-1 knockdown cells significantly retarded tumor growth in nude mice. Collectively, these results suggest that loss of HSulf-1 expression promotes tumorigenicity in ovarian cancer through regulating Bim expression. © 2014 UICC.

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Internal Wave-Convection-Mean Flow Interactions

NASA Astrophysics Data System (ADS)

Lecoanet, D.; Couston, L. A.; Favier, B.; Le Bars, M.

2017-12-01

We present a series of simulations of Boussinesq fluid with a nonlinear equation of state which in thermal equilibrium is convective in the bottom part of the domain, but stably stratified in the upper part of the domain. The stably stratified region supports internal gravity waves, which are excited by the convection. The convection can significantly affected by the stably stratified region. Furthermore, the waves in the stable region can interact nonlinearly to drive coherent mean flows which exhibit regular oscillations, similar to the QBO in the Earth's atmosphere. We will describe the dependence of the mean flow oscillations on the properties of the convection which generate the internal waves. This provides a novel framework for understanding mean flow oscillations in the Earth's atmosphere, as well as the atmospheres of giant planets.

African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

PubMed

Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2002-04-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

PubMed Central

Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

2002-01-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

Expression of a novel P22 ORFan gene reveals the phage carrier state in Salmonella typhimurium.

PubMed

Cenens, William; Mebrhatu, Mehari T; Makumi, Angella; Ceyssens, Pieter-Jan; Lavigne, Rob; Van Houdt, Rob; Taddei, François; Aertsen, Abram

2013-01-01

We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage-host interactions.

Expression of a Novel P22 ORFan Gene Reveals the Phage Carrier State in Salmonella Typhimurium

PubMed Central

Makumi, Angella; Ceyssens, Pieter-Jan; Lavigne, Rob; Van Houdt, Rob; Taddei, François

2013-01-01

We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage–host interactions. PMID:23483857

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio).

PubMed

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-10-09

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp ( Cyprinus carpio ) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.

Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain.

PubMed

Nandi, Somen; Yalda, Dorice; Lu, Stephen; Nikolov, Zivko; Misaki, Ryo; Fujiyama, Kazuhito; Huang, Ning

2005-06-01

In this paper, we show that recombinant human lactoferrin (rhLF) has been stably expressed at 0.5% brown rice flour weight for nine generations. Process development indicates that rhLF can be efficiently extracted from rice flour in 20 mM phosphate buffer (pH 7.0) containing up to 0.5 M NaCl and at a ratio of 1 kg flour to 10 L buffer. After solid/liquid separation, the extract can then be loaded directly onto an ion-exchange column and rhLF can be eluted using 0.8 M NaCl. The resulting rhLF is about 95% pure. A range of biochemical and biophysical analyses were carried out and results indicated that the purified rhLF was identical to its native human counterpart other than its glycosylation. Economic analysis shows that at 600 kg/year scale, the cash cost to produce 1 g of rhLF of pharmaceutical grade is US$ 5.90. Analysis also indicates that the expression level has profound impact on costs related to planting, milling, extraction and purification, thus high level expression of recombinant protein in plants is one of the key parameters for the success of plant made pharmaceuticals.

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

PubMed

Abouseadaa, Heba H; Osman, Gamal H; Ramadan, Ahmed M; Hassanein, Sameh E; Abdelsattar, Mohamed T; Morsy, Yasser B; Alameldin, Hussien F; El-Ghareeb, Doaa K; Nour-Eldin, Hanan A; Salem, Reda; Gad, Adel A; Elkhodary, Soheir E; Shehata, Maher M; Mahfouz, Hala M; Eissa, Hala F; Bahieldin, Ahmed

2015-07-22

Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. Transgenic wheat plants had improved resistance to Sitophilus granarius.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio)

PubMed Central

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-01-01

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro. PMID:27735853

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

PubMed

Ding, Jin; Feng, Yuan; Zheng, Zhao Hui; Li, Xue Yi; Wu, Zhen Biao; Zhu, Ping

2015-02-01

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or β2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis. © 2014 John Wiley & Sons Ltd.

Fast detection of extrasynaptic GABA with a whole-cell sniffer.

PubMed

Christensen, Rasmus K; Petersen, Anders V; Schmitt, Nicole; Perrier, Jean-François

2014-01-01

Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a "sniffer" allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

Fast detection of extrasynaptic GABA with a whole-cell sniffer

PubMed Central

Christensen, Rasmus K.; Petersen, Anders V.; Schmitt, Nicole; Perrier, Jean-François

2014-01-01

Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations. PMID:24860433

Angiogenesis characteristics of infantile hemangioma and feasibility observation of transplantation model of human hemangioma on mice.

PubMed

Fu, Y; Yang, Z-G; Zhao, L-Y

2017-03-01

To study pathogenic features of pediatric hemangiomas, we successfully established a model in mice, by transplanting human hemangioma tissues. The hemangioma from the leg of a two-month-old infant was dissected and sliced into several pieces. During a careful surgical procedure, the hemangioma tissues were individually transplanted into skin incisions in anesthetized mice. The volume of the transplanted tumors was measured and the changes in shape recorded at 1 day, and at 1, 2, 3, 4, 5 and 6 months after the transplantation. HE dyeing, CD31 and Glut1 IHC were applied to tumors in the proliferation and involuting phases. Also, 10 survival tumors and 10 normal tissues from infants undergoing circumcisions (control tissues) were used to determine their Angiotensin 1 (Ang1), Angiotensin 2 (Ang2), Tie2, and endothelium growth factor (VEGF) expression levels by IHC method. We observed all the tumors going through the same stages, where after two months their volumes increased sharply and then after 4 months they all began to recede. During the proliferative phase, newly born capillaries could be seen and the tumor elasticity increased (bright red color). During the involuting phase, the color faded away and the tumors became harder and were almost gone by 6 months. During the first two months after transplantation, HE dyeing showed hypertrophied and proliferating endothelial cells accumulating inside the tumors with irregular cavities inside blood vessels being filled by them. During the involuting phase (at 4 months), the lumen in blood vessels was distinctly enlarged while fiber and adipose tissue had significantly deposited. The transplanted and original tumors tested positive for CD31 and Glut1 dyeing, without significant differences. Compared with control samples, the Ang1 expression of the transplanted tumor in both the hyperplasia and proliferative phases was stably low (p<0.05), while expressions of Ang2 and Tie2 were both stably high (p<0.05). The VEGF expression in the tumors, however, was high during the proliferative phase (p<0.05), while the VEGF of the involuting phase showed no significant differences from that of the normal samples (p>0.05). We showed the reliability of the mouse model in reflecting the pathologic evolution of the proliferation and involuting phases of infantile hemangiomas. Angiogenic mediators Ang1, Ang2 and Tie2 may be abnormally expressed and play important roles in the development of this angiogenic disease.

Cysteine-rich secretory protein 3 plays a role in prostate cancer cell invasion and affects expression of PSA and ANXA1.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Apte, Snehal; Acharya, Kshitish; Mahale, Smita D

2016-01-01

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

2006-11-01

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

Transcriptional regulation by FOXP1, FOXP2, and FOXP4 dimerization.

PubMed

Sin, Cora; Li, Hongyan; Crawford, Dorota A

2015-02-01

FOXP1, FOXP2, and FOXP4 are three members of the FOXP gene subfamily of transcription factors involved in the development of the central nervous system. Previous studies have shown that the transcriptional activity of FOXP1/2/4 is regulated by homo- and heterodimerization. However, their transcriptional gene targets in the developing brain are still largely unknown. FOXP2 regulates the expression of many genes important in embryonic development, including WNT and Notch signaling pathways. In this study, we investigate whether dimerization of FOXP1/2/4 leads to differential expression of ten known FOXP2 target genes (CER1, SFRP4, WISP2, PRICKLE1, NCOR2, SNW1, NEUROD2, PAX3, EFNB3, and SLIT1). FOXP1/2/4 open-reading frames were stably transfected into HEK293 cells, and the expression level of these FOXP2 target genes was quantified using real-time polymerase chain reaction. Our results revealed that the specific combination of FOXP1/2/4 dimers regulates transcription of various FOXP2 target genes involved in early neuronal development.

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells

PubMed Central

Gonçalves da Silva, Patrícia Benites; Teixeira dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Leite Pereira, Márcia Cristina; Furukawa, Gabriela; Gimenes da Cruz, Daniel Sanzio; Goldfeder, Mauricio Barbugiani; Reily Rocha, Clarissa Ribeiro; Rosenberg, Carla; Okamoto, Oswaldo Keith

2017-01-01

Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. PMID:28186969

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells.

PubMed

da Silva, Patrícia Benites Gonçalves; Teixeira Dos Santos, Márcia Cristina; Rodini, Carolina Oliveira; Kaid, Carolini; Pereira, Márcia Cristina Leite; Furukawa, Gabriela; da Cruz, Daniel Sanzio Gimenes; Goldfeder, Mauricio Barbugiani; Rocha, Clarissa Ribeiro Reily; Rosenberg, Carla; Okamoto, Oswaldo Keith

2017-03-21

Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.

NR4A3 Suppresses Lymphomagenesis through Induction of Proapoptotic Genes.

PubMed

Deutsch, Alexander J A; Rinner, Beate; Pichler, Martin; Prochazka, Katharina; Pansy, Katrin; Bischof, Marco; Fechter, Karoline; Hatzl, Stefan; Feichtinger, Julia; Wenzl, Kerstin; Frisch, Marie-Therese; Stiegelbauer, Verena; Prokesch, Andreas; Krogsdam, Anne; Sill, Heinz; Thallinger, Gerhard G; Greinix, Hildegard T; Wang, Chenguang; Beham-Schmid, Christine; Neumeister, Peter

2017-05-01

Nuclear orphan receptor NR4A1 exerts an essential tumor suppressor function in aggressive lymphomas. In this study, we investigated the hypothesized contribution of the related NR4A family member NR4A3 to lymphomagenesis. In aggressive lymphoma patients, low expression of NR4A3 was associated with poor survival. Ectopic expression or pharmacological activation of NR4A3 in lymphoma cell lines led to a significantly higher proportion of apoptotic cells. In a mouse NSG xenograft model of lymphoma (stably transduced SuDHL4 cells), NR4A3 expression abrogated tumor growth, compared with vector control and uninduced cells that formed massive tumors. Transcript analysis of four different aggressive lymphoma cell lines overexpressing either NR4A3 or NR4A1 revealed that apoptosis was driven similarly by induction of BAK, Puma, BIK, BIM, BID, and Trail. Overall, our results showed that NR4A3 possesses robust tumor suppressor functions of similar impact to NR4A1 in aggressive lymphomas. Cancer Res; 77(9); 2375-86. ©2017 AACR . ©2017 American Association for Cancer Research.

RNAi Functions in Adaptive Reprogramming of the Genome | Center for Cancer Research

Cancer.gov

The regulation of transcribing DNA into RNA, including the production, processing, and degradation of RNA transcripts, affects the expression and the regulation of the genome in ways that are just beginning to be unraveled. A surprising discovery in recent years is that the vast majority of the genome is transcribed to yield an abundance of RNA transcripts. Many transcripts are regulated by the exosome, a multi-protein complex that degrades RNAs, and may also be targeted, under certain conditions, by the RNA interference (RNAi) pathway. These RNA degrading activities can recruit factors to silence certain regions of the genome by condensing the DNA into tightly-packed heterochromatin. For some chromosomal regions, such as centromeres and telomeres, which lie at the center and ends of chromosomes, respectively, silencing must be stably enforced through each cell generation. For other regions, silencing mechanisms must be easily reversible to activate gene expression in response to changing environmental or developmental conditions. Thus, the regulation of gene silencing is key to maintaining the integrity of the genome and proper cellular expression patterns, which, when disrupted can underlie many diseases, including cancer.

RNA Adenosine Deaminase ADAR1 Deficiency Leads to Increased Activation of Protein Kinase PKR and Reduced Vesicular Stomatitis Virus Growth Following Interferon Treatment

PubMed Central

Li, Zhiqun; Wolff, Karen C.; Samuel, Charles E.

2009-01-01

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells over expressing ADAR1 were utilized. Over expression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than ~10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was ~1 log10 further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment. PMID:19913273

Insulin induces alpha1B-adrenergic receptor phosphorylation and desensitization.

PubMed

García-Sáinz, J Adolfo; Romero-Avila, M Teresa; Molina-Muñoz, Tzindilú; Medina, Luz del Carmen

2004-09-03

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

Tafazzin (TAZ) promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis.

PubMed

Chen, Mei; Zhang, Yuan; Zheng, Peng-Sheng

2017-01-01

Tafazzin (TAZ) is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27), high-grade squamous intraepithelial lesions (HSIL, n = 26) and squamous cervical carcinoma (SCC, n = 41) by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. In vitro, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. In vivo, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis.

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase.

PubMed

Kassar, Telissa C; Magalhães, Tereza; S, José V J; Carvalho, Amanda G O; Silva, Andréa N M R DA; Queiroz, Sabrina R A; Bertani, Giovani R; Gil, Laura H V G

2017-01-01

Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

Tafazzin (TAZ) promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis

PubMed Central

Chen, Mei; Zhang, Yuan; Zheng, Peng-Sheng

2017-01-01

Tafazzin (TAZ) is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27), high-grade squamous intraepithelial lesions (HSIL, n = 26) and squamous cervical carcinoma (SCC, n = 41) by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. In vitro, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. In vivo, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis. PMID:28489874

Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1

PubMed Central

Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2016-01-01

Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). PMID:27292014

Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

PubMed

Ni-Komatsu, Li; Orlow, Seth J

2006-03-01

The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

Orbifold genera, product formulas and power operations

NASA Astrophysics Data System (ADS)

Ganter, Nora

2004-07-01

We generalize the definition of orbifold elliptic genus, and introduce orbifold genera of chromatic level h, using h-tuples rather than pairs of commuting elements. We show that our genera are in fact orbifold invariants, and we prove integrality results for them. If the genus arises from an H-infinity-map into the Morava-Lubin-Tate theory E_h, then we give a formula expressing the orbifold genus of the symmetric powers of a stably almost complex manifold M in terms of the genus of M itself. Our formula is the p-typical analogue of the Dijkgraaf-Moore-Verlinde-Verlinde formula for the orbifold elliptic genus. It depends only on h and not on the genus.

Functional antagonistic properties of clozapine at the 5-HT3 receptor.

PubMed

Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

1996-08-23

The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Fine Tuning Gene Expression: The Epigenome

PubMed Central

Mohtat, Davoud; Susztak, Katalin

2011-01-01

An epigenetic trait is a stably inherited phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic modifications, such as; DNA methylation, together with covalent modification of histones, are thought to alter chromatin density and accessibility of the DNA to cellular machinery, thereby modulating the transcriptional potential of the underlying DNA sequence. As epigenetic marks under environmental influence, epigenetics provides an added layer of variation that might mediate the relationship between genotype and internal and external environmental factors. Integration of our knowledge in genetics, epigenomics and genomics with the use of systems biology tools may present investigators with new powerful tools to study many complex human diseases such as kidney disease. PMID:21044758

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy.

PubMed

Ali, Eman Ali; Nakamura, Ryosuke; Falcone, Franco H

2017-01-01

Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

PubMed

Burlibașa, Liliana; Suciu, Ilinca

2015-12-01

Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

Post-mortem re-cloning of a transgenic red fluorescent protein dog

PubMed Central

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

2011-01-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid.

PubMed

Pelà, M; Del Zoppo, L; Allegri, L; Marzola, E; Ruzza, C; Calo, G; Perissutti, E; Frecentese, F; Salvadori, S; Guerrini, R

2014-07-01

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the lpv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.

H63D mutation in hemochromatosis alters cholesterol metabolism and induces memory impairment.

PubMed

Ali-Rahmani, Fatima; Grigson, Patricia S; Lee, Sang; Neely, Elizabeth; Connor, James R; Schengrund, Cara-Lynne

2014-06-01

The H63D variant of the hemochromatosis (HFE) gene, when expressed in carriers of the apolipoprotein E4 allele, is implicated as a risk factor for earlier onset of Alzheimer's disease (AD). We tested the hypothesis that like expression of apolipoprotein E4, expression of H63D-HFE disrupts cholesterol metabolism contributing to an increase in neurodegeneration and memory deficits. Analysis of SH-SY5Y human neuroblastoma cells transfected to stably express either wild type- (WT) or H63D-HFE indicated about a 50% reduction in cholesterol content in cells expressing H63D-HFE. This was accompanied by a significant decrease in expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and a significant increase in expression of cholesterol 24-hydroxylase. Consistent with these studies, H67D-HFE (orthologous to human H63D-HFE) knock-in mice, showed a greater age dependent decline in brain cholesterol than WT-HFE animals and changes in expression of proteins regulating cholesterol metabolism. Brains of aged H67D-HFE mice also exhibited a significant decrease in expression of synapse proteins and a significant increase in caspase-3 expression relative to WT-HFE controls. H67D-HFE mice also had a greater reduction in brain volume and poorer recognition and spatial memory than WT-HFE mice, symptoms associated with AD. These results indicate that the alterations in cholesterol metabolism associated with expression of H63D-HFE may contribute to the development of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

PubMed

Kitamura, M; Kawachi, H

1997-09-15

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Human cytomegalovirus UL76 induces chromosome aberrations

PubMed Central

2009-01-01

Background Human cytomegalovirus (HCMV) is known to induce chromosome aberrations in infected cells, which can lead to congenital abnormalities in infected fetuses. HCMV UL76 belongs to a conserved protein family from herpesviruses. Some reported roles among UL76 family members include involvement in virulence determination, lytic replication, reactivation of latent virus, modulation of gene expression, induction of apoptosis, and perturbation of cell cycle progression, as well as potential nuclease activity. Previously, we have shown that stable expression of UL76 inhibits HCMV replication in glioblastoma cells. Methods To examine chromosomal integrity and the DNA damage signal γ-H2AX in cells constitutively expressing UL76, immunofluorescent cell staining and Western blotting were performed. The comet assay was employed to assess DNA breaks in cells transiently expressing UL76. Results We report that stably transfected cells expressing UL76 developed chromosome aberrations including micronuclei and misaligned chromosomes, lagging and bridging. In mitotic cells expressing UL76, aberrant spindles were increased compared to control cells. However, cells with supernumerary centrosomes were marginally increased in UL76-expressing cells relative to control cells. We further demonstrated that UL76-expressing cells activated the DNA damage signal γ-H2AX and caused foci formation in nuclei. In addition, the number of cells with DNA breaks increased in proportion to UL76 protein levels. Conclusion Our findings suggest that the virus-associated protein UL76 induces DNA damage and the accumulation of chromosome aberrations. PMID:19930723

Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

PubMed

Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

2015-02-15

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

Intercellular Adhesion Molecule-1 Expression by Skeletal Muscle Cells Augments Myogenesis

PubMed Central

Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

2014-01-01

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. PMID:25281303

The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL).

PubMed

Guo, Y; Schoell, M C; Freeman, R S

2009-04-23

von Hippel-Lindau (VHL) disease is caused by germ-line mutations in the VHL tumor suppressor gene and is the most common cause of inherited renal cell carcinoma (RCC). Mutations in the VHL gene also occur in a large majority of sporadic cases of clear-cell RCC, which have high intrinsic resistance to chemotherapy and radiotherapy. Here we show that VHL-deficient RCC cells express lower levels of the proapoptotic Bcl-2 family protein BIM(EL) and are more resistant to etoposide and UV radiation-induced death compared to the same cells stably expressing the wild-type VHL protein (pVHL). Reintroducing pVHL into VHL-null cells increased the half-life of BIM(EL) protein without affecting its mRNA expression, and overexpressing pVHL inhibited BIM(EL) polyubiquitination. Suppressing pVHL expression with RNA interference resulted in a decrease in BIM(EL) protein and a corresponding decrease in the sensitivity of RCC cells to apoptotic stimuli. Directly inhibiting BIM(EL) expression in pVHL-expressing RCC cells caused a similar decrease in cell death. These results demonstrate that pVHL acts to promote BIM(EL) protein stability in RCC cells, and that destabilization of BIM(EL) in the absence of pVHL contributes to the increased resistance of VHL-null RCC cells to certain apoptotic stimuli.

Heterogeneous Nuclear Ribonucleoprotein F Suppresses Angiotensinogen Gene Expression and Attenuates Hypertension and Kidney Injury in Diabetic Mice

PubMed Central

Lo, Chao-Sheng; Chang, Shiao-Ying; Chenier, Isabelle; Filep, Janos G.; Ingelfinger, Julie R.; Zhang, Shao Ling; Chan, John S.D.

2012-01-01

We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes. PMID:22664958

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Epigenetics Research on the International Space Station

NASA Technical Reports Server (NTRS)

Love, John; Cooley, Vic

2016-01-01

The International Space Station (ISS) is a state-of-the orbiting laboratory focused on advancing science and technology research. Experiments being conducted on the ISS include investigations in the emerging field of Epigenetics. Epigenetics refers to stably heritable changes in gene expression or cellular phenotype (the transcriptional potential of a cell) resulting from changes in a chromosome without alterations to the underlying DNA nucleotide sequence (the genetic code), which are caused by external or environmental factors, such as spaceflight microgravity. Molecular mechanisms associated with epigenetic alterations regulating gene expression patterns include covalent chemical modifications of DNA (e.g., methylation) or histone proteins (e.g., acetylation, phorphorylation, or ubiquitination). For example, Epigenetics ("Epigenetics in Spaceflown C. elegans") is a recent JAXA investigation examining whether adaptations to microgravity transmit from one cell generation to another without changing the basic DNA of the organism. Mouse Epigenetics ("Transcriptome Analysis and Germ-Cell Development Analysis of Mice in Space") investigates molecular alterations in organ-specific gene expression patterns and epigenetic modifications, and analyzes murine germ cell development during long term spaceflight, as well as assessing changes in offspring DNA. NASA's first foray into human Omics research, the Twins Study ("Differential effects of homozygous twin astronauts associated with differences in exposure to spaceflight factors"), includes investigations evaluating differential epigenetic effects via comprehensive whole genome analysis, the landscape of DNA and RNA methylation, and biomolecular changes by means of longitudinal integrated multi-omics research. And the inaugural Genes in Space student challenge experiment (Genes in Space-1) is aimed at understanding how epigenetics plays a role in immune system dysregulation by assaying DNA methylation in immune cells directly in space using miniPCR technology. In addition, NASA's geneLAB campaign covers the epigenome as part of the "expressome", by employing an innovative open source science platform for multi-investigator high throughput utilization of the ISS. Earth benefits of Epigenetics research onboard the ISS range from contributions to the fundamental understanding of epigenetic phenomena with applications in countermeasure development for biomedical conditions, to the generation of integrated strategies for personalized medicine based on unique physiological responses.

Turbulent circulation above the surface heat source in stably stratified atmosphere

NASA Astrophysics Data System (ADS)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-10-01

The 3-level RANS approach for simulating a turbulent circulation over the heat island in a stably stratified environment under nearly calm conditions is formulated. The turbulent kinetic energy its spectral consumption (dissipation) and the dispersion of turbulent fluctuations of temperature are found from differential equations, thus the correct modeling of transport processes in the interface layer with the counter-gradient heat flux is assured. The three-parameter turbulence RANS approach minimizes difficulties in simulating the turbulent transport in a stably stratified environment and reduces efforts needed for the numerical implementation of the 3-level RANS approach. Numerical simulation of the turbulent structure of the penetrative convection over the heat island under conditions of stably stratified atmosphere demonstrates that the three-equation model is able to predict the thermal circulation induced by the heat island. The temperature distribution, root-mean-square fluctuations of the turbulent velocity and temperature fields and spectral turbulent kinetic energy flux are in good agreement with the experimental data. The model describes such thin physical effects, as a crossing of vertical profiles of temperature of a thermal plume with the formation of the negative buoyancy area testifying to development of the dome-shaped form at the top part of a plume in the form of "hat".

Comprehensive analysis of the MLH1 promoter region in 480 patients with colorectal cancer and 1150 controls reveals new variants including one with a heritable constitutional MLH1 epimutation.

PubMed

Morak, Monika; Ibisler, Ayseguel; Keller, Gisela; Jessen, Ellen; Laner, Andreas; Gonzales-Fassrainer, Daniela; Locher, Melanie; Massdorf, Trisari; Nissen, Anke M; Benet-Pagès, Anna; Holinski-Feder, Elke

2018-04-01

Germline defects in MLH1 , MSH2 , MSH6 and PMS2 predisposing for Lynch syndrome (LS) are mainly based on sequence changes, whereas a constitutional epimutation of MLH1 (CEM) is exceptionally rare. This abnormal MLH1 promoter methylation is not hereditary when arising de novo, whereas a stably heritable and variant-induced CEM was described for one single allele. We searched for MLH1 promoter variants causing a germline or somatic methylation induction or transcriptional repression. We analysed the MLH1 promoter sequence in five different patient groups with colorectal cancer (CRC) (n=480) composed of patients with i) CEM (n=16), ii) unsolved loss of MLH1 expression in CRC (n=37), iii) CpG-island methylator-phenotype CRC (n=102), iv) patients with LS (n=83) and v) MLH1-proficient CRC (n=242) as controls. 1150 patients with non-LS tumours also served as controls to correctly judge the results. We detected 10 rare MLH1 promoter variants. One novel, complex MLH1 variant c.-63_-58delins18 is present in a patient with CRC with CEM and his sister, both showing a complete allele-specific promoter methylation and transcriptional silencing. The other nine promoter variants detected in 17 individuals were not associated with methylation. For four of these, a normal, biallelic MLH1 expression was found in the patients' cDNA. We report the second promoter variant stably inducing a hereditary CEM. Concerning the classification of promoter variants, we discuss contradictory results from the literature for two variants, describe classification discrepancies between existing rules for five variants, suggest the (re-)classification of five promoter variants to (likely) benign and regard four variants as functionally unclear. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

PubMed

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-02-28

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

PubMed Central

Wang, Guirong; Christensen, Neil D; Wigdahl, Brian; Guttentag, Susan H; Floros, Joanna

2003-01-01

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions. PMID:12356334

Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

PubMed Central

Sun, Liying; Hao, Yanzhe; Wang, Zhan; Zeng, Yi

2018-01-01

Epstein-Barr virus (EBV) is related to a variety of malignant tumors, and its encoded protein, latent membrane protein 2 (LMP2), is an effective target antigen that is widely used to construct vector vaccines. However, the model cells carrying LMP2 have still not been established to assess the oncolytic effect of LMP2-related vaccines at present. In this study, TC-1-GLUC-LMP2 tumor cells were constructed as target cells to evaluate the anti-tumor effects of LMP2-assosiated vaccines. The results showed that both LMP2 and Gaussia luciferase (GLuc) genes could be detected by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) in TC-1-GLUC-LMP2 cells. Western blot results showed that the LMP2 and Gaussia luciferase proteins were stably expressed in tumor cells for at least 30 generations. We mixed 5 × 104 LMP2-specific mouse splenic lymphocytes with 5 × 103 TC-1-GLUC-LMP2 target cells and found that the target cells were killed as the specific killing effect was obviously enhanced by the increased quantities of LMP2-peptide stimulated spleens. Furthermore, the tumor cells could not be observed in the mice inoculated TC-1-GLUC-LMP2 cells after being immunized with vaccine-LMP2, while the vaccine-NULL immunized mice showed that tumor volume gradually grew with increased inoculation time. These results indicated that the TC-1-GLUC-LMP2 cells stably expressing LMP2 and GLuc produced tumors in mice, and that the LMP2-specific cytotoxic T lymphocyte (CTL) effectively killed the cells in vitro and in vivo, suggesting that TC-1-GLUC-LMP2 cells can be used as model cells to assess the immune and antitumor effects of LMP2-related vaccines. PMID:29570629

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy

PubMed Central

Zheng, Shu; Xiao, Zuo-Xiang; Pan, Yue-Long; Han, Ming-Yong; Dong, Qi

2003-01-01

AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene. METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated. RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-γ, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice. CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy. PMID:12717836

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

PubMed

Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2017-03-01

The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

PubMed

Tramontana, S; Bionaz, M; Sharma, A; Graugnard, D E; Cutler, E A; Ajmone-Marsan, P; Hurley, W L; Loor, J J

2008-08-01

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.

Generation of Referring Expressions: Assessing the Incremental Algorithm

ERIC Educational Resources Information Center

van Deemter, Kees; Gatt, Albert; van der Sluis, Ielka; Power, Richard

2012-01-01

A substantial amount of recent work in natural language generation has focused on the generation of "one-shot" referring expressions whose only aim is to identify a target referent. Dale and Reiter's Incremental Algorithm (IA) is often thought to be the best algorithm for maximizing the similarity to referring expressions produced by people. We…

Regulatory RNAs and chromatin modification in dosage compensation: a continuous path from flies to humans?

PubMed

Angelopoulou, Roxani; Lavranos, Giagkos; Manolakou, Panagiota

2008-03-20

Chromosomal sex determination is a widely distributed strategy in nature. In the most classic scenario, one sex is characterized by a homologue pair of sex chromosomes, while the other includes two morphologically and functionally distinct gonosomes. In mammalian diploid cells, the female is characterized by the presence of two identical X chromosomes, while the male features an XY pair, with the Y bearing the major genetic determinant of sex, i.e. the SRY gene. In other species, such as the fruitfly, sex is determined by the ratio of autosomes to X chromosomes. Regardless of the exact mechanism, however, all these animals would exhibit a sex-specific gene expression inequality, due to the different number of X chromosomes, a phenomenon inhibited by a series of genetic and epigenetic regulatory events described as "dosage compensation". Since adequate available data is currently restricted to worms, flies and mammals, while for other groups of animals, such as reptiles, fish and birds it is very limited, it is not yet clear whether this is an evolutionary conserved mechanism. However certain striking similarities have already been observed among evolutionary distant species, such as Drosophila melanogaster and Mus musculus. These mainly refer to a) the need for a counting mechanism, to determine the chromosomal content of the cell, i.e. the ratio of autosomes to gonosomes (a process well understood in flies, but still hypothesized in mammals), b) the implication of non-translated, sex-specific, regulatory RNAs (roX and Xist, respectively) as key elements in this process and the location of similar mediators in the Z chromosome of chicken c) the inclusion of a chromatin modification epigenetic final step, which ensures that gene expression remains stably regulated throughout the affected area of the gonosome. This review summarizes these points and proposes a possible role for comparative genetics, as they seem to constitute proof of maintained cell economy (by using the same basic regulatory elements in various different scenarios) throughout numerous centuries of evolutionary history.

Postmitotic Expression of SOD1G93A Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation

PubMed Central

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1G93A) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1G93A gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1G93A in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1G93A gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1G93A gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1G93A gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1G93A in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1G93A on myogenic program and disclosed potential signaling, activated by SOD1G93A, that affect the identity of the myogenic cell population. PMID:26491230

Postmitotic Expression of SOD1(G93A) Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation.

PubMed

Martini, Martina; Dobrowolny, Gabriella; Aucello, Michela; Musarò, Antonio

2015-01-01

To determine the role of mutant SOD1 gene (SOD1(G93A)) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1(G93A) gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1(G93A) in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1(G93A) gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1(G93A) gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1(G93A) gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1(G93A) in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1(G93A) on myogenic program and disclosed potential signaling, activated by SOD1(G93A), that affect the identity of the myogenic cell population.

Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.1[w

PubMed Central

Tapia, Gerardo; Verdugo, Isabel; Yañez, Mónica; Ahumada, Iván; Theoduloz, Cristina; Cordero, Cecilia; Poblete, Fernando; González, Enrique; Ruiz-Lara, Simón

2005-01-01

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5′-LTR region of this element can drive stress-induced transcriptional activation of the β-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the β-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense. PMID:16040666

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Guohua; Shi Lingfang; Qiu Daoming

2005-05-01

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mousemore » NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.« less

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediatedmore » toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.« less

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

[Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

PubMed

Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

2017-08-01

Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

Gastrin-releasing peptide-induced down-regulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) in neuroblastomas.

PubMed

Qiao, Jingbo; Kang, Junghee; Cree, Jeremy; Evers, B Mark; Chung, Dai H

2005-05-01

To evaluate whether aggressive, undifferentiated neuroblastomas express tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten) and to examine the effects of gastrin-releasing peptide (GRP) on PTEN gene and protein expression. We have previously shown that neuroblastomas secrete GRP, which binds to its cell surface receptor (GRP-R) to stimulate cell growth in an autocrine fashion. However, the effects of GRP on expression of the tumor suppressor gene PTEN have not been elucidated in neuroblastomas. Paraffin-embedded sections from human neuroblastomas were analyzed for PTEN and phospho-Akt protein expression by immunohistochemistry. Human neuroblastoma cell lines (SK-N-SH and SH-SY5Y) were stably transfected with the plasmid pEGFP-GRP-R to establish GRP-R overexpression cell lines, and the effects of GRP on PTEN gene and protein expression were determined. A decrease in the ratio of PTEN to phospho-Akt protein expression was identified in poorly differentiated neuroblastomas. An increase in GRP binding capacity was confirmed in GRP-R overexpressing cells, which demonstrated an accelerated constitutive cell growth rate. PTEN gene and protein expression was significantly decreased in GRP-R overexpressing cells when compared with controls. Our findings demonstrate decreased expression of the tumor suppressor protein PTEN in more aggressive undifferentiated neuroblastomas. An increase in GRP binding capacity, as a result of GRP-R overexpression, down-regulates PTEN expression. These findings suggest that an inhibition of the tumor suppressor gene PTEN may be an important regulatory mechanism involved in GRP-induced cell proliferation in neuroblastomas.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

PubMed

Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

2016-09-22

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

PubMed Central

Pateraki, Irini; Andersen-Ranberg, Johan; Jensen, Niels Bjerg; Wubshet, Sileshi Gizachew; Heskes, Allison Maree; Forman, Victor; Hallström, Björn; Hamberger, Britta; Motawia, Mohammed Saddik; Olsen, Carl Erik; Staerk, Dan; Hansen, Jørgen; Møller, Birger Lindberg; Hamberger, Björn

2017-01-01

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L. DOI: http://dx.doi.org/10.7554/eLife.23001.001 PMID:28290983

Improving the Safety of T Cell Therapies using an Inducible Caspase-9 Gene

PubMed Central

Zhou, Xiaoou; Brenner, Malcolm K.

2016-01-01

Adoptive transfer of T cells can be an effective anti-cancer treatment. However, uncontrolled or unpredictable immediate or persistent toxicities are a source of concern. The ability to conditionally eliminate aberrant cells in vivo therefore is becoming a critical step for the successful translation of this approach to the clinic. We review the evolution of safety systems, focusing on a suicide switch that can be expressed stably and efficiently in human T cells without impairing phenotype, function or antigen specificity. This system is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization in the presence of an otherwise bioinert small molecule drug. When exposed to the synthetic dimerizing drug, the inducible caspase 9 (iC9) becomes activated and leads to the rapid apoptosis of cells expressing this construct. We have demonstrated the clinical feasibility and efficacy of this approach after haploidentical hematopoietic stem cell transplant (haplo-HSCT). Here we review the benefits and limitations of the approach. PMID:27473568

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

PubMed

Keu, Khun Visith; Witney, Timothy H; Yaghoubi, Shahriar; Rosenberg, Jarrett; Kurien, Anita; Magnusson, Rachel; Williams, John; Habte, Frezghi; Wagner, Jamie R; Forman, Stephen; Brown, Christine; Allen-Auerbach, Martin; Czernin, Johannes; Tang, Winson; Jensen, Michael C; Badie, Behnam; Gambhir, Sanjiv S

2017-01-18

High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8 + cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [ 18 F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer. Copyright © 2017, American Association for the Advancement of Science.

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

PubMed

Wang, Quanfu; Hou, Yanhua; Shi, Yonglei; Han, Xiao; Chen, Qian; Hu, Zhiguo; Liu, Yuanping; Li, YuJin

2014-01-01

Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Phosphorylation status modulates Bcl-2 function during glucocorticoid-induced apoptosis in T lymphocytes.

PubMed

Huang, Se-Te J; Cidlowski, John A

2002-06-01

Glucocorticoids are known to induce apoptosis in lymphoid cells, and Bcl-2 overexpression can block the apoptosis-inducing action of glucocorticoids. Since phosphorylation of Bcl-2 is implicated in regulating Bcl-2 function, we considered the role of Bcl-2 phosphorylation in protecting lymphoid cells from glucocorticoid-induced cell death. Five stably transfected cell lines of WEHI 7.1 cells expressing either wild-type Bcl-2 or alanine mutants of Bcl-2 at amino acids threonine 56, serine 70, threonine 74, or serine 87 were created. Expression of the mutant Bcl-2 proteins was documented by flow cytometry and Western blot analysis. Mutation of Bcl-2 on T56 and S87 eliminated the ability of Bcl-2 to inhibit glucocorticoid-induced cell shrinkage, mitochondrial depolarization, DNA fragmentation, and cell death. Mutation of T74 only partially impaired the ability of Bcl-2 to block glucocorticoid-induced apoptosis whereas mutation of S70 in Bcl-2 did not alter its ability to block glucocorticoid-induced apoptosis.

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

PubMed

Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2007-12-01

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

PubMed

Lee, Suk Kyoo; Lee, Gyun Min

2003-06-30

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.

P16INK4a MEDIATED SUPPRESSION OF TELOMERASE IN NORMAL AND MALIGNANT HUMAN BREAST CELLS

PubMed Central

Bazarov, Alexey V.; van Sluis, Marjolein; Hines, Curtis; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W.; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-01-01

Summary The cyclin-dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. PMID:20569236

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

PubMed

Liao, Hsin-Kai; Gu, Ying; Diaz, Arturo; Marlett, John; Takahashi, Yuta; Li, Mo; Suzuki, Keiichiro; Xu, Ruo; Hishida, Tomoaki; Chang, Chan-Jung; Esteban, Concepcion Rodriguez; Young, John; Izpisua Belmonte, Juan Carlos

2015-03-10

To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.

Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

PubMed

Gao, Mengmeng; Liu, Yaping; Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

A trait stacking system via intra-genomic homologous recombination.

PubMed

Kumar, Sandeep; Worden, Andrew; Novak, Stephen; Lee, Ryan; Petolino, Joseph F

2016-11-01

A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3 % of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

The Induction of Serine/Threonine Protein Phosphorylations by a PDGFR/TrkA Chimera in Stably Transfected PC12 Cells*

PubMed Central

Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

2012-01-01

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping. PMID:22027198

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

PubMed

Lorenz, P; Koczan, D; Thiesen, H J

2001-04-01

The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative co-repressor TIF1beta were significantly changed. Furthermore, the TIF1beta/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1beta and HP1alpha was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1alpha was significantly affected. Thus, KRAB, TIF1beta and HP1alpha are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of

Uniform Recovery Bounds for Structured Random Matrices in Corrupted Compressed Sensing

NASA Astrophysics Data System (ADS)

Zhang, Peng; Gan, Lu; Ling, Cong; Sun, Sumei

2018-04-01

We study the problem of recovering an $s$-sparse signal $\\mathbf{x}^{\\star}\\in\\mathbb{C}^n$ from corrupted measurements $\\mathbf{y} = \\mathbf{A}\\mathbf{x}^{\\star}+\\mathbf{z}^{\\star}+\\mathbf{w}$, where $\\mathbf{z}^{\\star}\\in\\mathbb{C}^m$ is a $k$-sparse corruption vector whose nonzero entries may be arbitrarily large and $\\mathbf{w}\\in\\mathbb{C}^m$ is a dense noise with bounded energy. The aim is to exactly and stably recover the sparse signal with tractable optimization programs. In this paper, we prove the uniform recovery guarantee of this problem for two classes of structured sensing matrices. The first class can be expressed as the product of a unit-norm tight frame (UTF), a random diagonal matrix and a bounded columnwise orthonormal matrix (e.g., partial random circulant matrix). When the UTF is bounded (i.e. $\\mu(\\mathbf{U})\\sim1/\\sqrt{m}$), we prove that with high probability, one can recover an $s$-sparse signal exactly and stably by $l_1$ minimization programs even if the measurements are corrupted by a sparse vector, provided $m = \\mathcal{O}(s \\log^2 s \\log^2 n)$ and the sparsity level $k$ of the corruption is a constant fraction of the total number of measurements. The second class considers randomly sub-sampled orthogonal matrix (e.g., random Fourier matrix). We prove the uniform recovery guarantee provided that the corruption is sparse on certain sparsifying domain. Numerous simulation results are also presented to verify and complement the theoretical results.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter.

PubMed

Hwang, Daw-Yang; Ismail-Beigi, Faramarz

2002-03-15

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.

Circulating microRNAs (cmiRNAs) as novel potential biomarkers for hepatocellular carcinoma

PubMed Central

Qi, Jiahui; Wang, Jin; Katayama, Hiroshi; Sen, Subrata; Liu, Song-mei

2013-01-01

Incidence and mortality associated with hepatocellular carcinoma (HCC) is rising throughout the world. Accurate, noninvasive biomarkers for the early detection of HCC are urgently needed to reduce worldwide morbidity and mortality related to HCC. MicroRNAs (miRNAs), 17- to 25-nucleotide noncoding RNAs that are frequently dysregulated in HCC, have shown great promise as tissue-based markers for HCC diagnosis and prognosis. Moreover, they are stably expressed in serum and urine, and these circulating microRNAs (cmiRNAs) are emerging as novel noninvasive biomarkers for the early detection and prognosis of HCC. This article summarizes the latest findings on the role of circulating miRNAs as potential minimally invasive diagnostic and prognostic biomarkers for HCC. PMID:23259781

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins

PubMed Central

Zdobnova, Tatiana; Sokolova, Evgeniya; Stremovskiy, Oleg; Karpenko, Dmitry; Telford, William; Turchin, Ilya; Balalaeva, Irina; Deyev, Sergey

2015-01-01

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the “window of tissue transparency”. The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A. PMID:26436696

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.

The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here in this paper, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol andmore » sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.« less

Identification and comprehensive evaluation of reference genes for RT-qPCR analysis of host gene-expression in Brassica juncea-aphid interaction using microarray data.

PubMed

Ram, Chet; Koramutla, Murali Krishna; Bhattacharya, Ramcharan

2017-07-01

Brassica juncea is a chief oil yielding crop in many parts of the world including India. With advancement of molecular techniques, RT-qPCR based study of gene-expression has become an integral part of experimentations in crop breeding. In RT-qPCR, use of appropriate reference gene(s) is pivotal. The virtue of the reference genes, being constant in expression throughout the experimental treatments, needs to be validated case by case. Appropriate reference gene(s) for normalization of gene-expression data in B. juncea during the biotic stress of aphid infestation is not known. In the present investigation, 11 reference genes identified from microarray database of Arabidopsis-aphid interaction at a cut off FDR ≤0.1, along with two known reference genes of B. juncea, were analyzed for their expression stability upon aphid infestation. These included 6 frequently used and 5 newly identified reference genes. Ranking orders of the reference genes in terms of expression stability were calculated using advanced statistical approaches such as geNorm, NormFinder, delta Ct and BestKeeper. The analysis suggested CAC, TUA and DUF179 as the most suitable reference genes. Further, normalization of the gene-expression data of STP4 and PR1 by the most and the least stable reference gene, respectively has demonstrated importance and applicability of the recommended reference genes in aphid infested samples of B. juncea. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Blue light-mediated transcriptional activation and repression of gene expression in bacteria

PubMed Central

Jayaraman, Premkumar; Devarajan, Kavya; Chua, Tze Kwang; Zhang, Hanzhong; Gunawan, Erry; Poh, Chueh Loo

2016-01-01

Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes. PMID:27353329

Hepa1-6-FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability.

PubMed

Li, Yasha; Liu, Mengnan; Cui, Jiejie; Yang, Ke; Zhao, Li; Gong, Mengjia; Wang, Yi; He, Yun; He, Tongchuan; Bi, Yang

2018-05-01

Reliable animal models are required for the in vivo study of the molecular mechanisms and effects of chemotherapeutic drugs in hepatocarcinoma. In vivo tracing techniques based on firefly luciferase (FLuc) may optimize the non-invasive monitoring of experimental animals. The present study established a murine Hepa1-6-FLuc cell line that stably expressed a retrovirus-delivered FLuc protein gene. The cell morphology, proliferation, migration and invasion ability of Hepa1-6-FLuc cells were the same as that of the Hepa1-6 cells, and thus is suitable to replace Hepa1-6 cells in the construction of hepatocarcinoma animal models. No differences in subcutaneous tumor mass and its pathomorphology from implanted Hepa1-6-FLuc cells were observed compared with Hepa1-6 control tumors. Bioluminescence imaging indicated that the Luc signal of the Hepa1-6-FLuc cells was consistently strengthened with increases in tumor mass; however, the Luc signal of Hepa1-6-AdFLuc became weaker and eventually disappeared during tumor development. Therefore, compared with the transient expression by adenovirus, stable expression of the FLuc gene in Hepa1-6 cells may better reflect cell proliferation and survival in vivo , and provide a reliable source for the establishment of hepatocarcinoma models.

Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

PubMed

Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

2009-05-27

Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

PubMed Central

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-01-01

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

PubMed

Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

2013-12-01

Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1. Copyright © 2013 Elsevier Ltd. All rights reserved.

Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi.

PubMed

Freire, Anna Cláudia Guimarães; Alves, Ceres Luciana; Goes, Grazielle Ribeiro; Resende, Bruno Carvalho; Moretti, Nilmar Silvio; Nunes, Vinícius Santana; Aguiar, Pedro Henrique Nascimento; Tahara, Erich Birelli; Franco, Glória Regina; Macedo, Andréa Mara; Pena, Sérgio Danilo Junho; Gadelha, Fernanda Ramos; Guarneri, Alessandra Aparecida; Schenkman, Sergio; Vieira, Leda Quercia; Machado, Carlos Renato

2017-09-01

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma

PubMed Central

Young, Kon-Ji; Park, A-Reum; Choi, Ha-Rim; Lee, Hwa-Youn; Kim, Su-Man; Chung, Byung Yeoup; Park, Chul-Hong; Choi, Hyo Jin; Ko, Young-Hyeh; Bai, Hyoung-Woo; Kang, Hyung-Sik

2017-01-01

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma. PMID:28423548

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma.

PubMed

Lee, Eun-Hee; Kim, Eun-Mi; Ji, Kon-Young; Park, A-Reum; Choi, Ha-Rim; Lee, Hwa-Youn; Kim, Su-Man; Chung, Byung Yeoup; Park, Chul-Hong; Choi, Hyo Jin; Ko, Young-Hyeh; Bai, Hyoung-Woo; Kang, Hyung-Sik

2017-03-28

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma.

A multi-landing pad DNA integration platform for mammalian cell engineering

PubMed Central

Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

2018-01-01

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873