WorldWideScience

Sample records for reporter gene shows

  1. Gene Therapy Shows Promise for Aggressive Lymphoma

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_163824.html Gene Therapy Shows Promise for Aggressive Lymphoma Over one-third ... TUESDAY, Feb. 28, 2017 (HealthDay News) -- An experimental gene therapy for aggressive non-Hodgkin lymphoma beat back more ...

  2. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  3. A novel mutation in the calcium-sensing receptor gene in an Irish pedigree showing familial hypocalciuric hypercalcemia: a case report.

    LENUS (Irish Health Repository)

    Elamin, Wael F

    2010-01-01

    Familial hypocalciuric hypercalcemia is a rare autosomal dominant disorder characterized by asymptomatic and non-progressive hypercalcemia due to mutations of the calcium-sensing receptor gene. Disorders of calcium metabolism are very common in the elderly, and they can coexist with familial hypocalciuric hypercalcemia in affected families.

  4. Reports show fewer pallets entering landfills

    Science.gov (United States)

    Robert J. Bush; Philip A. Araman

    2010-01-01

    Information from a series of Virginia Tech studies reveals current trends in core utilization by the pallet industry. The most recent report released this year studied trends from 2006. Virginia Tech researchers compared these results to five previous studies going all the way back to 1992.

  5. Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

    Science.gov (United States)

    Mohan, Vijee; Gupta, Soni; Thomas, Sherinmol; Mickey, Hanjabam; Charakana, Chaitanya; Chauhan, Vineeta Singh; Sharma, Kapil; Kumar, Rakesh; Tyagi, Kamal; Sarma, Supriya; Gupta, Suresh Kumar; Kilambi, Himabindu Vasuki; Nongmaithem, Sapana; Kumari, Alka; Gupta, Prateek; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

  6. Pharmacogenomics genes show varying perceptibility to microRNA regulation

    DEFF Research Database (Denmark)

    Rukov, Jakob Lewin; Vinther, Jeppe; Shomron, Noam

    2011-01-01

    The aim of pharmacogenomics is to identify individual differences in genome and transcriptome composition and their effect on drug efficacy. MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression of the majority of animal genes, including many genes involved in drug...

  7. Chronic Bullying Can Show Up in Report Cards

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_163318.html Chronic Bullying Can Show Up in Report Cards Study found ... 2017 MONDAY, Jan. 30, 2017 (HealthDay News) -- Chronic bullying can take a toll on kids' grades. That's ...

  8. Imprinted genes show unique patterns of sequence conservation

    Directory of Open Access Journals (Sweden)

    Helms Volkhard

    2010-11-01

    Full Text Available Abstract Background Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse. Results Evolutionarily conserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA/(2·CpG ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements. Conclusions We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in

  9. The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression.

    Science.gov (United States)

    Mueller, Jacob L; Mahadevaiah, Shantha K; Park, Peter J; Warburton, Peter E; Page, David C; Turner, James M A

    2008-06-01

    According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)--the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells.

  10. A Technique for Showing Causal Arguments in Accident Reports

    Science.gov (United States)

    Holloway, C. M.; Johnson, C. W.

    2005-01-01

    In the prototypical accident report, specific findings, particularly those related to causes and contributing factors, are usually written out explicitly and clearly. Also, the evidence upon which these findings are based is typically explained in detail. Often lacking, however, is any explicit discussion, description, or depiction of the arguments that connect the findings and the evidence. That is, the reports do not make clear why the investigators believe that the specific evidence they found necessarily leads to the particular findings they enumerated. This paper shows how graphical techniques can be used to depict relevant arguments supporting alternate positions on the causes of a complex road-traffic accident.

  11. A de-novo STXBP1 gene mutation in a patient showing the Rett syndrome phenotype.

    Science.gov (United States)

    Romaniello, Romina; Saettini, Francesco; Panzeri, Elena; Arrigoni, Filippo; Bassi, Maria T; Borgatti, Renato

    2015-03-25

    This study reports on a 9-year-old girl who developed West syndrome and showed clinical features fulfilling the main revised diagnostic criteria for typical Rett syndrome (hand washing, severe cognitive impairment with absence of language, ataxic gait, progressive scoliosis and autistic features). Mutation analyses for methyl-CpG-binding protein 2 (MECP2), cyclin-dependent kinase-like 5 (CDKL5/STK9), ARX and Forkhead box G1 (FOXG1) genes were carried out, with negative results. A known de-novo c.1217G>A missense mutation in exon 14 leading to the substitution of a conserved residue, p.R406H in domain3b of the syntaxin-binding protein 1 (STXBP1) gene, was detected. The STXBP1 gene encodes the syntaxin-binding protein 1, a neuron-specific protein involved in synaptic vesicle release at both glutaminergic and GABAergic synapses. This function is also affected by MECP2 gene mutations, which are known to lead to a decrease in glutamate and GABA receptors' density. It is possible to speculate that the impairment in synaptic plasticity represents the pathogenic link between MECP2 and STXBP1 gene mutations. On reviewing the clinical features of the reported patients with the same mutation in the STXBP1 gene, it has been observed that poor eye contact, tremour, dyskinesia, head/hand stereotypies and both cognitive and motor progressive deterioration are common symptoms, although never considered as indicative of a Rett syndrome phenotype. In conclusion, the case described here suggests a relationship between the Rett syndrome and the STXBP1 gene not described so far, making the search for STXBP1 gene mutations advisable in patients with Rett syndrome and early onset of epilepsy.

  12. The drug target genes show higher evolutionary conservation than non-target genes.

    Science.gov (United States)

    Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-26

    Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

  13. Electricity show and related educational programming. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-03-19

    The on-site version of Balance of Power reached a total audience of 21,331 between November 10, 1994 and January 31, 1996; in addition, the Physics on Wheels van offered exhibits and programs to an additional 30,000 students in the 1995-1996 school year. The program provided a groundbreaking new approach to informal science education, combining a dynamic demonstration with an intensely interactive game show. Between the on-site programming and the Physics on Wheels van programs, 51,331 students were impacted by the activities, exhibits and energy-conservation message of Balance of Power.

  14. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

    Science.gov (United States)

    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A.; Woodman, Scott E.; Kwong, Lawrence N.

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy.

  15. TGFβ-dependent gene expression shows that senescence correlates with abortive differentiation along several lineages in Myc-induced lymphomas.

    Science.gov (United States)

    Müller, Judith; Samans, Birgit; van Riggelen, Jan; Fagà, Giovanni; Peh K N, Raquel; Wei, Chia-Lin; Müller, Heiko; Amati, Bruno; Felsher, Dean; Eilers, Martin

    2010-12-01

    Deregulated expression of Myc under the control of an immunoglobulin enhancer induces lymphoma formation in mice. The development of lymphomas is limited by TGFβ-dependent senescence and high levels of Myc expression are continuously required to antagonize senescence. The biological processes underlying senescence are not fully resolved. We report here a comprehensive analysis of TGFβ-dependent alterations in gene expression when the Myc transgene is switched off. Our data show that Myc-induced target genes are downregulated in a TGFβ-independent manner. In contrast, TGFβ is required to upregulate a broad spectrum of genes that are characteristic of different T-cell lineages when Myc is turned off. The analysis reveals a significant overlap between these Myc-repressed genes with genes that are targets of polycomb repressive complexes in embryonic stem cells. Therefore, TGFβ-dependent senescence is associated with gene expression patterns indicative of abortive cellular differentiation along several lineages.

  16. Detection of chromosomal regions showing differential gene expression in human skeletal muscle and in alveolar rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    Bortoluzzi Stefania

    2004-06-01

    Full Text Available Abstract Background Rhabdomyosarcoma is a relatively common tumour of the soft tissue, probably due to regulatory disruption of growth and differentiation of skeletal muscle stem cells. Identification of genes differentially expressed in normal skeletal muscle and in rhabdomyosarcoma may help in understanding mechanisms of tumour development, in discovering diagnostic and prognostic markers and in identifying novel targets for drug therapy. Results A Perl-code web client was developed to automatically obtain genome map positions of large sets of genes. The software, based on automatic search on Human Genome Browser by sequence alignment, only requires availability of a single transcribed sequence for each gene. In this way, we obtained tissue-specific chromosomal maps of genes expressed in rhabdomyosarcoma or skeletal muscle. Subsequently, Perl software was developed to calculate gene density along chromosomes, by using a sliding window. Thirty-three chromosomal regions harbouring genes mostly expressed in rhabdomyosarcoma were identified. Similarly, 48 chromosomal regions were detected including genes possibly related to function of differentiated skeletal muscle, but silenced in rhabdomyosarcoma. Conclusion In this study we developed a method and the associated software for the comparative analysis of genomic expression in tissues and we identified chromosomal segments showing differential gene expression in human skeletal muscle and in alveolar rhabdomyosarcoma, appearing as candidate regions for harbouring genes involved in origin of alveolar rhabdomyosarcoma representing possible targets for drug treatment and/or development of tumor markers.

  17. Human genes with a greater number of transcript variants tend to show biological features of housekeeping and essential genes

    DEFF Research Database (Denmark)

    Ryu, Jae Yong; Kim, Hyun Uk; Lee, Sang Yup

    2015-01-01

    64 vertebrate species as orthologs, subjected to regulations by transcription factors and microRNAs, and showed hub node-like properties in the human protein-protein interaction network. These findings were also confirmed by metabolic simulations of 60 cancer metabolic models. All these results......Alternative splicing is a process observed in gene expression that results in a multi-exon gene to produce multiple mRNA variants which might have different functions and activities. Although physiologically important, many aspects of genes with different number of transcript variants (or splice...... variants) still remain to be characterized. In this study, we provide bioinformatic evidence that genes with a greater number of transcript variants are more likely to play functionally important roles in cells, compared with those having fewer transcript variants. Among 21 983 human genes, 3728 genes were...

  18. Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

    Science.gov (United States)

    Jiang, S; Robertson, T; Mostajeran, M; Robertson, A J; Qiu, X

    2016-06-01

    Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees. © 2016 The Royal Entomological Society.

  19. Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

    Science.gov (United States)

    Delgado Sandoval, Silvia del Carmen; Abraham Juárez, María Jazmín; Simpson, June

    2012-03-01

    Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

  20. Genome-wide expression analysis of soybean MADS genes showing potential function in the seed development.

    Directory of Open Access Journals (Sweden)

    Cheng-Ming Fan

    Full Text Available The MADS family is an ancient and best-studied transcription factor and plays fundamental roles in almost every developmental process in plants. In the plant evolutionary history, the whole genome duplication (WGD events are important not only to the plant species evolution, but to expansion of members of the gene families. Soybean as a model legume crop has experience three rounds of WGD events. Members of some MIKC(C subfamilies, such as SOC, AGL6, SQUA, SVP, AGL17 and DEF/GLO, were expanded after soybean three rounds of WGD events. And some MIKC(C subfamilies, MIKC* and type I MADS families had experienced faster birth-and-death evolution and their traces before the Glycine WGD event were not found. Transposed duplication played important roles in tandem arrangements among the members of different subfamilies. According to the expression profiles of type I and MIKC paralog pair genes, the fates of MIKC paralog gene pairs were subfunctionalization, and the fates of type I MADS paralog gene pairs were nonfunctionalization. 137 out of 163 MADS genes were close to 186 loci within 2 Mb genomic regions associated with seed-relative QTLs, among which 115 genes expressed during the seed development. Although MIKC(C genes kept the important and conserved functions of the flower development, most MIKC(C genes showed potentially essential roles in the seed development as well as the type I MADS.

  1. Fifteen million years of evolution in the Oryza genus shows extensive gene family expansion.

    Science.gov (United States)

    Jacquemin, Julie; Ammiraju, Jetty S S; Haberer, Georg; Billheimer, Dean D; Yu, Yeisoo; Liu, Liana C; Rivera, Luis F; Mayer, Klaus; Chen, Mingsheng; Wing, Rod A

    2014-04-01

    In analyzing gene families in the whole-genome sequences available for O. sativa (AA), O. glaberrima (AA), and O. brachyantha (FF), we observed large size expansions in the AA genomes compared to FF genomes for the super-families F-box and NB-ARC, and five additional families: the Aspartic proteases, BTB/POZ proteins (BTB), Glutaredoxins, Trypsin α-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.

  2. Identification of a set of genes showing regionally enriched expression in the mouse brain

    Directory of Open Access Journals (Sweden)

    Marra Marco A

    2008-07-01

    Full Text Available Abstract Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters ( Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.

  3. MRI Reporter Genes for Noninvasive Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Caixia Yang

    2016-05-01

    Full Text Available Magnetic resonance imaging (MRI is one of the most important imaging technologies used in clinical diagnosis. Reporter genes for MRI can be applied to accurately track the delivery of cell in cell therapy, evaluate the therapy effect of gene delivery, and monitor tissue/cell-specific microenvironments. Commonly used reporter genes for MRI usually include genes encoding the enzyme (e.g., tyrosinase and β-galactosidase, the receptor on the cells (e.g., transferrin receptor, and endogenous reporter genes (e.g., ferritin reporter gene. However, low sensitivity limits the application of MRI and reporter gene-based multimodal imaging strategies are common including optical imaging and radionuclide imaging. These can significantly improve diagnostic efficiency and accelerate the development of new therapies.

  4. The sex determination gene shows no founder effect in the giant honey bee, Apis dorsata.

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    Zhi Yong Liu

    Full Text Available BACKGROUND: All honey bee species (Apis spp share the same sex determination mechanism using the complementary sex determination (csd gene. Only individuals heterogeneous at the csd allele develop into females, and the homozygous develop into diploid males, which do not survive. The honeybees are therefore under selection pressure to generate new csd alleles. Previous studies have shown that the csd gene is under balancing selection. We hypothesize that due to the long separation from the mainland of Hainan Island, China, that the giant honey bees (Apis dorsata should show a founder effect for the csd gene, with many different alleles clustered together, and these would be absent on the mainland. METHODOLOGY/PRINCIPAL FINDINGS: We sampled A. dorsata workers from both Hainan and Guangxi Provinces and then cloned and sequenced region 3 of the csd gene and constructed phylogenetic trees. We failed to find any clustering of the csd alleles according to their geographical origin, i.e. the Hainan and Guangxi samples did not form separate clades. Further analysis by including previously published csd sequences also failed to show any clade-forming in both the Philippines and Malaysia. CONCLUSIONS/SIGNIFICANCE: Results from this study and those from previous studies did not support the expectations of a founder effect. We conclude that because of the extremely high mating frequency of A. dorsata queens, a founder effect does not apply in this species.

  5. In planta Transformed Cumin (Cuminum cyminum L. Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    Directory of Open Access Journals (Sweden)

    Sonika Pandey

    Full Text Available Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13, overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid, and lower electrolytic leakage, lipid peroxidation (MDA content and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  6. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    Science.gov (United States)

    Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  7. The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    周斌; 彭开蔓; 储昭晖; 王石平; 张启发

    2002-01-01

    Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with genes in databases which encode or putatively encode transcription regulating proteins, translation regulating proteins, transport proteins, kinases, metabolic enzymes, and proteins involved in other functions. Most of the genes have not been previously reported as being involved in the disease resistance response in rice. The results from cDNA arrays, reverse transcription-polymerase chain reaction, and RNA gel blot analysis suggest that activation or repression of most of these genes might occur commonly in the defense response.

  8. Human retinal gene therapy for Leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement.

    Science.gov (United States)

    Cideciyan, Artur V; Jacobson, Samuel G; Beltran, William A; Sumaroka, Alexander; Swider, Malgorzata; Iwabe, Simone; Roman, Alejandro J; Olivares, Melani B; Schwartz, Sharon B; Komáromy, András M; Hauswirth, William W; Aguirre, Gustavo D

    2013-02-05

    Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5-11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term.

  9. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

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    Stephen eDownes

    2014-08-01

    Full Text Available Thymidine kinase 1 (TK1 is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumour suppressor (TP53 and human telomerase reverse transcriptase (hTERT gene regions, over 1 hour after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, while levels of genomic DNA repair were consistant between the two cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 minute repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

  10. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Science.gov (United States)

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  11. Transcriptome profiling in conifers and the PiceaGenExpress database show patterns of diversification within gene families and interspecific conservation in vascular gene expression

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    Raherison Elie

    2012-08-01

    Full Text Available Abstract Background Conifers have very large genomes (13 to 30 Gigabases that are mostly uncharacterized although extensive cDNA resources have recently become available. This report presents a global overview of transcriptome variation in a conifer tree and documents conservation and diversity of gene expression patterns among major vegetative tissues. Results An oligonucleotide microarray was developed from Picea glauca and P. sitchensis cDNA datasets. It represents 23,853 unique genes and was shown to be suitable for transcriptome profiling in several species. A comparison of secondary xylem and phelloderm tissues showed that preferential expression in these vascular tissues was highly conserved among Picea spp. RNA-Sequencing strongly confirmed tissue preferential expression and provided a robust validation of the microarray design. A small database of transcription profiles called PiceaGenExpress was developed from over 150 hybridizations spanning eight major tissue types. In total, transcripts were detected for 92% of the genes on the microarray, in at least one tissue. Non-annotated genes were predominantly expressed at low levels in fewer tissues than genes of known or predicted function. Diversity of expression within gene families may be rapidly assessed from PiceaGenExpress. In conifer trees, dehydrins and late embryogenesis abundant (LEA osmotic regulation proteins occur in large gene families compared to angiosperms. Strong contrasts and low diversity was observed in the dehydrin family, while diverse patterns suggested a greater degree of diversification among LEAs. Conclusion Together, the oligonucleotide microarray and the PiceaGenExpress database represent the first resource of this kind for gymnosperm plants. The spruce transcriptome analysis reported here is expected to accelerate genetic studies in the large and important group comprised of conifer trees.

  12. The ARMC5 gene shows extensive genetic variance in primary macronodular adrenocortical hyperplasia

    Science.gov (United States)

    Correa, Ricardo; Zilbermint, Mihail; Berthon, Annabel; Espiard, Stephanie; Batsis, Maria; Papadakis, Georgios Z.; Xekouki, Paraskevi; Lodish, Maya B.; Bertherat, Jerome; Faucz, Fabio R.; Stratakis, Constantine A.

    2015-01-01

    Objective Primary macronodular adrenal hyperplasia (PMAH) is a rare type of Cushing’s syndrome (CS) that results in increased cortisol production and bilateral enlargement of the adrenal glands. Recent work showed that the disease may be caused by germline and somatic mutations in the ARMC5 gene, a likely tumor-suppressor gene (TSG). We investigated 20 different adrenal nodules from one patient with PMAH for ARMC5 somatic sequence changes. Design All of the nodules where obtained from a single patient who underwent bilateral adrenalectomy. DNA was extracted by standard protocols and the ARMC5 sequence was determined by the Sanger method. Results Sixteen of 20 adrenocortical nodules harbored, in addition to what appeared to be the germline mutation, a second somatic variant. The p.Trp476* sequence change was present in all 20 nodules, as well as in normal tissue from the adrenal capsule, identifying it as the germline defect; each of the 16 other variants were found in different nodules: 6 were frame shift, 4 were missense, 3 were nonsense, and 1 was a splice site variation. Allelic losses were confirmed in 2 of the nodules. Conclusion This is the most genetic variance of the ARMC5 gene ever described in a single patient with PMAH: each of 16 adrenocortical nodules had a second new, “private”, and -in most cases- completely inactivating ARMC5 defect, in addition to the germline mutation. The data support the notion that ARMC5 is a TSG that needs a second, somatic hit, to mediate tumorigenesis leading to polyclonal nodularity; however, the driver of this extensive genetic variance of the second ARMC5 allele in adrenocortical tissue in the context of a germline defect and PMAH remains a mystery. PMID:26162405

  13. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    Science.gov (United States)

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  14. Reporter Gene Imaging in Therapy and Diagnosis

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    Pritha Ray, Abhijit De

    2012-01-01

    Full Text Available Noninvasive molecular imaging using reporter genes is a relatively recent field in biomedical imaging that holds great promises for disease diagnosis and therapy. As modern medicine is moving towards personalized medicine, targeted biomolecule based therapies is gaining popularity that requires careful and systematic validation. Reporter genes have emerged as important generalizable tools to overcome the shortcomings of direct evaluation of individual biomolecules and are being applied in various fields such as cell therapy, stem cell therapy, immune therapy, viral gene delivery through optical, radionuclide, magnetic resonance imaging techniques. New approaches to image protein-protein interaction, protein phosphorylation, protein folding that are crucial parameters for theranostic study using reporter genes are being developed. All these new technologies and relevant preclinical and clinical researches will determine the success of early detection and personalized therapy in the future.

  15. Analysis of genetic interaction networks shows that alternatively spliced genes are highly versatile.

    Science.gov (United States)

    Talavera, David; Sheoran, Ritika; Lovell, Simon C

    2013-01-01

    Alternative splicing has the potential to increase the diversity of the transcriptome and proteome. Where more than one transcript arises from a gene they are often so different that they are quite unlikely to have the same function. However, it remains unclear if alternative splicing generally leads to a gene being involved in multiple biological processes or whether it alters the function within a single process. Knowing that genetic interactions occur between functionally related genes, we have used them as a proxy for functional versatility, and have analysed the sets of genes of two well-characterised model organisms: Caenorhabditis elegans and Drosophila melanogaster. Using network analyses we find that few genes are functionally homogenous (only involved in a few functionally-related biological processes). Moreover, there are differences between alternatively spliced genes and genes with a single transcript; specifically, genes with alternatively splicing are, on average, involved in more biological processes. Finally, we suggest that factors other than specific functional classes determine whether a gene is alternatively spliced.

  16. Live Reporting in a News / Current Affairs TV Show as a Factor of (Non Credibility

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    Tena Perišin

    2010-12-01

    Full Text Available Only a perfunctory glance at the content of current aff airs/news programs makes it clear that in just a few years time, live reporting on Croatian TV news shows has increased dramatically. Technologicaladvancements, the equipping of TV stations with mobile satellite vans, and the pressure of competition have all resulted in increased instances of live reporting. When investigating news values, pictureand sound, as well as the possibility of immediate, timely reporting represent the key characteristics of TV journalism. In this context, live reports, as part of a news segment, should add to the authenticity and credibility of the program. Currently, however, TV broadcasting houses attempt to best one another in the number of live broadcasts as a means to purport a higher quality of their program. The direct address to the camera turns a reporter from an anonymous bearer of information into the “main star”. The figures accumulated on the extent of this form used in a news show do not determine the professional level of the editorial policy. Live reporting cannot be regarded as news value if other news values are neglected in the process. Recent research shows that live reports, in most cases, have been stripped of the initial notion of reporting on important and recent events. As such, they are becoming less of a justifi ed element in the creation of news.

  17. Transcriptome profiling shows gene regulation patterns in a flavonoid pathway in response to exogenous phenylalanine in Boesenbergia rotunda cell culture.

    Science.gov (United States)

    Md-Mustafa, Noor Diyana; Khalid, Norzulaani; Gao, Huan; Peng, Zhiyu; Alimin, Mohd Firdaus; Bujang, Noraini; Ming, Wong Sher; Mohd-Yusuf, Yusmin; Harikrishna, Jennifer A; Othman, Rofina Yasmin

    2014-11-18

    Panduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal indications which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory properties. Boesenbergia rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from Boesenbergia rotunda over the last few years has resulted in rising demands for panduratin A. However large scale extraction has been hindered by the naturally low abundance of the compound and limited knowledge of its biosynthetic pathway. Transcriptome sequencing and digital gene expression (DGE) analysis of native and phenylalanine treated Boesenbergia rotunda cell suspension cultures were carried out to elucidate the key genes differentially expressed in the panduratin A biosynthetic pathway. Based on experiments that show increase in panduratin A production after 14 days post treatment with exogenous phenylalanine, an aromatic amino acid derived from the shikimic acid pathway, total RNA of untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. The transcriptome data generated 101, 043 unigenes with 50, 932 (50.41%) successfully annotated in the public protein databases; including 49.93% (50, 447) in the non-redundant (NR) database, 34.63% (34, 989) in Swiss-Prot, 24,07% (24, 316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16, 426) in Clusters of Orthologous Groups (COG). Through DGE analysis, we found that 14, 644 unigenes were up-regulated and 14, 379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the phenylpropanoid pathway leading to the proposed panduratin A production, 2 up-regulated phenylalanine ammonia-lyase (PAL), 3 up-regulated 4-coumaroyl

  18. Amerindians show no association of PC-1 gene Gln121 allele and obesity: a thrifty gene population genetics.

    Science.gov (United States)

    Rey, Diego; Fernandez-Honrado, Mercedes; Areces, Cristina; Algora, Manuel; Abd-El-Fatah-Khalil, Sedeka; Enriquez-de-Salamanca, Mercedes; Coca, Carmen; Arribas, Ignacio; Arnaiz-Villena, Antonio

    2012-07-01

    PC-1 Gln121 gene is a risk factor for type 2 diabetes, obesity and insulin resistance in European/American Caucasoids and Orientals. We have aimed to correlate for the first time this gene in Amerindians with obesity and their corresponding individuals genotypes with obesity in order to establish preventive medicine programs for this population and also studying the evolution of gene frequencies in world populations. Central obesity was diagnosed by waist circumference perimeter and food intake independent HDL-cholesterol plasma levels were measured. HLA genes were determined in order to more objectively ascertain participants Amerindians origin. 321 Amerindian blood donors who were healthy according to the blood doning parameters were studied. No association was found between PC-1 Gln121 variant and obesity. Significant HDL-cholesterol lower values were found in the PC-1 Lys121 bearing gene individuals versus PC-1 Gln121 bearing gene ones (45.1 ± 12.7 vs. 48.7 ± 15.2 mg/dl, p gene is found with obesity in Amerindians when association is well established in Europeans. (2) PC-1 Gln121 gene is associated to higher levels of HDL-cholesterol than the alternative PC-1 Lys121 allele. This may be specific for Amerindians. (3) Amerindians have an intermediate frequency of this possible PC-1 Gln121 thrifty gene when compared with Negroid African Americans (78.5%) or Han Chinese (7.5%, p gene.

  19. Addiction and Reward-related Genes Show Altered Expression in the Postpartum Nucleus Accumbens

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    Changjiu eZhao

    2014-11-01

    Full Text Available Motherhood involves a switch in natural rewards, whereby offspring become highly rewarding. Nucleus accumbens (NAC is a key CNS region for natural rewards and addictions, but to date no study has evaluated on a large scale the events in NAC that underlie the maternal change in natural rewards. In this study we utilized microarray and bioinformatics approaches to evaluate postpartum NAC gene expression changes in mice. Modular Single-set Enrichment Test (MSET indicated that postpartum (relative to virgin NAC gene expression profile was significantly enriched for genes related to addiction and reward in 5 of 5 independently curated databases (e.g., Malacards, Phenopedia. Over 100 addiction/reward related genes were identified and these included: Per1, Per2, Arc, Homer2, Creb1, Grm3, Fosb, Gabrb3, Adra2a, Ntrk2, Cry1, Penk, Cartpt, Adcy1, Npy1r, Htr1a, Drd1a, Gria1, and Pdyn. ToppCluster analysis found maternal NAC expression profile to be significantly enriched for genes related to the drug action of nicotine, ketamine, and dronabinol. Pathway analysis indicated postpartum NAC as enriched for RNA processing, CNS development/differentiation, and transcriptional regulation. Weighted Gene Coexpression Network Analysis identified possible networks for transcription factors, including Nr1d1, Per2, Fosb, Egr1, and Nr4a1. The postpartum state involves increased risk for mental health disorders and MSET analysis indicated postpartum NAC to be enriched for genes related to depression, bipolar disorder, and schizophrenia. Mental health related genes included: Fabp7, Grm3, Penk, and Nr1d1. We confirmed via quantitative PCR Nr1d1, Per2, Grm3, Penk, Drd1a, and Pdyn. This study indicates for the first time that postpartum NAC involves large scale gene expression alterations linked to addiction and reward. Because the postpartum state also involves decreased response to drugs, the findings could provide insights into how to mitigate addictions.

  20. Mice lacking pituitary tumor transforming gene show elevated exposure of DGalNAc carbohydrate determinants

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    Lutsyk A. D.

    2012-04-01

    Full Text Available Aim. To investigate the influence of pituitary tumor transforming gene (pttg-1 knockout on glycome of parenchimal organs by means of lectin histochemistry. Methods. DGalNAc, DGlcNAc, NeuNAc carbohydrate determinants were labelled with soybean agglutinin (SBA and wheat germ agglutinin (WGA, conjugated to peroxidase, with subsequent visualization of the lectin-binding sites with diaminobenzidine. The testes and kidneys of murine strain BL6/C57 with the pttg-1 gene knockout (PTTG-KO were compared to the wild type (PTTG-WT animals, both groups 1 month of age. Results. Knockout of the pttg-1 gene was accompanied by enhanced exposure of the DGalNAc sugar residues within the Golgi complex of secondary spermatocytes, in a brush border of renal tubules and on the lumenal surface of collecting ducts. Conclusions. This study suggests that knockout of the pttg-1 gene may lead to the changes in carbohydrate processing in mammalian organism.

  1. SINORHIZOBIUM MELILOTI ELECTROTRANSPORANT CONTAINING ORTHO-DECHLORINATION GENE SHOWS ENHANCED PCB-DECHLORINATION. (R828770)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. A single-nucleotide polymorphism of human neuropeptide s gene originated from Europe shows decreased bioactivity.

    Directory of Open Access Journals (Sweden)

    Cheng Deng

    Full Text Available Using accumulating SNP (Single-Nucleotide Polymorphism data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS, a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L(6-NPS causing non-synonymous substitution on the 6(th position (V to L of the NPS mature peptide region. L(6-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ~25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L(6-NPS exhibits a significant lower bioactivity than the wild type NPS, with ~20-fold higher EC50 values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6(th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.

  3. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    Science.gov (United States)

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  4. Ceruloplasmin gene-deficient mice with experimental autoimmune encephalomyelitis show attenuated early disease evolution.

    Science.gov (United States)

    Gresle, Melissa M; Schulz, Katrin; Jonas, Anna; Perreau, Victoria M; Cipriani, Tania; Baxter, Alan G; Miranda-Hernandez, Socorro; Field, Judith; Jokubaitis, Vilija G; Cherny, Robert; Volitakis, Irene; David, Samuel; Kilpatrick, Trevor J; Butzkueven, Helmut

    2014-06-01

    We conducted a microarray study to identify genes that are differentially regulated in the spinal cords of mice with the inflammatory disease experimental autoimmune encephalomyelitis (EAE) relative to healthy mice. In total 181 genes with at least a two-fold increase in expression were identified, and most of these genes were associated with immune function. Unexpectedly, ceruloplasmin (Cp), a ferroxidase that converts toxic ferrous iron to its nontoxic ferric form and also promotes the efflux of iron from astrocytes in the CNS, was shown to be highly upregulated (13.2-fold increase) in EAE spinal cord. Expression of Cp protein is known to be increased in several neurological conditions, but the role of Cp regulation in CNS autoimmune disease is not known. To investigate this, we induced EAE in Cp gene knockout, heterozygous, and wild-type mice. Cp knockout mice were found to have slower disease evolution than wild-type mice (EAE days 13-17; P = 0.05). Interestingly, Cp knockout mice also exhibited a significant increase in the number of astrocytes with reactive morphology in early EAE compared with wild-type mice at the same stage of disease. CNS iron levels were not increased with EAE in these mice. Based on these observations, we propose that an increase in Cp expression could contribute to tissue damage in early EAE. In addition, endogenous CP either directly or indirectly inhibits astrocyte reactivity during early disease, which could also worsen early disease evolution.

  5. Interleukin-18 gene-deficient mice show enhanced defense and reduced inflammation during pneumococcal meningitis.

    NARCIS (Netherlands)

    Zwijnenburg, P.J.G.; Poll, van der T.; Florquin, S; Akira, S; Takeda, K; Roord, J.J.; Furth, van A.M.

    2003-01-01

    To determine the role of endogenous interleukin-18 (IL-18) in pneumococcal meningitis, meningitis was induced in IL-18 gene-deficient (IL-18(-/-)) and wild-type (WT) mice by intranasal inoculation of Streptococcus pneumoniae with hyaluronidase. Induction of meningitis resulted in an upregulation of

  6. Angiosarcoma of Anterior Mandibular Gingiva Showing Recurrence – A Case Report with Immunohistochemistry

    Science.gov (United States)

    Koneru, Anila; Vanishree, M.; Manvikar, Vardendra

    2016-01-01

    Angiosarcomas of oral cavity and salivary gland represent 1% of all cases reported in the literature and are therefore considered as extremely rare. To the best of our knowledge very few cases of angiosarcomas involving mandibular gingiva have been reported previously. Here, we report a case of angiosarcoma occurring in the gingiva with review of literature on clinical features. A 30-year-old female patient presented with a complaint of a small growing mass in relation to lower front teeth. Intraoral examination revealed a soft sessile growth arising from the labial gingiva in relation to 31 and 41 on the labial aspect extending distally to 32. The lesion was locally excised. Histolopathological analysis showed that the tumour was composed of spindle shaped to polygonal cells with hyperchromatic nuclei, conspicuous nucleoli and intracytoplasmic vacuoles, mitotic figure were also scattered. Immunohistochemical staining revealed that the tumour cells was positive for factor VIII-related antigen, CD31 and CD34. An excisional biopsy showed a diagnosis of angiosarcoma. After two months patient reported back with the same chief complaint. This present case is a 17th case report of angiosarcoma arising in anterior mandiblular gingiva. PMID:27630964

  7. Angiosarcoma of Anterior Mandibular Gingiva Showing Recurrence - A Case Report with Immunohistochemistry.

    Science.gov (United States)

    Hunasgi, Santosh; Koneru, Anila; Vanishree, M; Manvikar, Vardendra

    2016-07-01

    Angiosarcomas of oral cavity and salivary gland represent 1% of all cases reported in the literature and are therefore considered as extremely rare. To the best of our knowledge very few cases of angiosarcomas involving mandibular gingiva have been reported previously. Here, we report a case of angiosarcoma occurring in the gingiva with review of literature on clinical features. A 30-year-old female patient presented with a complaint of a small growing mass in relation to lower front teeth. Intraoral examination revealed a soft sessile growth arising from the labial gingiva in relation to 31 and 41 on the labial aspect extending distally to 32. The lesion was locally excised. Histolopathological analysis showed that the tumour was composed of spindle shaped to polygonal cells with hyperchromatic nuclei, conspicuous nucleoli and intracytoplasmic vacuoles, mitotic figure were also scattered. Immunohistochemical staining revealed that the tumour cells was positive for factor VIII-related antigen, CD31 and CD34. An excisional biopsy showed a diagnosis of angiosarcoma. After two months patient reported back with the same chief complaint. This present case is a 17(th) case report of angiosarcoma arising in anterior mandiblular gingiva.

  8. Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes

    Directory of Open Access Journals (Sweden)

    Shunsuke Iizuka

    2017-09-01

    Full Text Available Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX. An adenovirus (Ad vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX (Ad-E4-122aT-AHAFIX maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

  9. Gene expression profiling in the Cynomolgus macaque Macaca fascicularis shows variation within the normal birth range

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    Vickers Mark H

    2011-10-01

    Full Text Available Abstract Background Although an adverse early-life environment has been linked to an increased risk of developing the metabolic syndrome, the molecular mechanisms underlying altered disease susceptibility as well as their relevance to humans are largely unknown. Importantly, emerging evidence suggests that these effects operate within the normal range of birth weights and involve mechanisms of developmental palsticity rather than pathology. Method To explore this further, we utilised a non-human primate model Macaca fascicularis (Cynomolgus macaque which shares with humans the same progressive history of the metabolic syndrome. Using microarray we compared tissues from neonates in the average birth weight (50-75th centile to those of lower birth weight (5-25th centile and studied the effect of different growth trajectories within the normal range on gene expression levels in the umbilical cord, neonatal liver and skeletal muscle. Results We identified 1973 genes which were differentially expressed in the three tissue types between average and low birth weight animals (P Conclusion These differences in gene expression levels between animals in the upper and lower percentiles of the normal birth weight range may point towards early life metabolic adaptations that in later life result in differences in disease risk.

  10. Alcohol-related genes show an enrichment of associations with a persistent externalizing factor.

    Science.gov (United States)

    Ashenhurst, James R; Harden, K Paige; Corbin, William R; Fromme, Kim

    2016-10-01

    Research using twins has found that much of the variability in externalizing phenotypes-including alcohol and drug use, impulsive personality traits, risky sex, and property crime-is explained by genetic factors. Nevertheless, identification of specific genes and variants associated with these traits has proven to be difficult, likely because individual differences in externalizing are explained by many genes of small individual effect. Moreover, twin research indicates that heritable variance in externalizing behaviors is mostly shared across the externalizing spectrum rather than specific to any behavior. We use a longitudinal, "deep phenotyping" approach to model a general externalizing factor reflecting persistent engagement in a variety of socially problematic behaviors measured at 11 assessment occasions spanning early adulthood (ages 18 to 28). In an ancestrally homogenous sample of non-Hispanic Whites (N = 337), we then tested for enrichment of associations between the persistent externalizing factor and a set of 3,281 polymorphisms within 104 genes that were previously identified as associated with alcohol-use behaviors. Next, we tested for enrichment among domain-specific factors (e.g., property crime) composed of residual variance not accounted for by the common factor. Significance was determined relative to bootstrapped empirical thresholds derived from permutations of phenotypic data. Results indicated significant enrichment of genetic associations for persistent externalizing, but not for domain-specific factors. Consistent with twin research findings, these results suggest that genetic variants are broadly associated with externalizing behaviors rather than unique to specific behaviors. (PsycINFO Database Record

  11. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

    Science.gov (United States)

    Markholt, S; Grøndahl, M L; Ernst, E H; Andersen, C Yding; Ernst, E; Lykke-Hartmann, K

    2012-02-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

  12. Comparison of Affymetrix Gene Array with the Exon Array shows potential application for detection of transcript isoform variation

    Directory of Open Access Journals (Sweden)

    Coulombe-Huntington Jasmin

    2009-11-01

    Full Text Available Abstract Background The emergence of isoform-sensitive microarrays has helped fuel in-depth studies of the human transcriptome. The Affymetrix GeneChip Human Exon 1.0 ST Array (Exon Array has been previously shown to be effective in profiling gene expression at the isoform level. More recently, the Affymetrix GeneChip Human Gene 1.0 ST Array (Gene Array has been released for measuring gene expression and interestingly contains a large subset of probes from the Exon Array. Here, we explore the potential of using Gene Array probes to assess expression variation at the sub-transcript level. Utilizing datasets of the high quality Microarray Quality Control (MAQC RNA samples previously assayed on the Exon Array and Gene Array, we compare the expression measurements of the two platforms to determine the performance of the Gene Array in detecting isoform variations. Results Overall, we show that the Gene Array is comparable to the Exon Array in making gene expression calls. Moreover, to examine expression of different isoforms, we modify the Gene Array probe set definition file to enable summarization of probe intensity values at the exon level and show that the expression profiles between the two platforms are also highly correlated. Next, expression calls of previously known differentially spliced genes were compared and also show concordant results. Splicing index analysis, representing estimates of exon inclusion levels, shows a lower but good correlation between platforms. As the Gene Array contains a significant subset of probes from the Exon Array, we note that, in comparison, the Gene Array overlaps with fewer but still a high proportion of splicing events annotated in the Known Alt Events UCSC track, with abundant coverage of cassette exons. We discuss the ability of the Gene Array to detect alternative splicing and isoform variation and address its limitations. Conclusion The Gene Array is an effective expression profiling tool at gene and

  13. Tolerogenic dendritic cells show gene expression profiles that are different from those of immunogenic dendritic cells in DBA/1 mice.

    Science.gov (United States)

    Lee, Eun Gae; Jung, Nam-Chul; Lee, Jun-Ho; Song, Jie-Young; Ryu, Sang-Young; Seo, Han Geuk; Han, Sung Gu; Ahn, Keun Jae; Hong, Kwan Soo; Choi, Jinjung; Lim, Dae-Seog

    2016-01-01

    Tolerogenic dendritic cells (tDCs) play an important role in inducing peripheral tolerance; however, few tDC-specific markers have been identified. The aims of this study were to examine whether tDCs show a different gene expression profile from that of immunogenic DCs and identify specific gene markers of each cell type, in DBA/1 mice. tDCs were generated by treating immature DCs (imDCs) with TNF-α and type II collagen. The gene expression profiles of mature (m)DCs and tDCs were then investigated by microarray analysis and candidate markers were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Supervised selection identified 75 gene signatures, 63 of which were consistently upregulated in mDCs and 12 of which were upregulated only in tDCs. Additionally, 10 genes were overexpressed or equally expressed in both tDCs and mDCs. Scin (tDC-specific genes) and Orm1, Pdlim4 and Enpp2 (mDC-specific genes) were validated by real-time qRT-PCR. Taken together, these results clearly show that tDCs and mDCs can be identified according to their expression of specific gene markers.

  14. Norwegian farmers' vigilance in reporting sheep showing scrapie-associated signs

    Directory of Open Access Journals (Sweden)

    Jarp Jorun

    2007-12-01

    Full Text Available Abstract Background Scrapie is a chronic neurodegenerative disease affecting small ruminants and belongs to the transmissible spongiform encephalopathies. Scrapie is considered a serious animal disease and it has been notifiable in Norway since 1965. The clinical signs of scrapie might be vague and the farmers, if familiar with the signs of scrapie, are often in the best position for detecting scrapie suspects. In 2002, an anonymous questionnaire survey was conducted in order to assess Norwegian sheep farmers' vigilance of scrapie. Results Although the potential detection of a scrapie-positive animal would lead to the destruction of the sheep flock concerned, almost all the farmers (97 % expressed their willingness to report scrapie suspects. This was most certainly dependent on the Government taking the economic responsibility for the control programme as nearly all the farmers responded that this was an important condition. Listeriosis is relatively common disease in Norwegian sheep and a differential diagnosis for scrapie. In a multinomial logistic regression the reporting behaviour for non-recovering listeriosis cases, used as a measurement of willingness to report scrapie, was examined. The reporting of non-recovering listeriosis cases increased as the knowledge of scrapie-associated signs increased, and the reporting behaviour was dependent on both economic and non-economic values. Conclusion The results indicate that in 2002 almost all sheep farmers showed willingness to report any scrapie suspects. Nevertheless there is an underreporting of scrapie suspects and the farmers' awareness and hence their vigilance of scrapie could be improved. Furthermore, the results suggest that to ensure the farmers' compliance to control programmes for serious infectious diseases, the farmers' concerns of non-economic as well as economic values should be considered.

  15. Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

    Directory of Open Access Journals (Sweden)

    Jan Salomonsen

    2014-06-01

    Full Text Available Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC, and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region, and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells, correlating with two different kinds of promoters and 5' untranslated regions (5'UTR. However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

  16. Identification and analysis of unitary loss of long-established protein-coding genes in Poaceae shows evidences for biased gene loss and putatively functional transcription of relics.

    Science.gov (United States)

    Zhao, Yi; Tang, Liang; Li, Zhe; Jin, Jinpu; Luo, Jingchu; Gao, Ge

    2015-04-18

    Long-established protein-coding genes may lose their coding potential during evolution ("unitary gene loss"). Members of the Poaceae family are a major food source and represent an ideal model clade for plant evolution research. However, the global pattern of unitary gene loss in Poaceae genomes as well as the evolutionary fate of lost genes are still less-investigated and remain largely elusive. Using a locally developed pipeline, we identified 129 unitary gene loss events for long-established protein-coding genes from four representative species of Poaceae, i.e. brachypodium, rice, sorghum and maize. Functional annotation suggested that the lost genes in all or most of Poaceae species are enriched for genes involved in development and response to endogenous stimulus. We also found that 44 mutated genomic loci of lost genes, which we referred as relics, were still actively transcribed, and of which 84% (37 of 44) showed significantly differential expression across different tissues. More interestingly, we found that there were totally five expressed relics may function as competitive endogenous RNA in brachypodium, rice and sorghum genome. Based on comparative genomics and transcriptome data, we firstly compiled a comprehensive catalogue of unitary gene loss events in Poaceae species and characterized a statistically significant functional preference for these lost genes as well showed the potential of relics functioning as competitive endogenous RNAs in Poaceae genomes.

  17. Markers for sebaceoma show a spectrum of cell cycle regulators, tumor suppressor genes, and oncogenes

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2015-01-01

    Full Text Available Background: Sebaceoma is a tumor for which the causative oncogenes are not well-understood. Sebaceomas demonstrate some histopathologic features similar to basal cell carcinoma (BCC, such as palisading borders and basaloid cells with additional features, including foamy cytoplasm and indented nuclei. Aims: We examine multiple cell-cycle, oncogene, and tumor suppressor gene markers in sebaceomas, to try to find some suitable biological markers for this tumor, and compare with other published studies. Materials and Methods: We investigated a panel of immunohistochemical (IHC stains that are important for cellular signaling, including a cell cycle regulator, tumor suppressor gene, oncogene, hormone receptor, and genomic stability markers in our cohort of sebaceomas. We collected 30 sebaceomas from three separate USA dermatopathology laboratories. The following IHC panel: Epithelial membrane antigen (EMA/CD227, cytokeratin AE1/AE3, cyclin D1, human breast cancer 1 protein (BRCA-1, C-erb-2, Bcl-2, human androgen receptor (AR, cyclin-dependent kinase inhibitor 1B (p27 kip1 , p53, topoisomerase II alpha, proliferating cell nuclear antigen, and Ki-67 were tested in our cases. Results: EMA/CD227 was positive in the well-differentiated sebaceomas (13/30. Cyclin-dependent kinase inhibitor 1B was positive in tumors with intermediate differentiation (22/30. The less well-differentiated tumors failed to stain with EMA and AR. Most of the tumors with well-differentiated palisaded areas demonstrated positive staining for topoisomerase II alpha, p27 kip1 , and p53, with positive staining in tumoral basaloid areas (22/30. Numerous tumors were focally positive with multiple markers, indicating a significant degree of variability in the complete group. Conclusions: Oncogenes, tumor suppressor genes, cell cycle regulators, and hormone receptors are variably expressed in sebaceomas. Our results suggest that in these tumors, selected marker staining seems to correlate

  18. Asthma families show transmission disequilibrium of gene variants in the vitamin D metabolism and signalling pathway

    Directory of Open Access Journals (Sweden)

    Bahnweg Margret

    2006-04-01

    Full Text Available Abstract The vitamin D prophylaxis of rickets in pregnant women and newborns may play a role in early allergic sensitization. We now asked if an already diseased population may have inherited genetic variants in the vitamin D turnover or signalling pathway. Serum levels of calcidiol (25-OH-D3 and calcitriol (1,25-(OH2-D3 were retrospectively assessed in 872 partipants of the German Asthma Family Study. 96 DNA single base variants in 13 different genes were genotyped with MALDI-TOF and a bead array system. At least one positive SNP with a TDT of p Genetic analysis of biological pathways seem to be a promising approach where this may be a first entry point into effects of a polygenic inherited vitamin D sensitivity that may affect also other metabolic, immunological and cancerous diseases.

  19. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction

    DEFF Research Database (Denmark)

    Markholt, Sara; Grøndahl, M L; Ernst, Erik

    2012-01-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood...... but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis...

  20. Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew.

    Science.gov (United States)

    Hurni, Severine; Brunner, Susanne; Buchmann, Gabriele; Herren, Gerhard; Jordan, Tina; Krukowski, Patricia; Wicker, Thomas; Yahiaoui, Nabila; Mago, Rohit; Keller, Beat

    2013-12-01

    The improvement of wheat through breeding has relied strongly on the use of genetic material from related wild and domesticated grass species. The 1RS chromosome arm from rye was introgressed into wheat and crossed into many wheat lines, as it improves yield and fungal disease resistance. Pm8 is a powdery mildew resistance gene on 1RS which, after widespread agricultural cultivation, is now widely overcome by adapted mildew races. Here we show by homology-based cloning and subsequent physical and genetic mapping that Pm8 is the rye orthologue of the Pm3 allelic series of mildew resistance genes in wheat. The cloned gene was functionally validated as Pm8 by transient, single-cell expression analysis and stable transformation. Sequence analysis revealed a complex mosaic of ancient haplotypes among Pm3- and Pm8-like genes from different members of the Triticeae. These results show that the two genes have evolved independently after the divergence of the species 7.5 million years ago and kept their function in mildew resistance. During this long time span the co-evolving pathogens have not overcome these genes, which is in strong contrast to the breakdown of Pm8 resistance since its introduction into commercial wheat 70 years ago. Sequence comparison revealed that evolutionary pressure acted on the same subdomains and sequence features of the two orthologous genes. This suggests that they recognize directly or indirectly the same pathogen effectors that have been conserved in the powdery mildews of wheat and rye.

  1. Two double heterozygous mutations in the F7 gene show different manifestations.

    Science.gov (United States)

    Nagaizumi, Keiko; Inaba, Hiroshi; Suzuki, Takashi; Hatta, Yoshihiro; Hagiwara, Takeshi; Amano, Kagehiro; Arai, Morio; Fukutake, Katsuyuki

    2002-12-01

    We sequenced the factor VII gene (F7) in two unrelated Japanese patients with factor VII (FVII) deficiency. In the first (an asymptomatic 46-year-old man with FVII activity and antigen levels of 1.2% and 21% of normal respectively), novel E25K and H348Q mutations were identified in the doubly heterozygous state. In transiently transfected HEK293 cells, the level of FVII-E25K mutant activity in the culture media was significantly lower than that of FVII wild type, whereas the antigen levels of both proteins were similar. This suggests that the E25K mutation is associated with a dysfunctional FVII molecule. In the second patient (a 47-year-old woman with FVII activity and antigen levels of less than 1% and 6% respectively), an IVS4+1 mutation and a novel -96C to T transition were detected in the double heterozygous state. In electrophoretic mobility shift assays, the -96T mutation was shown to disrupt binding of Sp1.

  2. Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig.

    Science.gov (United States)

    Komatsu, Yuuta; Sukegawa, Shin; Yamashita, Mai; Katsuda, Naoki; Tong, Bin; Ohta, Takeshi; Kose, Hiroyuki; Yamada, Takahisa

    2016-06-01

    Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 colocalized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanism underlying growth rate in Landrace pig breed.

  3. Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer

    Directory of Open Access Journals (Sweden)

    Ramenghi Ugo

    2009-09-01

    Full Text Available Abstract Background Diamond-Blackfan anaemia (DBA is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. Patients have an increased risk of solid tumors. Mutations have been found in several ribosomal protein (RP genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A. Studies in haematopoietic progenitors from patients show that haplo-insufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes show reduced protein synthesis and fibroblasts display abnormal rRNA processing and impaired proliferation. Results To evaluate the involvement of non-haematopoietic tissues in DBA, we have analysed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are differentially expressed in DBA patient fibroblasts. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. Conclusion This analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in DBA. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis.

  4. The electron transfer flavoprotein fixABCX gene products from Azospirillum brasilense show a NifA-dependent promoter regulation.

    Science.gov (United States)

    Sperotto, Raul Antonio; Gross, Jeferson; Vedoy, Cleber; Passaglia, Luciane Maria Pereira; Schrank, Irene Silveira

    2004-10-01

    The complete nucleotide sequence of the A. brasilense fixA, fixB, fixC, and fixX genes is reported here. Sequence similarities between the protein sequences deduced from fixABCX genes and many electron transfer flavoproteins (ETFs) have been noted. Comparison of the amino acid sequences of both subunits of ETF with the A. brasilense fixA and fixB gene products exhibits an identity of 30%. The amino acid sequence of the other two genes, fixC and fixX, revealed similarity with the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Using site-directed mutagenesis, mutations were introduced in the fixA promoter element of the A. brasilense fixABCX operon and chimeric p fixA-lacZ reporter gene fusions were constructed. The activation of the fixA promoter of A. brasilense is dependent upon the presence of the NifA protein being approximately 7 times less active than the A. brasilense nifH promoter. These results indicate that NifA from Klebsiella pneumoniae activates the fix promoter of A. brasilense and provide further evidence in support of the regulatory model of NifA activation in A. brasilense. Although no specific function has been assigned to the fixABCX gene products they are apparently required for symbiotic nitrogen fixation. An electron-transferring capacity in the nitrogen fixation pathway has been suggested for the fix gene products based on sequence homologies to the ETFs and ETF-QO proteins and by the absence of orthologous electron transfer proteins NifJ and NifF in A. brasilense.

  5. Polymorphism and gene organization of water buffalo MHC-DQB genes show homology to the BoLA DQB region.

    Science.gov (United States)

    Sena, L; Schneider, M P C; Brenig, B B; Honeycutt, R L; Honeycutt, D A; Womack, J E; Skow, L C

    2011-08-01

    In cattle (Bos taurus), there is evidence of more than 50 alleles of BoLA-DQB (bovine lymphocyte antigen DQB) that are distributed across at least five DQB loci, making this region one of the most complex in the BoLA gene family. In this study, DQB alleles were analysed for the water buffalo (Bubalus bubalis), another economically important bovine species. Twelve alleles for Bubu-DQB (Bubalis bubalis DQB) were determined by nucleotide sequence analysis. A phylogenetic analysis revealed numerous trans-species polymorphisms, with alleles from water buffalo assigned to at least three different loci (BoLA-DQB1, BoLA-DQB3 and BoLA-DQB4) that are also found in cattle. These presumptive loci were analysed for patterns of synonymous (d(S)) and non-synonymous (d(N)) substitution. Like BoLA-DQB1, Bubu-DQB1 was observed to be under strong positive selection for polymorphism. We conclude that water buffalo and cattle share the current arrangement of their DQB region because of their common ancestry. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

  6. Cases report of ossifying fibroma showing various radiographic appearances in posterior mandible

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byung Do; Oh, Seung Hwan [School of Dentistry, Wonkwang University, Seoul (Korea, Republic of); Son, Hyun Jin [Department of Pathology, School of Medicine, Eulji University, Daejeon (Korea, Republic of)

    2010-06-15

    Common radiographic appearances of ossifying fibroma (OF) are well demarcated margin, radiolucent or mixed lesion. Lesions for the radiographic differential diagnosis with OF include fibrous dysplasia, focal cemento-osseous dysplasia. Other confusing lesions might be the mixed lesions such as calcifying odontogenic cyst, adenomatoid odontogenic tumor, calcifying epithelial odontogenic tumor, and benign cementoblastoma. We reported three cases of OF in posterior mandible. These cases showed a little distinguished radiographic features of OF and diagnosed from a combination of clinical, radiographic, and histopathologic information. We need to further refine radiographic and histopathological features of OF and other confusing lesions with literatures review because some cases of these lesions are not easily differentiated radiographically and histopathologically.

  7. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    Directory of Open Access Journals (Sweden)

    Silvija Miosic

    Full Text Available During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin and two (cyanidin hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring, whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  8. A Pelargonium ARGONAUTE4 gene shows organ-specific expression and differences in RNA and protein levels.

    Science.gov (United States)

    He, Jie; Gray, John; Leisner, Scott

    2010-03-01

    RNAi-induced gene silencing plays a role in plant DNA methylation and defense. While most gene silencing studies have been performed on annuals, little is known about the expression of key components of this process (like ARGONAUTE proteins) in ornamentals. Using a combination of polymerase chain reaction techniques, an ARGONAUTE4 gene, PhAGO4, was isolated from Pelargonium. PhAGO4 encodes a predicted product of 934 amino acids that contains the PAZ and PIWI domains typical of ARGONAUTE (AGO) proteins. Phylogenetic analyses indicate that PhAGO4 clusters with other plant AGO4 proteins. Organ expression patterns of the AGO4 genes in Pelargonium and Arabidopsis show intriguing differences. AGO4 RNA levels decline with leaf age in both Arabidopsis and Pelargonium. In contrast AGO4 RNA levels in roots relative to leaves are higher in Pelargonium than in Arabidopsis. Both Arabidopsis and Pelargonium AGO4 showed higher RNA levels in flowers than leaves or roots. Even though flowers show higher levels of PhAGO4 RNA when compared to leaves and roots, protein gel blot analysis shows that at the protein level, the reverse is true. This suggests that PhAGO4 expression may be regulated at the translational or post-translational level in Pelargonium flowers.

  9. Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig

    Indian Academy of Sciences (India)

    YUUTA KOMATSU; SHIN SUKEGAWA; MAIYAMA SHITA; NAOKI KATSUDA; BIN TONG; TAKESHI OHTA; HIROYUKI KOSE; TAKAHISA YAMADA

    2016-06-01

    Suppression subtractive hybridization was used to identify genes showing differential expression profile associated withgrowth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from mus-culus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregu-lated genes (EEF1A2 ,TSG101andTTN) and six downregulated genes (ATP5B ,ATP5C1 ,COQ3 ,HADHA ,MYH1andMYH7)in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clonesselected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis con-firmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Lan-drace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identifiedgenes except for theTTNwas validated by Western blot analysis. Additionally, the eight genes other than theATP5C1co-localized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally,the changes of expression predicted from gene function suggested association of upregulation of expression of theEEF1A2 ,TSG101andTTNgenes and downregulation of theATP5B ,ATP5C1 ,COQ3 ,HADHA ,MYH1andMYH7gene expressionwith increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanismunderlying growth rate in Landrace pig breed.

  10. Members of the barley NAC transcription factor gene family show differential co-regulation with senescence-associated genes during senescence of flag leaves

    DEFF Research Database (Denmark)

    Christiansen, Michael W; Gregersen, Per L.

    2014-01-01

    The senescence process of plants is important for the completion of their life cycle, particularly for crop plants, it is essential for efficient nutrient remobilization during seed filling. It is a highly regulated process, and in order to address the regulatory aspect, the role of genes...... in the NAC transcription factor family during senescence of barley flag leaves was studied. Several members of the NAC transcription factor gene family were up-regulated during senescence in a microarray experiment, together with a large range of senescence-associated genes, reflecting the coordinated...... activation of degradation processes in senescing barley leaf tissues. This picture was confirmed in a detailed quantitative reverse transcription–PCR (qRT–PCR) experiment, which also showed distinct gene expression patterns for different members of the NAC gene family, suggesting a group of ~15 out of the 47...

  11. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

    Directory of Open Access Journals (Sweden)

    Wentao Wu

    2017-10-01

    Full Text Available The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens to 63 (Glycine max. The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

  12. Xyloglucan endotransglycosylase/hydrolase genes from a susceptible and resistant jute species show opposite expression pattern following Macrophomina phaseolina infection.

    Science.gov (United States)

    Sharmin, Sazia; Azam, Muhammad Shafiul; Islam, Md Shahidul; Sajib, Abu Ashfaqur; Mahmood, Niaz; Hasan, A M Mahedi; Ahmed, Razib; Sultana, Kishwar; Khan, Haseena

    2012-11-01

    Two of the most widely and intensively cultivated jute species, Corchorus capsularis and Corchorus olitorius, suffer severely from a stem rot disease caused by the fungus Macrophomina phaseolina. Wild jute species, C. trilocularis, shows resistance to this pathogenic fungus. In this study, the technique of differential display was applied to identify genes which are differentially expressed, under both infected and un-infected conditions, between C. trilocularis and C. olitorius var O-72. Two xyloglucan endotransglycosylase/hydrolase (XTH) genes designated CoXTH1 (from Corchorus olitorius) and CtXTH1 (from C.trilocularis) were identified from each of the two species which show different expression patterns upon fungal infection. A steady rise in the expression of CtXTH1 in response to infection was observed by quantitative real time PCR whereas the expression of CoXTH1 was found to be downregulated. Full length sequences of these two genes were determined using primer based gene walking and RACE PCR. This study confirms the involvement of XTH in molecular interactions between M. phaseolina and jute. However, it remains to be explored whether XTH is an essential component of the signaling pathway involved in plant-fungal interaction.

  13. Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion

    Science.gov (United States)

    2010-01-01

    Introduction Circulating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis. Methods We enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study. Results The number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts. Conclusions We report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of

  14. Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications.

    Science.gov (United States)

    Yaghoubi, Shahriar S; Campbell, Dean O; Radu, Caius G; Czernin, Johannes

    2012-01-01

    Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

  15. Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

    Directory of Open Access Journals (Sweden)

    Shahriar S. Yaghoubi, Dean O. Campbell, Caius G. Radu, Johannes Czernin

    2012-01-01

    Full Text Available Positron emission tomography (PET imaging reporter genes (IRGs and PET reporter probes (PRPs are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

  16. Two iron-regulated transporter (IRT) genes showed differential expression in poplar trees under iron or zinc deficiency.

    Science.gov (United States)

    Huang, Danqiong; Dai, Wenhao

    2015-08-15

    Two iron-regulated transporter (IRT) genes were cloned from the iron chlorosis resistant (PtG) and susceptible (PtY) Populus tremula 'Erecta' lines. Nucleotide sequence analysis showed no significant difference between PtG and PtY. The predicted proteins contain a conserved ZIP domain with 8 transmembrane (TM) regions. A ZIP signature sequence was found in the fourth TM domain. Phylogenetic analysis revealed that PtIRT1 was clustered with tomato and tobacco IRT genes that are highly responsible to iron deficiency. The PtIRT3 gene was clustered with the AtIRT3 gene that was related to zinc and iron transport in plants. Tissue specific expression indicated that PtIRT1 only expressed in the root, while PtIRT3 constitutively expressed in all tested tissues. Under iron deficiency, the expression of PtIRT1 was dramatically increased and a significantly higher transcript level was detected in PtG than in PtY. Iron deficiency also enhanced the expression of PtIRT3 in PtG. On the other hand, zinc deficiency down-regulated the expression of PtIRT1 and PtIRT3 in both PtG and PtY. Zinc accumulated significantly under iron-deficient conditions, whereas the zinc deficiency showed no significant effect on iron accumulation. A yeast complementation test revealed that the PtIRT1 and PtIRT3 genes could restore the iron uptake ability under the iron uptake-deficiency condition. The results will help understand the mechanisms of iron deficiency response in poplar trees and other woody species.

  17. Pseudoscorpion mitochondria show rearranged genes and genome-wide reductions of RNA gene sizes and inferred structures, yet typical nucleotide composition bias

    Directory of Open Access Journals (Sweden)

    Ovchinnikov Sergey

    2012-03-01

    Full Text Available Abstract Background Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes. Results We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic

  18. Specific genes involved in synthesis and editing of heparan sulfate proteoglycans show altered expression patterns in breast cancer

    Directory of Open Access Journals (Sweden)

    Fernández-Vega Iván

    2013-01-01

    Full Text Available Abstract Background The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs, which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer. Methods Twenty-three infiltrating ductal adenocarcinomas (IDCs, both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest. Results No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic, while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of

  19. Spinal meningeal melanocytoma with benign histology showing leptomeningeal spread: Case report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ok Hwa; Kim, Seon Jeong; Choo, Hye Jung; Lee, Sun Joo; Kim, Ji Yeon; Kim, Hoon [Inje University Haeundae Paik Hospital, Busan (Korea, Republic of); Lee, In Sook [Dept. of Radiology, Busan National University Hospital, Busan (Korea, Republic of)

    2013-06-15

    Meningeal melanocytoma is a rare benign tumor with relatively good prognosis. However, local aggressive behavior of meningeal melanocytoma has been reported, especially in cases of incomplete surgical resection. Malignant transformation was raised as possible cause by prior reports to explain this phenomenon. We present an unusual case of meningeal melanocytoma associated with histologically benign leptomeningeal spread and its subsequent aggressive clinical course, and describe its radiological findings.

  20. Finasteride and male breast cancer: Does the MHRA report show a link?

    Directory of Open Access Journals (Sweden)

    Niraj K Shenoy

    2010-01-01

    Full Text Available Finasteride is an important drug for the management of androgenetic alopecia. However, there are concerns about the possible side effects of the drug such as impotence. Recently stray reports have appeared about the occurrence of male breast carcinoma in patients who received the drug. These have been looked in to by Medicines and Health care products Regulatory Agency (MHRA. This article summarizes the MHRA report.

  1. Hippocampal gene expression analysis using the ORESTES methodology shows that homer 1a mRNA is upregulated in the acute period of the pilocarpine epilepsy model.

    Science.gov (United States)

    Avedissian, Marcelo; Longo, Beatriz M; Jaqueta, Carolina B; Schnabel, Beatriz; Paiva, Paulo B; Mello, Luiz Eugênio A M; Briones, Marcelo R S

    2007-01-01

    In the study of temporal lobe epilepsy (TLE) the characterization of genes expressed in the hippocampus is of central importance for understanding their roles in epileptogenic mechanisms. Although several large-scale studies on TLE gene expression have been reported, precise assignment of individual genes associated with this syndrome is still debatable. Here we investigated differentially expressed genes by comparison of mRNAs from normal and epileptic rat hippocampus in the pilocarpine model of epilepsy. For this we used a powerful EST sequencing methodology, ORESTES (Open Reading frame Expressed Sequence Tags), which generates sequence datasets enriched for mRNAs open reading frames (ORFs) rather than simple 5' and 3' ends of mRNAs. Analysis of our sequences shows that ORESTES readily enables the identification of epilepsy associated ORFs. PFAM analysis of protein motifs present in our ORESTES epilepsy database revealed diverse important protein family domains, such as cytoskeletal, cell signaling and protein kinase domains, which could be involved in processes underlying epileptogenesis. More importantly, we show that the expression of homer 1a, known to be coupled to mGluR and NMDA synaptic transmission, is associated with pilocarpine induced status epilepticus (SE). The combined use of the pilocarpine model of epilepsy with the ORESTES technique can significantly contribute to the identification of specific genes and proteins related to TLE. This is the first study applying a large-scale method for rapid shotgun sequencing directed to ORFs in epilepsy research.

  2. HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains.

    Science.gov (United States)

    Lundström, A M; Blom, K; Sundaeus, V; Bölin, I

    2001-11-01

    Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath. Copyright 2001 Academic Press.

  3. Dendritic cell subtypes from lymph nodes and blood show contrasted gene expression programs upon Bluetongue virus infection.

    Science.gov (United States)

    Ruscanu, Suzana; Jouneau, Luc; Urien, Céline; Bourge, Mickael; Lecardonnel, Jérôme; Moroldo, Marco; Loup, Benoit; Dalod, Marc; Elhmouzi-Younes, Jamila; Bevilacqua, Claudia; Hope, Jayne; Vitour, Damien; Zientara, Stéphan; Meyer, Gilles; Schwartz-Cornil, Isabelle

    2013-08-01

    Human and animal hemorrhagic viruses initially target dendritic cells (DCs). It has been proposed, but not documented, that both plasmacytoid DCs (pDCs) and conventional DCs (cDCs) may participate in the cytokine storm encountered in these infections. In order to evaluate the contribution of DCs in hemorrhagic virus pathogenesis, we performed a genome-wide expression analysis during infection by Bluetongue virus (BTV), a double-stranded RNA virus that induces hemorrhagic fever in sheep and initially infects cDCs. Both pDCs and cDCs accumulated in regional lymph nodes and spleen during BTV infection. The gene response profiles were performed at the onset of the disease and markedly differed with the DC subtypes and their lymphoid organ location. An integrative knowledge-based analysis revealed that blood pDCs displayed a gene signature related to activation of systemic inflammation and permeability of vasculature. In contrast, the gene profile of pDCs and cDCs in lymph nodes was oriented to inhibition of inflammation, whereas spleen cDCs did not show a clear functional orientation. These analyses indicate that tissue location and DC subtype affect the functional gene expression program induced by BTV and suggest the involvement of blood pDCs in the inflammation and plasma leakage/hemorrhage during BTV infection in the real natural host of the virus. These findings open the avenue to target DCs for therapeutic interventions in viral hemorrhagic diseases.

  4. Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

    Science.gov (United States)

    van Es, S; Kooistra, R A; Schaap, P

    1997-03-10

    The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

  5. Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

    Directory of Open Access Journals (Sweden)

    Cervigne Nilva K

    2010-05-01

    Full Text Available Abstract Background Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Methods Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts. Results RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. Conclusion These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma.

  6. Whole genome expression profiling shows that BRG1 transcriptionally regulates UV inducible genes and other novel targets in human cells.

    Science.gov (United States)

    Zhang, Ling; Nemzow, Leah; Chen, Hua; Hu, Jennifer J; Gong, Feng

    2014-01-01

    UV irradiation is known to cause cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), and plays a large role in the development of cancer. Tumor suppression, through DNA repair and proper cell cycle regulation, is an integral factor in maintaining healthy cells and preventing development of cancer. Transcriptional regulation of the genes involved in the various tumor suppression pathways is essential for them to be expressed when needed and to function properly. BRG1, an ATPase catalytic subunit of the SWI/SNF chromatin remodeling complex, has been identified as a tumor suppressor protein, as it has been shown to play a role in Nucleotide Excision Repair (NER) of CPDs, suppress apoptosis, and restore checkpoint deficiency, in response to UV exposure. Although BRG1 has been shown to regulate transcription of some genes that are instrumental in proper DNA damage repair and cell cycle maintenance in response to UV, its role in transcriptional regulation of the whole genome in response to UV has not yet been elucidated. With whole genome expression profiling in SW13 cells, we show that upon UV induction, BRG1 regulates transcriptional expression of many genes involved in cell stress response. Additionally, our results also highlight BRG1's general role as a master regulator of the genome, as it transcriptionally regulates approximately 4.8% of the human genome, including expression of genes involved in many pathways. RT-PCR and ChIP were used to validate our genome expression analysis. Importantly, our study identifies several novel transcriptional targets of BRG1, such as ATF3. Thus, BRG1 has a larger impact on human genome expression than previously thought, and our studies will provide inroads for future analysis of BRG1's role in gene regulation.

  7. Human reporter genes: potential use in clinical studies

    Energy Technology Data Exchange (ETDEWEB)

    Serganova, Inna [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Ponomarev, Vladimir [Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Blasberg, Ronald [Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States)], E-mail: blasberg@neuro1.mskcc.org

    2007-10-15

    The clinical application of positron-emission-tomography-based reporter gene imaging will expand over the next several years. The translation of reporter gene imaging technology into clinical applications is the focus of this review, with emphasis on the development and use of human reporter genes. Human reporter genes will play an increasingly more important role in this development, and it is likely that one or more reporter systems (human gene and complimentary radiopharmaceutical) will take leading roles. Three classes of human reporter genes are discussed and compared: receptors, transporters and enzymes. Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). In addition, we also discuss the nonhuman dopamine 2 receptor and two viral reporter genes, the wild-type herpes simplex virus 1 thymidine kinase (HSV1-tk) gene and the HSV1-tk mutant (HSV1-sr39tk). Initial applications of reporter gene imaging in patients will be developed within two different clinical disciplines: (a) gene therapy and (b) adoptive cell-based therapies. These studies will benefit from the availability of efficient human reporter systems that can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies

  8. Generation of a transformant showing higher manganese peroxidase (Mnp) activity by overexpression of Mnp gene in Trametes versicolor.

    Science.gov (United States)

    Yeo, Sumin; Park, Nammee; Song, Hong-Gyu; Choi, Hyoung T

    2007-06-01

    Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.

  9. Imaging reporter gene for monitoring gene therapy; Imagerie par gene rapporteur: un atout pour la therapie genique

    Energy Technology Data Exchange (ETDEWEB)

    Beco, V. de; Baillet, G.; Tamgac, F.; Tofighi, M.; Weinmann, P.; Vergote, J.; Moretti, J.L. [Centre Hospitalier Universitaire Avicenne, Service Central de Medecine Nucleaire et Biophysique, UPRES 2360, 93 - Bobigny (France); Tamgac, G. [Univetsite d' Uludag, Service de Medecine Nucleaire, Bursa (Turkey)

    2002-06-01

    Scintigraphic images can be obtained to document gene function at cellular level. This approach is presented here and the use of a reporter gene to monitor gene therapy is described. Two main ways are presented: either the use of a reporter gene coding for an enzyme the action of which will be monitored by radiolabeled pro-drug, or a cellular receptor gene, the action of which is documented by a radio labeled cognate receptor ligand. (author)

  10. Maxillary plexiform ameloblastoma showing basaloid differentiation: Report of a rare case with review of literature.

    Science.gov (United States)

    Ghattamaneni, Sravani; Guttikonda, Venkateswara Rao; Kumari, M Geetha; Kumar, D Rakesh

    2015-01-01

    Ameloblastoma is a benign, locally aggressive tumor originating from the odontogenic epithelium. It manifests as a slow growing swelling, causing expansion of the jaw bones. Radiologically, it presents as a unilocular or multilocular radiolucency exhibiting a characteristic soap bubble or honeycomb appearance. Ameloblastoma exhibits several histologic patterns of which basal cell variant is a rare entity. The present case report is that of a maxillary ameloblastoma exhibiting a basaloid differentiation that may put one in the mind of a basaloid squamous cell carcinoma or a basal cell carcinoma. Confirmation of such rare variants should be done not only based on histopathology but with the help of supplemental immunohistochemical analysis. The present case report helps in exposing a rare variant of ameloblastoma and emphasizes the role of advanced diagnostic aids such as immunohistochemistry in establishing the diagnosis.

  11. Nondisease genetic testing: reporting of muscle SNPs shows effects on self-concept and health orientation scales.

    Science.gov (United States)

    Gordon, Erynn S; Gordish-Dressman, Heather A; Devaney, Joseph; Clarkson, Priscilla; Thompson, Paul; Gordon, Paul; Pescatello, Linda S; Hubal, Monica J; Pistilli, Emidio E; Gianetti, Gary; Kelsey, Bethany; Hoffman, Eric P

    2005-09-01

    The purpose of this study was to assess the impact of genetic self-knowledge (nondisease genotype information) on individual self-concept and Health Orientation Scale (HOS). Adult volunteers (n=257) were recruited from an ongoing genetic association study identifying muscle quantitative trait loci (QTLs). Participants completed psychosocial assessments before and after 12 weeks of resistance training of the nondominant arm. At study exit, a genetic counselor informed participants of genetic test results on three to four genes that have an association with muscle-related traits, and counseled subjects on the potential significance of these findings. The second psychosocial assessment was performed immediately following this counseling session. The Tennessee Self-Concept Scale v.2 (TSCS:2) and the HOS showed female subjects to have a significantly greater positive change between first and second assessments, relative to male subjects. Most self-concept subscales improved significantly, when 'neutral' genotypes (no anticipated beneficial or deleterious impact) were reported, compared to positive genotypes. TSCS:2 subscales showing improvement included: total (P=0.013); physical (P=0.004); satisfaction (P=0.019); and behavioral (P=0.047). HOS subscales showing improvement included health image concern (P=0.006); and health expectations (P=0.047). In conclusion, these results suggest that genetic self-knowledge affects self-concept, consistent with the 'attribution' theory. Individuals who received neutral genetic information attributed positive changes from the exercise program to their own abilities, while those who received positive information were more likely to attribute positive changes to their genetics. This study is limited by the ability to determine the direction of the impact of nondisease genetic information presented to participants.

  12. Solitary eccrine syringofibroadenoma: a case report showing papillary tubular adenoma-like features

    Directory of Open Access Journals (Sweden)

    Toshiyuki Yamamoto

    2016-10-01

    Full Text Available We herein describe a case showing eccrine syringofibroadenoma occurred on the dorsum of the right foot of a 46-year-old Japanese female. Histopathologic examination revealed anastomosing cords and strands of cuboidal epithelial cells extending from the epidermis to the upper dermis, with a number of well-defined ducts suggesting eccrine ductal origin. In addition, there were papillary tubular adenoma-like ductal structures lined by a few rows of epithelial cells with papillary projections into the lumen surrounded by fibrous stroma in the mid-dermis. It is of note that various histologic features showing different differentiation were seen in a single lesion of eccrine syringofibroadenoma.

  13. Transgenic tobacco plants expressing BoRS1 gene from Brassica oleracea var. acephala show enhanced tolerance to water stress

    Indian Academy of Sciences (India)

    Dongqin Tang; Hongmei Qian; Lingxia Zhao; Danfeng Huang; Kexuan Tang

    2005-12-01

    Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through Agrobacterium-mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows that BoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.

  14. Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis

    Directory of Open Access Journals (Sweden)

    João Miguel Carvas

    2012-08-01

    Full Text Available Period2 (Per2 is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial NO production in response to insulin in the mPer2Brdm1 mice. We show that mPer2Brdm1 mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated endothelial NO release in the aortas in both active and inactive phases of the animals. As compared to wild type mice, the mPer2Brdm1 mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2Brdm1 mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to wild type controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated endothelial NO release, independently of obesity.

  15. Mice heterozygous for the oxytocin receptor gene (Oxtr(+/-)) show impaired social behaviour but not increased aggression or cognitive inflexibility: evidence of a selective haploinsufficiency gene effect.

    Science.gov (United States)

    Sala, M; Braida, D; Donzelli, A; Martucci, R; Busnelli, M; Bulgheroni, E; Rubino, T; Parolaro, D; Nishimori, K; Chini, B

    2013-02-01

    We characterised the behavioural phenotype of mice heterozygous (Oxtr(+/-)) for the oxytocin receptor gene (Oxtr) and compared it with that of Oxtr null mice (Oxtr(-/-)), which display autistic-like behaviours, including impaired sociability and preference for social novelty, impaired cognitive flexibility, and increased aggression. Similar to Oxtr(-/-) mice, the Oxtr(+/-) showed impaired sociability and preference for social novelty but, unlike the null genotype, their cognitive flexibility and aggression were normal. By autoradiography, Oxtr(+/-) mice were found to have approximately 50% fewer oxytocin receptors (OXTRs) in all of the examined brain regions. Thus, because a partial reduction in Oxtr gene expression is sufficient to compromise social behaviour, the Oxtr acts as a haploinsufficient gene. Furthermore, the inactivation of the Oxtr gene affects specific behaviours in a dose-dependent manner: social behaviour is sensitive to even a partial reduction in Oxtr gene expression, whereas defects in aggression and cognitive flexibility require the complete inactivation of the Oxtr gene to emerge. We then investigated the rescue of the Oxtr(+/-) social deficits by oxytocin (OT) and Thr(4)Gly(7)OT (TGOT) administered i.c.v. at different doses. TGOT was more potent than OT in rescuing sociability and social novelty in both genotypes. Furthermore, the TGOT doses that reverted impaired sociability and preference for social novelty in Oxtr(+/-) were lower than those required in Oxtr(-/-), thus suggesting that the rescue effect is mediated by OXTR in Oxtr(+/-) and by other receptors (presumably vasopressin V1a receptors) in Oxtr(-/-). In line with this, a low dose of the selective oxytocin antagonist desGlyDTyrOVT blocks the rescue effect of TGOT only in the Oxtr(+/-) genotype, whereas the less selective antagonist SR49059 blocks rescue in both genotypes. In conclusion, the Oxtr(+/-) mouse is a unique animal model for investigating how partial loss of the Oxtr gene

  16. Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD

    Directory of Open Access Journals (Sweden)

    Navarro Arcadi

    2009-07-01

    Full Text Available Abstract Background It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8. Results Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively. Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia and the Surui (America. Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. Conclusion The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in

  17. Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD

    Science.gov (United States)

    Bosch, Elena; Laayouni, Hafid; Morcillo-Suarez, Carlos; Casals, Ferran; Moreno-Estrada, Andrés; Ferrer-Admetlla, Anna; Gardner, Michelle; Rosa, Araceli; Navarro, Arcadi; Comas, David; Graffelman, Jan; Calafell, Francesc; Bertranpetit, Jaume

    2009-01-01

    Background It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP) pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8. Results Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively). Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia) and the Surui (America). Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. Conclusion The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in genetic epidemiology. PMID

  18. An Inflammatory Dentigerous Cyst Shows Rim Uptake on Bone Scan: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Son, Hye Joo; Jeong, Young Jin; Jeong, Jin Sook; Kang, Doyoung [Dong-A Univ. Medical Center, Busan (Korea, Republic of)

    2014-03-15

    Dentigerous cysts are developmental odontogenic jaw cysts, commonly manifesting in the 2nd and 3rd decades of life. This article presents an extremely rare case of dentigerous cyst showing increased uptake in the peripheral rim on bone scan. Herein, we discuss the clinical, radiographic, and histologic features of dentigerous cysts as well as the pathological mechanism underlying their activities on the bone scan. Bone scan was a sensitive tool for detecting the biologic activity of dentigerous cyst in our case.

  19. Autoimmune Hepatitis and Celiac Disease: Case Report Showing an Entero-Hepatic Link

    Directory of Open Access Journals (Sweden)

    Francesco Tovoli

    2010-10-01

    Full Text Available Celiac disease is an autoimmune disorder primarily targeting the small bowel, although extraintestinal extensions have been reported. The autoimmune processes can affect the liver with manifestations such as primary biliary cirrhosis and autoimmune hepatitis. We describe a 61-year-old woman with celiac disease and an increased levels of aminotransferases. The persistence of increased levels of aminotransferases after 1 year of gluten-free diet and the positivity for an anti-nuclear and anti-double-strand DNA antibodies led to a misdiagnosis of systemic lupus erythematosus-related hepatitis. Based on these findings the patient was placed on steroids, which after a few months were stopped because of the onset of diabetes mellitus. Soon after steroid withdrawal, the patient had a marked increase in aminotransferases and γ-globulins, and a liver biopsy revealed chronic active hepatitis. A course of three months of steroids and azathioprine normalized both biochemical and clinical parameters. Currently the patient is symptom-free and doing well. In conclusion, a hypertransaminasemia persisting after a gluten-free diet should be interpreted as a sign of coexisting autoimmune liver disease. Any autoantibody positivity (in this case to ANA and anti-dsDNA should be carefully considered in order to avoid misdiagnosis delaying appropriate clinical management.

  20. Tolosa-Hunt syndrome: is it really necessary to show granuloma? - The report of eight cases

    Directory of Open Access Journals (Sweden)

    Podgorac Ana

    2017-01-01

    Full Text Available Introduction. Tolosa–Hunt syndrome (THS is a rare entity, characterized by unilateral orbital pain associated with paresis of one or more of the oculomotor cranial nerves and caused by a granulomatous inflammation in the cavernous sinus, superior orbital fissure or orbit. The low prevalence of THS with a broad spectrum of other disorders that could cause painful ophtalmoplegia resulted in a stricter diagnostic criteria of THS in the latest edition of the International Classification of Headache Disorders. Current criteria require demonstration of granuloma by magnetic resonance imaging or biopsy. The diagnosis could be difficult and the initiation of treatment delayed due to a high variablity of clinical presentation of TSH. Reducing the number of patients that, based on clinical presentation, could be classified as having THS, but do not fullfil all diagnostic criteria further complicates establishing of correct diagnosis. Case report. Hereby we presented eight patients diagnosed with and treated for THS. Inspite the exclusion of other causes of painful ophtalmoplegia, granuloma could not be demonstrated in a half of patients. Clinical presentation of THS in patients with and without shown granuloma, did not significantly differ concerning headache characteristics (localization, intensity, quality, duration preceding cranial nerve palsy, response to steroids, the affected cranial nerve, disease course and response to the treatment, as well as types of diagnostic procedures that were performed in ruling out other diseases from the extensive differential diagnosis of painful ophthalmoplegia. Conclusion. There is no significant difference between the THS patients with and without demonstrated granuloma.

  1. Differential expression of hypothalamic fear- and stress-related genes in broiler chickens showing short or long tonic immobility.

    Science.gov (United States)

    Wang, S; Ni, Y; Guo, F; Sun, Z; Ahmed, A; Zhao, R

    2014-04-01

    The serotonin system and the hypothalamic-pituitary-adrenal axis play important roles in modulating fear and stress-coping characteristics. Tonic immobility (TI) is a fear-related phenotype, and previously we have shown that broiler chickens showing short TI (STI) duration experience better growth performance and higher adaptability to stress. Here, we sought to further elucidate the central mechanisms underlying the phenotypic differences between chickens showing STI and long TI duration, by comparing the hypothalamic expression of genes in the serotonergic system and the hypothalamic-pituitary-adrenal axis under basal and corticosterone-exposed situations. The STI broilers had significantly lower (P chickens at the level of both mRNA (P chickens, but the protein content was approximately 2 times higher (P chickens. The uncoupled expression of GR mRNA and protein was associated with significantly lower (P chickens. Corticosterone administration reduced the mRNA expression of postsynaptic serotonin receptors, 5-hydroxytryptamine receptor 1B (P = 0.059) and 5-hydroxytryptamine receptor 7 (P stress-related genes.

  2. Sacrococcygeal chordoma in infancy showing an aggressive clinical course: an autopsy case report.

    Science.gov (United States)

    Shinmura, Yuichiro; Miura, Katsutoshi; Yajima, Shuhei; Tsutsui, Yoshihiro

    2003-07-01

    The autopsy case of a 3-year 6-month-old boy with chordoma arising in the sacrococcygeal region is presented. The primary lesion of the sacrococcygeal area was unresectable and lung metastasis was detected. He was treated with multi-agent systemic chemotherapy and radiation therapy, but the tumor was less responsive to these therapies. He died about one year after first admission. An autopsy revealed a massive sacrococcygeal mass and metastasis in the thoracic and lumbar vertebrae, retroperitoneal and mediastinal lymph nodes, and also in the bilateral lungs and liver. Histologically, the tumor was composed of 'pink' cells and scattered 'physaliphorous' cells with a myxoid matrix. Sacrococcygeal chordoma in infancy is very rare. Our case showed a highly aggressive clinical course.

  3. Astrocyte Cultures Mimicking Brain Astrocytes in Gene Expression, Signaling, Metabolism and K(+) Uptake and Showing Astrocytic Gene Expression Overlooked by Immunohistochemistry and In Situ Hybridization.

    Science.gov (United States)

    Hertz, Leif; Chen, Ye; Song, Dan

    2017-01-01

    Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K(+) concentrations and for Ca(2+) homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K(+) uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K(+) effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.

  4. Agricultural Education. Missouri's Show-Me Standards and Vocational Education Competencies. Cross Reference. Main Report [and] Mini Report.

    Science.gov (United States)

    Missouri Univ., Columbia. Instructional Materials Lab.

    These two documents deal with the relationship between Missouri's Show-Me Standards (the standards defining what all Missouri students should know upon graduation from high school) with the vocational competencies taught in secondary-level agricultural education courses. The first document, which is a database documenting the common ground that…

  5. Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns

    DEFF Research Database (Denmark)

    Salomonsen, Jan; Chattaway, John A.; Chan, Andrew C. Y.

    2014-01-01

    Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility c...... the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood....

  6. EPA Report Shows Progress on E-Recycling and Identifies Opportunity to Advance G7s Recognition of Circular Economy

    Science.gov (United States)

    WASHINGTON - The U.S. Environmental Protection Agency (EPA) today released the Advancing Sustainable Materials Management (SMM) Facts and Figures report showing progress in consumer electronics recycling in the United States. Consumer electronics re

  7. Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns

    DEFF Research Database (Denmark)

    Salomonsen, Jan; Chattaway, John A; Chan, Andrew C Y;

    2014-01-01

    Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility c...

  8. SMARCB1/INI1-deficient sinonasal carcinoma shows methylation of RASSF1 gene: A clinicopathological, immunohistochemical and molecular genetic study of a recently described entity.

    Science.gov (United States)

    Laco, Jan; Chmelařová, Marcela; Vošmiková, Hana; Sieglová, Kateřina; Bubancová, Ivana; Dundr, Pavel; Němejcová, Kristýna; Michálek, Jaroslav; Čelakovský, Petr; Mottl, Radovan; Sirák, Igor; Vošmik, Milan; Ryška, Aleš

    2017-02-01

    The aim of the study was detailed clinicopathological investigation of SMARCB1/INI1-deficient sinonasal carcinomas, including molecular genetic analysis of mutational status and DNA methylation of selected protooncogenes and tumor suppressor genes by means of next generation sequencing (NGS) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). A total of 4/56 (7%) cases of SMARCB1/INI1-deficient carcinomas were detected among 56 sinonasal carcinomas diagnosed over a 19year period using immunohistochemical screening. The series comprised 3 males and 1 female, aged 27-76 years (median 64 years). All tumors arose in the nasal cavity. Three neoplasms were diagnosed in advanced stage pT4. During the follow-up period (range 14-111 months (median 72 months)), three tumors recurred locally, but none of the patients developed regional or distant metastases. Ultimately, two patients died due to the tumor. Microscopically, all tumors consisted of infiltrating nests of polygonal basaloid cells with a variable component of rhabdoid cells with eosinophilic cytoplasm. Immunohistochemically, there was almost diffuse expression of cytokeratins (CK), p16, p40 and p63 in all cases, while expression of CK5/6, CK7 and vimentin was only focal or absent. The detection of NUT gave negative results. In three cases, the absence of SMARCB1/INI1 expression was due to deletion of SMARCB1/INI1 gene. Methylation of SMARCB1/INI1 gene was not found. One tumor harbored HPV18 E6/E7 mRNA. All 12 genes (BRAF, BRCA1, BRCA2, KIT, EGFR, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, RET, and ROS1) tested for mutations using NGS were wild-type. Regarding DNA methylation, all four SMARCB1/INI1-deficient tumors showed methylation of RASSF1 gene by means of MS-MLPA. There was a statistically significant difference in RASSF1 gene methylation between SMARCB1/INI1-deficient and SMARCB1/INI1-positive tumors (p=0.0095). All other examined genes (ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDKN1B

  9. Candidate genes for obesity-susceptibility show enriched association within a large genome-wide association study for BMI

    Science.gov (United States)

    Vimaleswaran, Karani S.; Tachmazidou, Ioanna; Zhao, Jing Hua; Hirschhorn, Joel N.; Dudbridge, Frank; Loos, Ruth J.F.

    2012-01-01

    Before the advent of genome-wide association studies (GWASs), hundreds of candidate genes for obesity-susceptibility had been identified through a variety of approaches. We examined whether those obesity candidate genes are enriched for associations with body mass index (BMI) compared with non-candidate genes by using data from a large-scale GWAS. A thorough literature search identified 547 candidate genes for obesity-susceptibility based on evidence from animal studies, Mendelian syndromes, linkage studies, genetic association studies and expression studies. Genomic regions were defined to include the genes ±10 kb of flanking sequence around candidate and non-candidate genes. We used summary statistics publicly available from the discovery stage of the genome-wide meta-analysis for BMI performed by the genetic investigation of anthropometric traits consortium in 123 564 individuals. Hypergeometric, rank tail-strength and gene-set enrichment analysis tests were used to test for the enrichment of association in candidate compared with non-candidate genes. The hypergeometric test of enrichment was not significant at the 5% P-value quantile (P = 0.35), but was nominally significant at the 25% quantile (P = 0.015). The rank tail-strength and gene-set enrichment tests were nominally significant for the full set of genes and borderline significant for the subset without SNPs at P < 10−7. Taken together, the observed evidence for enrichment suggests that the candidate gene approach retains some value. However, the degree of enrichment is small despite the extensive number of candidate genes and the large sample size. Studies that focus on candidate genes have only slightly increased chances of detecting associations, and are likely to miss many true effects in non-candidate genes, at least for obesity-related traits. PMID:22791748

  10. Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma show alterations of genes encoding the NADPH oxidase complex and impaired reactive oxygen species synthesis capacity.

    Science.gov (United States)

    Giefing, Maciej; Winoto-Morbach, Supandi; Sosna, Justyna; Döring, Claudia; Klapper, Wolfram; Küppers, Ralf; Böttcher, Sebastian; Adam, Dieter; Siebert, Reiner; Schütze, Stefan

    2013-01-01

    The membrane bound NADPH oxidase involved in the synthesis of reactive oxygen species (ROS) is a multi-protein enzyme encoded by CYBA, CYBB, NCF1, NCF2 and NCF4 genes. Growing evidence suggests a role of ROS in the modulation of signaling pathways of non-phagocytic cells, including differentiation and proliferation of B-cell progenitors. Transcriptional downregulation of the CYBB gene has been previously reported in cell lines of the B-cell derived classical Hodgkin lymphoma (cHL). Thus, we explored functional consequences of CYBB downregulation on the NADPH complex. Using flow cytometry to detect and quantify superoxide anion synthesis in cHL cell lines we identified recurrent loss of superoxide anion production in all stimulated cHL cell lines in contrast to stimulated non-Hodgkin lymphoma cell lines. As CYBB loss proved to exert a deleterious effect on the NADPH oxidase complex in cHL cell lines, we analyzed the CYBB locus in Hodgkin and Reed-Sternberg (HRS) cells of primary cHL biopsies by in situ hybridisation and identified recurrent deletions of the gene in 8/18 cases. Immunohistochemical analysis to 14 of these cases revealed a complete lack of detectable CYBB protein expression in all HRS cells in all cases studied. Moreover, by microarray profiling of cHL cell lines we identified additional alterations of NADPH oxidase genes including CYBA copy number loss in 3/7 cell lines and a significant downregulation of the NCF1 transcription (p=0.006) compared to normal B-cell subsets. Besides, NCF1 protein was significantly downregulated (p<0.005) in cHL compared to other lymphoma cell lines. Together this findings show recurrent alterations of the NADPH oxidase encoding genes that result in functional inactivation of the enzyme and reduced production of superoxide anion in cHL.

  11. Repeat length variation in the dopamine D4 receptor gene shows no evidence of association with schizophrenia

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, J.; Williams, J.; Asherson, P. [Univ. of Wales College of Medicine, Cardiff (United Kingdom)] [and others

    1994-09-15

    The D4 receptor has been shown to exist in several allelic forms reflecting variation in the number of 48 base-pair sequence repeats in the putative cytoplasmic loop. We report a comparison of repeat length variation between schizophrenic patients and controls. Our sample of 106 unrelated schizophrenic cases and 119 controls showed no significant differences in allele or genotype distribution between patients and controls. In particular, we were unable to support the previous observation of an excess of 4-repeat homozygotes in patients. 16 refs., 2 tabs.

  12. hNIS-IRES-eGFP Dual Reporter Gene Imaging

    Directory of Open Access Journals (Sweden)

    Jiantu Che

    2005-04-01

    Full Text Available The human and rodent sodium iodide symporters (NIS have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES-linked human NIS (hNIS-enhanced green fluorescent protein (eGFP hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131I-iodide and 99mTcO4-pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na131I and 99mTcO4– to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging.

  13. Post genome-wide association studies of novel genes associated with type 2 diabetes show gene-gene interaction and high predictive value.

    Directory of Open Access Journals (Sweden)

    Stéphane Cauchi

    Full Text Available BACKGROUND: Recently, several Genome Wide Association (GWA studies in populations of European descent have identified and validated novel single nucleotide polymorphisms (SNPs, highly associated with type 2 diabetes (T2D. Our aims were to validate these markers in other European and non-European populations, then to assess their combined effect in a large French study comparing T2D and normal glucose tolerant (NGT individuals. METHODOLOGY/PRINCIPAL FINDINGS: In the same French population analyzed in our previous GWA study (3,295 T2D and 3,595 NGT, strong associations with T2D were found for CDKAL1 (OR(rs7756992 = 1.30[1.19-1.42], P = 2.3x10(-9, CDKN2A/2B (OR(rs10811661 = 0.74[0.66-0.82], P = 3.5x10(-8 and more modestly for IGFBP2 (OR(rs1470579 = 1.17[1.07-1.27], P = 0.0003 SNPs. These results were replicated in both Israeli Ashkenazi (577 T2D and 552 NGT and Austrian (504 T2D and 753 NGT populations (except for CDKAL1 but not in the Moroccan population (521 T2D and 423 NGT. In the overall group of French subjects (4,232 T2D and 4,595 NGT, IGFBP2 and CXCR4 synergistically interacted with (LOC38776, SLC30A8, HHEX and (NGN3, CDKN2A/2B, respectively, encoding for proteins presumably regulating pancreatic endocrine cell development and function. The T2D risk increased strongly when risk alleles, including the previously discovered T2D-associated TCF7L2 rs7903146 SNP, were combined (8.68-fold for the 14% of French individuals carrying 18 to 30 risk alleles with an allelic OR of 1.24. With an area under the ROC curve of 0.86, only 15 novel loci were necessary to discriminate French individuals susceptible to develop T2D. CONCLUSIONS/SIGNIFICANCE: In addition to TCF7L2, SLC30A8 and HHEX, initially identified by the French GWA scan, CDKAL1, IGFBP2 and CDKN2A/2B strongly associate with T2D in French individuals, and mostly in populations of Central European descent but not in Moroccan subjects. Genes expressed in the pancreas interact together and their

  14. A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

    Science.gov (United States)

    Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

    2011-09-01

    A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

  15. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367.

    Science.gov (United States)

    Tolonen, Andrew C; Chilaka, Amanda C; Church, George M

    2009-12-01

    Summary Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.

  16. Cell type-specific genes show striking and distinct patterns of spatial expression in the mouse brain.

    Science.gov (United States)

    Ko, Younhee; Ament, Seth A; Eddy, James A; Caballero, Juan; Earls, John C; Hood, Leroy; Price, Nathan D

    2013-02-19

    To characterize gene expression patterns in the regional subdivisions of the mammalian brain, we integrated spatial gene expression patterns from the Allen Brain Atlas for the adult mouse with panels of cell type-specific genes for neurons, astrocytes, and oligodendrocytes from previously published transcriptome profiling experiments. We found that the combined spatial expression patterns of 170 neuron-specific transcripts revealed strikingly clear and symmetrical signatures for most of the brain's major subdivisions. Moreover, the brain expression spatial signatures correspond to anatomical structures and may even reflect developmental ontogeny. Spatial expression profiles of astrocyte- and oligodendrocyte-specific genes also revealed regional differences; these defined fewer regions and were less distinct but still symmetrical in the coronal plane. Follow-up analysis suggested that region-based clustering of neuron-specific genes was related to (i) a combination of individual genes with restricted expression patterns, (ii) region-specific differences in the relative expression of functional groups of genes, and (iii) regional differences in neuronal density. Products from some of these neuron-specific genes are present in peripheral blood, raising the possibility that they could reflect the activities of disease- or injury-perturbed networks and collectively function as biomarkers for clinical disease diagnostics.

  17. Amerindians show association to obesity with adiponectin gene SNP45 and SNP276: population genetics of a food intake control and "thrifty" gene.

    Science.gov (United States)

    Arnaiz-Villena, Antonio; Fernández-Honrado, Mercedes; Rey, Diego; Enríquez-de-Salamanca, Mercedes; Abd-El-Fatah-Khalil, Sedeka; Arribas, Ignacio; Coca, Carmen; Algora, Manuel; Areces, Cristina

    2013-02-01

    Adiponectin gene polymorphisms SNP45 and SNP276 have been related to metabolic syndrome (MS) and related pathologies, including obesity. However results of associations are contradictory depending on which population is studied. In the present study, these adiponectin SNPs are for the first time studied in Amerindians. Allele frequencies are obtained and comparison with obesity and other MS related parameters are performed. Amerindians were also defined by characteristic HLA genes. Our main results are: (1) SNP276 T is associated to low diastolic blood pressure in Amerindians, (2) SNP45 G allele is correlated with obesity in female but not in male Amerindians, (3) SNP45/SNP276 T/G haplotype in total obese/non-obese subjects tends to show a linkage with non-obese Amerindians, (4) SNP45/SNP276 T/T haplotype is linked to obese Amerindian males. Also, a world population study is carried out finding that SNP45 T and SNP276 T alleles are the most frequent in African Blacks and are found significantly in lower frequencies in Europeans and Asians. This together with the fact that there is a linkage of this haplotype to obese Amerindian males suggest that evolutionary forces related to famine (or population density in relation with available food) may have shaped world population adiponectin polymorphism frequencies.

  18. Cloning-free regulated monitoring of reporter and gene expression

    Directory of Open Access Journals (Sweden)

    Demirkaya Omer

    2009-03-01

    Full Text Available Abstract Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

  19. Chemotherapy Agents Alter Plasma Lipids in Breast Cancer Patients and Show Differential Effects on Lipid Metabolism Genes in Liver Cells.

    Science.gov (United States)

    Sharma, Monika; Tuaine, Jo; McLaren, Blair; Waters, Debra L; Black, Katherine; Jones, Lynnette M; McCormick, Sally P A

    2016-01-01

    Cardiovascular complications have emerged as a major concern for cancer patients. Many chemotherapy agents are cardiotoxic and some appear to also alter lipid profiles, although the mechanism for this is unknown. We studied plasma lipid levels in 12 breast cancer patients throughout their chemotherapy. Patients received either four cycles of doxorubicin and cyclophosphamide followed by weekly paclitaxel or three cycles of epirubicin, cyclophosphamide and 5'-fluorouracil followed by three cycles of docetaxel. Patients demonstrated a significant reduction (0.32 mmol/L) in high density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (apoA1) levels (0.18 g/L) and an elevation in apolipoprotein B (apoB) levels (0.15 g/L) after treatment. Investigation of the individual chemotherapy agents for their effect on genes involved in lipoprotein metabolism in liver cells showed that doxorubicin decreased ATP binding cassette transporter A1 (ABCA1) via a downregulation of the peroxisomal proliferator activated receptor γ (PPARγ) and liver X receptor α (LXRα) transcription factors. In contrast, ABCA1 levels were not affected by cyclophosphamide or paclitaxel. Likewise, apoA1 levels were reduced by doxorubicin and remained unaffected by cyclophosphamide and paclitaxel. Doxorubicin and paclitaxel both increased apoB protein levels and paclitaxel also decreased low density lipoprotein receptor (LDLR) protein levels. These findings correlate with the observed reduction in HDL-C and apoA1 and increase in apoB levels seen in these patients. The unfavourable lipid profiles produced by some chemotherapy agents may be detrimental in the longer term to cancer patients, especially those already at risk of cardiovascular disease (CVD). This knowledge may be useful in tailoring effective follow-up care plans for cancer survivors.

  20. SINORHIZOBIUM MELILOTI ELECTROTRANSPORANT CONTAINING ORTHO-DECHLORINATION GENE SHOWS ENHANCED PCB-DECHLORINATION. (R828770C008)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  1. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments

    Directory of Open Access Journals (Sweden)

    Gyöngyi Munkácsy

    2016-01-01

    Full Text Available No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06. Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04. There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.

  2. Effect of SNPs in protein kinase Czgene on gene expression in the reporter gene detection system

    Institute of Scientific and Technical Information of China (English)

    Zhuo Liu; Hong-Xia Sun; Yong-Wei Zhang; Yun-Feng Li; Jin Zuo; Yan Meng; Fu-De Fang

    2004-01-01

    AIM: To investigated the effects of the SNPs (rs411021,rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes.METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above.These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity of the luciferase was assayed. Effects of the SNPs on RNA splicing were analyzed by bioinformatics.RESULTS: rs427811T allele and rs809912G allele enhanced the activity of the reporter gene expression. None of the 5 SNPs affected RNA splicing.CONCLUSION: SNPs in protein kinase Cz (PKCZ) gene probably play a role in the susceptibility to type 2 diabetes by affecting the expression level of the relevant genes.

  3. Structure, variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence.

    Science.gov (United States)

    Wang, Kai; Zhang, Xue; Zhao, Ying; Chen, Fanguo; Xia, Guangmin

    2013-09-25

    In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2,554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> -700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5'-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.

  4. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  5. The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation

    DEFF Research Database (Denmark)

    Harris, Abigail K P; Williamson, Neil R; Slater, Holly

    2004-01-01

    The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and...

  6. Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content.

    Science.gov (United States)

    Jube, Sandro L R; Borthakur, Dulal

    2010-04-01

    The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

  7. Commonly studied polymorphisms in inflammatory cytokine genes show only minor effects on mortality and related risk factors in nonagenarians

    DEFF Research Database (Denmark)

    Dato, Serena; Krabbe, Karen S; Thinggaard, Mikael;

    2010-01-01

    , in nonagenarian men, the balance of pro- and anti-inflammatory activity at IL18 and IL10 loci is protective against cognitive decline. In conclusion, in this large study with virtually complete follow-up, commonly studied polymorphisms in cytokine genes do not have a major impact on late-life survival...

  8. Feasibility of sodium/iodide symporter gene as a new imaging reporter gene: comparison with HSV1-tk

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jae Hoon; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea); Department of Tumor Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Laboratory of Molecular Imaging and Therapy, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Kang, Joo Hyun; Jeong, Jae Min; Lee, Dong Soo [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea); Kim, Chul Woo [Department of Pathology, Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea); Lee, Myung Chul [Department of Nuclear Medicine, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, 110-744, Chongno-gu, Seoul (Korea)

    2004-03-01

    Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D{sub 2} receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter (NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([{sup 125}I]IVDU) and {sup 125}I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [{sup 125}I]IVDU and {sup 131}I, and {sup 131}I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of {sup 125}I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [{sup 125}I]IVDU than CT-26 and CTN. NIS gene expression and {sup 125}I accumulation were found to be directly correlated (R{sup 2}=0.923), as were HSV1-tk gene expression and [{sup 125}I]IVDU accumulation (R{sup 2}=0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240{+-}3,755 cpm and 34,039{+-}5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of {sup 131}I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [{sup 125}I]IVDU than CT-26 and CTN. Scintigraphy using {sup 131}I easily visualised CTN and

  9. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    Science.gov (United States)

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  10. Commonly studied polymorphisms in inflammatory cytokine genes show only minor effects on mortality and related risk factors in nonagenarians.

    Science.gov (United States)

    Dato, Serena; Krabbe, Karen S; Thinggaard, Mikael; Pedersen, Bente K; Christensen, Kaare; Bruunsgaard, Helle; Christiansen, Lene

    2010-03-01

    Systemic low-grade inflammation is consistently associated with functional status, cognitive functioning, multimorbidity, and survival in oldest olds. If inflammation is either a cause or a consequence of age-related pathology, genetic determinants of late-life survival can reside in cytokine genes polymorphisms, regulating inflammatory responses. The aim of this study was to test associations between commonly studied polymorphisms in interleukin (IL)6, IL10, IL15, and IL18, and tumor necrosis factor-alpha genes and late-life survival in a longitudinal cohort of nonagenarians: the Danish 1905 cohort. Additionally, associations were investigated between inflammatory markers and major predictors of mortality as cognitive and functional status. Modest sex-specific associations were found with survival, cognitive functioning, and handgrip strength. Evaluation of combined genotypes indicated that, in nonagenarian men, the balance of pro- and anti-inflammatory activity at IL18 and IL10 loci is protective against cognitive decline. In conclusion, in this large study with virtually complete follow-up, commonly studied polymorphisms in cytokine genes do not have a major impact on late-life survival or associated risk phenotypes.

  11. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  12. Gene expression profiles of mouse aorta and cultured vascular smooth muscle cells differ widely, yet show common responses to dioxin exposure.

    Science.gov (United States)

    Puga, Alvaro; Sartor, Maureen A; Huang, Ming-Ya; Kerzee, J Kevin; Wei, Yu-Dan; Tomlinson, Craig R; Baxter, C Stuart; Medvedovic, Mario

    2004-01-01

    Exposure to environmental toxicants may play a role in the onset and progression of cardiovascular disease. Many environmental agents, such as dioxin, are risk factors for atherosclerosis because they may exacerbate an underlying disease by altering gene expression patterns. Expression profiling of vascular tissues allows the simultaneous analysis of thousands of genes and may provide predictive information particularly useful in early disease stages. Often, however, in vivo experiments are unfeasible for material or ethical reasons, and data from cultured cells must be used instead, even though it may not be known whether cultured cells and live tissues share common global responses to the same toxicant. In a search for genes responsive to dioxin exposure, we used oligonucleotide microarrays with DNA sequences from 13,433 genes to compare global gene expression profiles of C57BL/6 mice aortas with cultured vascular smooth muscle cells (vSMCs) of the same mice. Aorta segments and vSMCs differed in the expression of more than 4500 genes, many showing expression differences greater than 1000-fold. Integration of microarray data into Gene Ontology Project annotations showed that many of the genes differentially expressed belonged to the same biological process or metabolic pathway. Notwithstanding these results, a subset of 35 genes responded in the same fashion to dioxin exposure in both systems. Genes in this subset encoded phase I and phase II detoxification enzymes, signal transduction kinases and phosphatases, and proteins involved in DNA repair and the cell cycle. We conclude that vSMCS may be useful aorta surrogates to study early gene expression responses to dioxin exposure, provided that analyses focus on this subset of genes.

  13. Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence

    DEFF Research Database (Denmark)

    Christiansen, Michael W.; Matthewman, Colette; Podzimska-Sroka, Dagmara Agata;

    2016-01-01

    -expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling......The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005...

  14. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    Science.gov (United States)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K.; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D.; de Ángeles Granados-Silvestre, Ma; Montufar-Robles, Isela; Tito-Alvarez, Ana M.; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P.; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L.; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A.; Lisker, Ruben; Moises, Regina S.; Menjivar, Marta; Salzano, Francisco M.; Knowler, William C.; Bortolini, M. Cátira; Hayden, Michael R.; Baier, Leslie J.; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 × 10−11) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations. PMID:20418488

  15. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans.

    Science.gov (United States)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-07-15

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Americans and 863 individuals from other ethnic groups to investigate genetic evidence of positive selection, to assess its functional effect in vitro and to explore associations with HDL-C levels and other metabolic traits. The C230 allele was found in 29 of 36 Native American groups, but not in European, Asian or African individuals. C230 was observed on a single haplotype, and C230-bearing chromosomes showed longer relative haplotype extension compared with other haplotypes in the Americas. Additionally, single-nucleotide polymorphism data from the Human Genome Diversity Panel Native American populations were enriched in significant integrated haplotype score values in the region upstream of the ABCA1 gene. Cells expressing the C230 allele showed a 27% cholesterol efflux reduction (P< 0.001), confirming this variant has a functional effect in vitro. Moreover, the C230 allele was associated with lower HDL-C levels (P = 1.77 x 10(-11)) and with higher body mass index (P = 0.0001) in the combined analysis of Native American populations. This is the first report of a common functional variant exclusive to Native American and descent populations, which is a major determinant of HDL-C levels and may have contributed to the adaptive evolution of Native American populations.

  16. Report of a chimeric origin of transposable elements in a bovine-coding gene.

    Science.gov (United States)

    Almeida, L M; Amaral, M E J; Silva, I T; Silva, W A; Riggs, P K; Carareto, C M

    2008-02-01

    Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is approximately 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.

  17. Repetitive Imaging of Reporter Gene Expression in the Lung

    Directory of Open Access Journals (Sweden)

    Jean-Christophe Richard

    2003-10-01

    Full Text Available Positron emission tomographic imaging is emerging as a powerful technology to monitor reporter transgene expression in the lungs and other organs. However, little information is available about its usefulness for studying gene expression over time. Therefore, we infected 20 rats with a replication-deficient adenovirus containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase and an enhanced green fluorescent protein. Five additional rats were infected with a control virus. Pulmonary gene transfer was performed via intratracheal administration of vector using a surfactant-based method. Imaging was performed 4–6 hr, and 4, 7, and 10 days after gene transfer, using 9-(4-[18F]-fluoro-3-hydroxymethylbutylguanine, an imaging substrate for the mutant kinase. Lung tracer uptake assessed with imaging was moderately but significantly increased 4–6 hr after gene transfer, was maximal after 4 days, and was no longer detectable by 10 days. The temporal pattern of transgene expression measured ex vivo with in vitro assays of thymidine kinase activity and green fluorescent protein was similar to imaging. In conclusion, positron emission tomography is a reliable new tool to evaluate the onset and duration of reporter gene expression noninvasively in the lungs of intact animals.

  18. Genome-wide analysis of AP2/ERF family genes from Lotus corniculatus shows LcERF054 enhances salt tolerance.

    Science.gov (United States)

    Sun, Zhan-Min; Zhou, Mei-Liang; Xiao, Xing-Guo; Tang, Yi-Xiong; Wu, Yan-Min

    2014-09-01

    Lotus corniculatus is used in agriculture as a main forage plant. Members of the Apetala2/ethylene response factor (AP2/ERF) family play important roles in regulating gene expression in response to many forms of stress, including drought and salt. Here, starting from database of the L. corniculatus var. japonicus genome, we identified 127 AP2/ERF genes by insilico cloning method. The phylogeny, gene structures, and putative conserved motifs in L. corniculatus var. japonicus ERF proteins were analyzed. Based on the number of AP2/ERF domains and the function of the genes, 127 AP2/ERF genes from L. corniculatus var. japonicus were classified into five subfamilies named the AP2, dehydration-responsive element binding factor (DREB), ERF, RAV, and a soloist. Outside the AP2/ERF domain, many L. corniculatus var. japonicus-specific conserved motifs were detected. Expression profile analysis of AP2/ERF genes by quantitative real-time PCR revealed that 19 LcERF genes, including LcERF054 (KJ004728), were significantly induced by salt stress. The results showed that the LcERF054 gene encodes a nuclear transcription activator. Overexpression of LcERF054 in Arabidopsis enhanced the tolerances to salt stress, showed higher germination ratio of seeds, and had elevated levels of relative moisture contents, soluble sugars, proline, and lower levels of malondialdehyde under stress conditions compared to wild-type plants. The expression of hyperosmotic salinity response genes COR15A, LEA4-5, P5CS1, and RD29A was found to be elevated in the LcERF054-overexpressing Arabidopsis plants compared to wild type. These results revealed that the LcERF genes play important roles in L. corniculatus cv Leo under salt stress and that LcERFs are attractive engineering targets in applied efforts to improve abiotic stress tolerances in L. corniculatus cv Leo or other crops.

  19. Non-invasive imaging using reporter genes altering cellular water permeability

    Science.gov (United States)

    Mukherjee, Arnab; Wu, Di; Davis, Hunter C.; Shapiro, Mikhail G.

    2016-12-01

    Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

  20. Gene network analysis shows immune-signaling and ERK1/2 as novel genetic markers for multiple addiction phenotypes: alcohol, smoking and opioid addiction.

    Science.gov (United States)

    Reyes-Gibby, Cielito C; Yuan, Christine; Wang, Jian; Yeung, Sai-Ching J; Shete, Sanjay

    2015-06-05

    Addictions to alcohol and tobacco, known risk factors for cancer, are complex heritable disorders. Addictive behaviors have a bidirectional relationship with pain. We hypothesize that the associations between alcohol, smoking, and opioid addiction observed in cancer patients have a genetic basis. Therefore, using bioinformatics tools, we explored the underlying genetic basis and identified new candidate genes and common biological pathways for smoking, alcohol, and opioid addiction. Literature search showed 56 genes associated with alcohol, smoking and opioid addiction. Using Core Analysis function in Ingenuity Pathway Analysis software, we found that ERK1/2 was strongly interconnected across all three addiction networks. Genes involved in immune signaling pathways were shown across all three networks. Connect function from IPA My Pathway toolbox showed that DRD2 is the gene common to both the list of genetic variations associated with all three addiction phenotypes and the components of the brain neuronal signaling network involved in substance addiction. The top canonical pathways associated with the 56 genes were: 1) calcium signaling, 2) GPCR signaling, 3) cAMP-mediated signaling, 4) GABA receptor signaling, and 5) G-alpha i signaling. Cancer patients are often prescribed opioids for cancer pain thus increasing their risk for opioid abuse and addiction. Our findings provide candidate genes and biological pathways underlying addiction phenotypes, which may be future targets for treatment of addiction. Further study of the variations of the candidate genes could allow physicians to make more informed decisions when treating cancer pain with opioid analgesics.

  1. The human norepinephrine transporter in combination with C-11-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy

    NARCIS (Netherlands)

    Buursma, A.R.; Beerens, Antoine; de Vries, E.F J; van Waarde, Aaren; Rots, Marianne; Hospers, G.A.P.; Vaalburg, W.; Haisma, H.J.

    2005-01-01

    Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility o

  2. Synonymous Codon Usage Bias in the Plastid Genome is Unrelated to Gene Structure and Shows Evolutionary Heterogeneity.

    Science.gov (United States)

    Qi, Yueying; Xu, Wenjing; Xing, Tian; Zhao, Mingming; Li, Nana; Yan, Li; Xia, Guangmin; Wang, Mengcheng

    2015-01-01

    Synonymous codon usage bias (SCUB) is the nonuniform usage of codons, occurring often in nearly all organisms. Our previous study found that SCUB is correlated with intron number, is unequal among exons in the plant nuclear genome, and mirrors evolutionary specialization. However, whether this rule exists in the plastid genome has not been addressed. Here, we present an analysis of SCUB in the plastid genomes of 25 species from lower to higher plants (algae, bryophytes, pteridophytes, gymnosperms, and spermatophytes). We found NNA and NNT (A- and T-ending codons) are preferential in the plastid genomes of all plants. Interestingly, this preference is heterogeneous among taxonomies of plants, with the strongest preference in bryophytes and the weakest in pteridophytes, suggesting an association between SCUB and plant evolution. In addition, SCUB frequencies are consistent among genes with varied introns and among exons, indicating that the bias of NNA and NNT is unrelated to either intron number or exon position. Further, SCUB is associated with DNA methylation-induced conversion of cytosine to thymine in the vascular plants but not in algae or bryophytes. These data demonstrate that these SCUB profiles in the plastid genome are distinctly different compared with the nuclear genome.

  3. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  4. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    Science.gov (United States)

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A

    2013-10-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  5. Clusters of ancestrally related genes that show paralogy in whole or in part are a major feature of the genomes of humans and other species.

    Directory of Open Access Journals (Sweden)

    Michael B Walker

    Full Text Available Arrangements of genes along chromosomes are a product of evolutionary processes, and we can expect that preferable arrangements will prevail over the span of evolutionary time, often being reflected in the non-random clustering of structurally and/or functionally related genes. Such non-random arrangements can arise by two distinct evolutionary processes: duplications of DNA sequences that give rise to clusters of genes sharing both sequence similarity and common sequence features and the migration together of genes related by function, but not by common descent. To provide a background for distinguishing between the two, which is important for future efforts to unravel the evolutionary processes involved, we here provide a description of the extent to which ancestrally related genes are found in proximity.Towards this purpose, we combined information from five genomic datasets, InterPro, SCOP, PANTHER, Ensembl protein families, and Ensembl gene paralogs. The results are provided in publicly available datasets (http://cgd.jax.org/datasets/clustering/paraclustering.shtml describing the extent to which ancestrally related genes are in proximity beyond what is expected by chance (i.e. form paraclusters in the human and nine other vertebrate genomes, as well as the D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae genomes. With the exception of Saccharomyces, paraclusters are a common feature of the genomes we examined. In the human genome they are estimated to include at least 22% of all protein coding genes. Paraclusters are far more prevalent among some gene families than others, are highly species or clade specific and can evolve rapidly, sometimes in response to environmental cues. Altogether, they account for a large portion of the functional clustering previously reported in several genomes.

  6. Oxytocin receptor gene sequences in owl monkeys and other primates show remarkable interspecific regulatory and protein coding variation.

    Science.gov (United States)

    Babb, Paul L; Fernandez-Duque, Eduardo; Schurr, Theodore G

    2015-10-01

    The oxytocin (OT) hormone pathway is involved in numerous physiological processes, and one of its receptor genes (OXTR) has been implicated in pair bonding behavior in mammalian lineages. This observation is important for understanding social monogamy in primates, which occurs in only a small subset of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential relationship between social monogamy and OXTR variation, we sequenced its 5' regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the 1000 Genomes Project. We also assessed interspecific variation of OXTR in 25 primate and rodent species that represent a set of phylogenetically and behaviorally disparate taxa. Our analysis revealed substantial variation in the putative 5' regulatory region of OXTR, with marked structural differences across primate taxa, particularly for humans and chimpanzees, which exhibited unique patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5' UTR. In addition, we observed a large number of amino acid substitutions in the OXTR CDS region among New World primate taxa that distinguish them from Old World primates. Furthermore, primate taxa traditionally defined as socially monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all exhibited different amino acid motifs for their respective OXTR protein coding sequences. These findings support the notion that monogamy has evolved independently in Old World and New World primates, and that it has done so through different molecular mechanisms, not exclusively through the oxytocin pathway.

  7. Fetal and neonatal anemia associated with anti-Jr(a) : a case report showing a poorly hemolytic mechanism.

    Science.gov (United States)

    Sasamoto, Naoko; Tomimatsu, Takuji; Nagamine, Keisuke; Oshida, Machiko; Kashiwagi, Hirokazu; Koyama, Shinsuke; Kanagawa, Takeshi; Arahori, Hitomi; Tomiyama, Yoshiaki; Kimura, Tadashi

    2011-08-01

    Although recently published case reports suggest the significance of Jr(a) alloimmunization in the obstetric setting, the involved mechanism still remains unclear. Here we report a case of severe fetal and neonatal anemia associated with anti-Jr(a) alloimmunization, which was successfully managed using Doppler assessment of peak systolic velocity of the fetal middle cerebral artery (MCA-PSV). A Japanese woman with anti-Jr(a) (titer 1024) was referred to our department at 20 weeks' gestation. As fetal MCA-PSV exceeded 1.5 multiple of median, labor was induced and a female neonate of 1998 g was delivered vaginally at 33 weeks and 5 days of gestation. The infant's hematocrit and hemoglobin levels were 25.4% and 82 g/L, respectively, but her total bilirubin level (15 µmol/L; 0.9 mg/dL) and reticulocyte counts (4.5%) were low. During the course, the infant showed no apparent signs of hemolysis. Jr(a) alloimmunization should be recognized as a possible cause of fetal anemia with no direct hemolytic process. © 2011 The Authors. Journal of Obstetrics and Gynaecology Research © 2011 Japan Society of Obstetrics and Gynecology.

  8. Sequencing of Sylvilagus VDJ genes reveals a new VHa allelic lineage and shows that ancient VH lineages were retained differently in leporids.

    Science.gov (United States)

    Pinheiro, Ana; Melo-Ferreira, José; Abrantes, Joana; Martinelli, Nicola; Lavazza, Antonio; Alves, Paulo C; Gortázar, Christian; Esteves, Pedro J

    2014-12-01

    Antigen recognition by immunoglobulins depends upon initial rearrangements of heavy chain V, D, and J genes. In leporids, a unique system exists for the VH genes usage that exhibit highly divergent lineages: the VHa allotypes, the Lepus sL lineage and the VHn genes. For the European rabbit (Oryctolagus cuniculus), four VHa lineages have been described, the a1, a2, a3 and a4. For hares (Lepus sp.), one VHa lineage was described, the a2L, as well as a more ancient sL lineage. Both genera use the VHn genes in a low frequency of their VDJ rearrangements. To address the hypothesis that the VH specificities could be associated with different environments, we sequenced VDJ genes from a third leporid genus, Sylvilagus. We found a fifth and equally divergent VHa lineage, the a5, and an ancient lineage, the sS, related to the hares' sL, but failed to obtain VHn genes. These results show that the studied leporids employ different VH lineages in the generation of the antibody repertoire, suggesting that the leporid VH genes are subject to strong selective pressure likely imposed by specific pathogens.

  9. Long-running German panel survey shows that personal and economic choices, not just genes, matter for happiness

    Science.gov (United States)

    Headey, Bruce; Muffels, Ruud; Wagner, Gert G.

    2010-01-01

    Psychologists and economists take contradictory approaches to research on what psychologists call happiness or subjective well-being, and economists call subjective utility. A direct test of the most widely accepted psychological theory, set-point theory, shows it to be flawed. Results are then given, using the economists’ newer “choice approach”—an approach also favored by positive psychologists—which yields substantial payoffs in explaining long-term changes in happiness. Data come from the German Socio-Economic Panel (1984–2008), a unique 25-y prospective longitudinal survey. This dataset enables direct tests of theories explaining long-term happiness. PMID:20921399

  10. Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1.

    Science.gov (United States)

    Singh, M; Kreutzer, R; Acker, G; Klingmüller, W

    1988-01-01

    A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.

  11. Intra-articular epithelioid sarcoma showing mixed classic and proximal-type features: report of 2 cases, with immunohistochemical and molecular cytogenetic INI-1 study.

    Science.gov (United States)

    Kosemehmetoglu, Kemal; Kaygusuz, Gulsah; Bahrami, Armita; Raimondi, Susana C; Kilicarslan, Kasim; Yildiz, Yusuf; Folpe, Andrew L

    2011-06-01

    Epithelioid sarcoma, a rare sarcoma with epithelial differentiation, most often occurs in the distal extremities; however, it may occur in essentially any location. With the recent recognition that the loss of expression of the tumor-suppressor gene INI-1 may be associated with epithelioid sarcoma, it has become clear that epithelioid sarcoma may occur in previously unsuspected locations such as bone. Only 2 cases of intra-articular epithelioid sarcoma have been previously reported. We retrieved 2 intra-articular cases coded as epithelioid sarcoma from our archives. Both expressed cytokeratins (AE1/AE3 and OSCAR), CD34, vimentin, and epithelial membrane antigen, and showed complete loss of expression of INI-1. Fluorescence in situ hybridization was performed on formalin-fixed, paraffin-embedded sections by using a laboratory-developed dual-color probe containing INI1 (CTD-2511E13 and CTD-2034E7) (22q11.2) (OR) and PANX2 (RPCI3-402G11) (22q13.33) (GR) probes as control. Both cases occurred in a clearly intra-articular location in the knee. Case 1 was that of a 19-year-old man with a long-standing history of pain and limited joint function. This patient was disease free after amputation. Case 2 was that of a 60-year-old woman. Follow-up information available for this patient showed bilateral subpleural metastases. Morphologically, case 1 showed features of proximal-type epithelioid sarcoma, whereas case 2 showed mixed features of classic and proximal-type epithelioid sarcoma. Immunohistochemistry showed complete loss of INI-1 protein in both cases; fluorescence in situ hybridization analyses were negative for INI-1 gene deletion. Herein, we have reported 2 cases of intra-articular epithelioid sarcoma, showing morphologic and immunohistochemical features identical to those of epithelioid sarcoma in conventional locations, including loss of INI-1 expression. Intra-articular epithelioid sarcoma should be distinguished from malignant pigmented villonodular synovitis and

  12. Analysis of Gene Targeting & Nonhomologous End-joining. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Haber, J. E.

    2002-11-30

    Overall, we identified a number of new proteins that participate in nonhomologous end-joining and also in telomere addition to the ends of broken chromosomes. We showed that NHEJ is severely reduced in cells expressing both yeast mating-type genes and then went on to identify the NEJ1 gene that was under this control. We showed the epistasis relations among a set of mutations that impair telomere addition and we showed that there are in fact two pathways to repair broken chromosomes in the absence of telomerase. We characterized the DNA damage checkpoint pathway in response to a single broken chromosome and characterized especially the adaptation of cells arrested by an unrepaired DSB. We demonstrated that the DNA damage response is nuclear-limited. We showed adaptation defects for Tid1and Srs2 proteins and showed that Srs2 was also recovery-defective, even when DNA was repaired.

  13. Aegilops tauschii Accessions with Geographically Diverse Origin Show Differences in Chromosome Organization and Polymorphism of Molecular Markers Linked to Leaf Rust and Powdery Mildew Resistance Genes

    Science.gov (United States)

    Majka, Maciej; Kwiatek, Michał T.; Majka, Joanna; Wiśniewska, Halina

    2017-01-01

    Aegilops tauschii (2n = 2x = 14) is a diploid wild species which is reported as a donor of the D-genome of cultivated bread wheat. The main goal of this study was to examine the differences and similarities in chromosomes organization among accessions of Ae. tauschii with geographically diversed origin, which is believed as a potential source of genes, especially determining resistance to fungal diseases (i.e., leaf rust and powdery mildew) for breeding of cereals. We established and compared the fluorescence in situ hybridization patterns of 21 accessions of Ae. tauschii using various repetitive sequences mainly from the BAC library of wheat cultivar Chinese Spring. Results obtained for Ae. tauschii chromosomes revealed many similarities between analyzed accessions, however, some hybridization patterns were specific for accessions, which become from cognate regions of the World. The most noticeable differences were observed for accessions from China which were characterized by presence of distinct signals of pTa-535 in the interstitial region of chromosome 3D, less intensity of pTa-86 signals in chromosome 2D, as well as lack of additional signals of pTa-86 in chromosomes 1D, 5D, or 6D. Ae. tauschii of Chinese origin appeared homogeneous and separate from landraces that originated in western Asia. Ae. tauschii chromosomes showed similar hybridization patterns to wheat D-genome chromosomes, but some differences were also observed among both species. What is more, we identified reciprocal translocation between short arm of chromosome 1D and long arm of chromosome 7D in accession with Iranian origin. High polymorphism between analyzed accessions and extensive allelic variation were revealed using molecular markers associated with resistance genes. Majority of the markers localized in chromosomes 1D and 2D showed the diversity of banding patterns between accessions. Obtained results imply, that there is a moderate or high level of polymorphism in the genome of Ae

  14. Wong-Type Dermatomyositis Showing Porokeratosis-Like Changes (Columnar Dyskeratosis: A Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Nicole Umanoff

    2015-01-01

    Full Text Available Background: Wong-type dermatomyositis (DM exhibits simultaneous pityriasis rubra pilaris (PRP features. Case Report: A 50-year-old woman presented with a heliotrope rash, Gottron's papules, and a poikilodermic, erythematous rash in shawl distribution without evidence of muscle weakness. Despite topical corticosteroids, the eruption progressed 9 months later to include generalized hyperkeratotic follicular papules, islands of sparing, and atrophic macules with a collarette of scale suggestive of porokeratosis. Mild dysphonia was the only sign of muscle weakness. Serology showed positive ANA. Histopathology revealed interface dermatitis with dermal mucin and melanophages, irregular psoriasiform hyperplasia, alternating mounds of para- and orthokeratosis, and tiers of dyskeratotic cells (columnar dyskeratosis. Systemic corticosteroid therapy was not tolerated; acitretin diminished the hyperkeratosis. While hyperpigmentation persisted, no progression of cutaneous or muscular symptoms has occurred after 22 months of follow-up and cessation of the therapy. Overall, her course did not differ from the natural history documented in the literature review of Wong-type DM. The most similar case also exhibited pseudocornoid lamella changes. Conclusion: Wong-type DM is a clinicopathologic DM-PRP hybrid that can also exhibit porokeratosis-like features best described as columnar dyskeratosis. Recognizing these types of lesions in DM is warranted in order to make an accurate assessment of their prognostic significance.

  15. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  16. Signal transduction pathways that regulate CAB gene expression. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Chory, J.

    1993-12-31

    We have completed the initial genetic and phenotypic characterization of several classes of new mutants that affect CAB gene expression. The doc mutants (for dark overexpression of cab) are characterized by elevated levels of CAB gene expression in the dark; however, unlike the previously isolated de-etiolated mutants (also isolated in my lab), the doc mutants still appear etiolated. The doc alleles define 3 loci, each of which maps to a separate chromosome. The details of the mutant isolation scheme and the genetic and phenotypic description of these new mutants are described. The second class of mutants, the gun mutants (for genomes uncoupled) show accumulation of CAB mRNA in the absence of chloroplast gene expression and development. Thus, the normally tightly coordinated expression between the chloroplast and nuclear genes that encode chloroplast-destined proteins has been uncoupled. We have shown that the Arabidopsis HY3 locus encodes the type B phytochrome apoprotein gene and have characterized the phenotypes of null hy3 alleles to ascertain a role for this phytochrome in Arabidopsis development. We have also isolated and characterized a number of alleles of the phytochrome A gene.

  17. SPECT imaging of cardiac reporter gene expression in living rabbits

    Institute of Scientific and Technical Information of China (English)

    LIU Ying; LAN Xiaoli; ZHANG Liang; WU Tao; JIANG Rifeng; ZHANG Yongxue

    2009-01-01

    This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine ki-nase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-β-D- arabi-nofuranosyl-5-iodouracil (131I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1×109, 5×108, 1×108, 5×107 and 1×107 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1×109, 5×108, 1×108, 5×107, 1×107 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse tran-scription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSV1-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images qual-ity was obtained at 24~48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5×107 pfu of virus titer. The result could be set better in 1~5×108 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSV1-tk/131I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter gene imaging

  18. Gene Knockout Shows That PML (TRIM19) Does Not Restrict the Early Stages of HIV-1 Infection in Human Cell Lines.

    Science.gov (United States)

    Masroori, Nasser; Cherry, Pearl; Merindol, Natacha; Li, Jia-Xin; Dufour, Caroline; Poulain, Lina; Plourde, Mélodie B; Berthoux, Lionel

    2017-01-01

    The PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. In contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNA interference, with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used the clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9) system to knock out its gene in epithelial, lymphoid, and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Overexpression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α (another human TRIM protein), and TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages. IMPORTANCE PML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide

  19. SNA Releases Back to School Nutrition Trends Report: Results Show What Schools Are Doing to Increase Healthy Options for Kids

    Science.gov (United States)

    Curriculum Review, 2008

    2008-01-01

    This article talks about the School Nutrition Association's 2008 Back to School Nutrition Trends Report that was released on August 19. According to the report, the trend towards more healthful school meal choices continues this fall with district nutrition programs emphasizing whole grains, fruits, and vegetables while cutting back on trans fats,…

  20. 3p22.1p21.31 microdeletion identifies CCK as Asperger syndrome candidate gene and shows the way for therapeutic strategies in chromosome imbalances.

    Science.gov (United States)

    Iourov, Ivan Y; Vorsanova, Svetlana G; Voinova, Victoria Y; Yurov, Yuri B

    2015-01-01

    In contrast to other autism spectrum disorders, chromosome abnormalities are rare in Asperger syndrome (AS) or high-functioning autism. Consequently, AS was occasionally subjected to classical positional cloning. Here, we report on a case of AS associated with a deletion of the short arm of chromosome 3. Further in silico analysis has identified a candidate gene for AS and has suggested a therapeutic strategy for manifestations of the chromosome rearrangement. Using array comparative genomic hybridization, an interstitial deletion of 3p22.1p21.31 (~2.5 Mb in size) in a child with Asperger's syndrome, seborrheic dermatitis and chronic pancreatitis was detected. Original bioinformatic approach to the prioritization of candidate genes/processes identified CCK (cholecystokinin) as a candidate gene for AS. In addition to processes associated with deleted genes, bioinformatic analysis of CCK gene interactome indicated that zinc deficiency might be a pathogenic mechanism in this case. This suggestion was supported by plasma zinc concentration measurements. The increase of zinc intake produced a rise in zinc plasma concentration and the improvement in the patient's condition. Our study supported previous linkage findings and had suggested a new candidate gene in AS. Moreover, bioinformatic analysis identified the pathogenic mechanism, which was used to propose a therapeutic strategy for manifestations of the deletion. The relative success of this strategy allows speculating that therapeutic or dietary normalization of metabolic processes altered by a chromosome imbalance or genomic copy number variations may be a way for treating at least a small proportion of cases of these presumably incurable genetic conditions.

  1. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung [School of Medicine, Chonnam National University, Gwangju (Korea, Republic of); Kim, Yeon Soo [Schoole of Medicine, Inje University, Seoul (Korea, Republic of)

    2004-07-01

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells.

  2. Show Time

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    <正> Story: Show Time!The whole class presents the story"Under the Sea".Everyone is so excited and happy.Both Leo and Kathy show their parentsthe characters of the play."Who’s he?"asks Kathy’s mom."He’s the prince."Kathy replies."Who’s she?"asks Leo’s dad."She’s the queen."Leo replieswith a smile.

  3. Snobbish Show

    Institute of Scientific and Technical Information of China (English)

    YIN PUMIN

    2010-01-01

    @@ The State Administration of Radio,Film and Television (SARFT),China's media watchdog,issued a new set of mles on June 9 that strictly regulate TV match-making shows,which have been sweeping the country's primetime programming. "Improper social and love values such as money worship should not be presented in these shows.Humiliation,verbal attacks and sex-implied vulgar content are not allowed" the new roles said.

  4. A simple method for analyzing exome sequencing data shows distinct levels of nonsynonymous variation for human immune and nervous system genes.

    Directory of Open Access Journals (Sweden)

    Jan Freudenberg

    Full Text Available To measure the strength of natural selection that acts upon single nucleotide variants (SNVs in a set of human genes, we calculate the ratio between nonsynonymous SNVs (nsSNVs per nonsynonymous site and synonymous SNVs (sSNVs per synonymous site. We transform this ratio with a respective factor f that corrects for the bias of synonymous sites towards transitions in the genetic code and different mutation rates for transitions and transversions. This method approximates the relative density of nsSNVs (rdnsv in comparison with the neutral expectation as inferred from the density of sSNVs. Using SNVs from a diploid genome and 200 exomes, we apply our method to immune system genes (ISGs, nervous system genes (NSGs, randomly sampled genes (RSGs, and gene ontology annotated genes. The estimate of rdnsv in an individual exome is around 20% for NSGs and 30-40% for ISGs and RSGs. This smaller rdnsv of NSGs indicates overall stronger purifying selection. To quantify the relative shift of nsSNVs towards rare variants, we next fit a linear regression model to the estimates of rdnsv over different SNV allele frequency bins. The obtained regression models show a negative slope for NSGs, ISGs and RSGs, supporting an influence of purifying selection on the frequency spectrum of segregating nsSNVs. The y-intercept of the model predicts rdnsv for an allele frequency close to 0. This parameter can be interpreted as the proportion of nonsynonymous sites where mutations are tolerated to segregate with an allele frequency notably greater than 0 in the population, given the performed normalization of the observed nsSNV to sSNV ratio. A smaller y-intercept is displayed by NSGs, indicating more nonsynonymous sites under strong negative selection. This predicts more monogenically inherited or de-novo mutation diseases that affect the nervous system.

  5. Genome-Wide Identification of Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D and D-E-L Motifs in EF-Hand Domain.

    Science.gov (United States)

    Mohanta, Tapan K; Mohanta, Nibedita; Mohanta, Yugal K; Bae, Hanhong

    2015-01-01

    Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca(2+) concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca(2+) ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins.

  6. The first report of the vanC₁ gene in Enterococcus faecium isolated from a human clinical specimen.

    Science.gov (United States)

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-09-01

    The vanC₁ gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC₁gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC₁ and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC₁ gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC₁gene. However, this study is the first to report the presence of the vanC₁gene in E. faecium of human origin. Additionally, our research showed the vanC₁gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC₁gene from different species.

  7. Primary microcephaly gene MCPH1 shows signatures of tumor suppressors and is regulated by miR-27a in oral squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Thejaswini Venkatesh

    Full Text Available Mutations in the MCPH1 (microcephalin 1 gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC samples, and observed that 14/71 (19.72% informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22% and 19/25 (76% OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10% tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

  8. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    Science.gov (United States)

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  9. EROBATIC SHOW

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    Visitors look at plane models of the Commercial Aircraft Corp. of China, developer of the count,s first homegrown large passenger jet C919, during the Singapore Airshow on February 16. The biennial event is the largest airshow in Asia and one of the most important aviation and defense shows worldwide. A number of Chinese companies took part in the event during which Okay Airways, the first privately owned aidine in China, signed a deal to acquire 12 Boeing 737 jets.

  10. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  11. Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group.

    Science.gov (United States)

    Kreuze, J F; Karyeija, R F; Gibson, R W; Valkonen, J P

    2000-01-01

    Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) infects sweet potatoes (Ipomoea batatas) worldwide, but no sequence data on isolates from Africa are available. Coat protein (CP) gene sequences from eight East African isolates from Madagascar and different districts of Uganda (the second biggest sweet potato producer in the world) and two West African isolates from Nigeria and Niger were determined. They were compared by phylogenetic analysis with the previously reported sequences of ten SPFMV isolates from other continents. The East African SPFMV isolates formed a distinct cluster, whereas the other isolates were not clustered according to geographic origin. These data indicate that East African isolates of SPFMV form a genetically unique group.

  12. Delusion of pregnancy in a drug-naïve young woman showing hyperprolactinemia and hypothyroidism: a case report.

    Science.gov (United States)

    Penta, Elena; Lasalvia, Antonio

    2013-01-01

    The present article reports the case of a delusional pregnancy that developed in a drug-naïve young woman affected by paranoid schizophrenia and Hashimoto-related hyperprolactinemia. The literature in this field has addressed the phenomenon of distorted thoughts of pregnancy being elicited by physical changes due to drug-induced hyperprolactinemia. The present report gives account of a case of delusional pregnancy that developed in a schizophrenic patient with concurrent hyperprolactinemia induced by a primary endocrine disease. In this instance, however, amelioration of delusional beliefs did not ensue from hyperprolactinemia normalization, but was mostly due to cognitive restructuring of distorted thinking (together with the antipsychotic treatment). This finding confirms the importance of considering the key role of the interaction of biological, cognitive and psychological mechanisms in the construction of inaccurate beliefs and feelings about pregnancy.

  13. Conservative treatment for lumbar compartment syndrome shows efficacy over 2-year follow-up: a case report and literature review.

    Science.gov (United States)

    Kanaya, Haruhisa; Enokida, Makoto; Tanishima, Shinji; Hayashi, Ikuta; Tanida, Atsushi; Nagashima, Hideki

    2017-06-20

    Since in all studies of conservative treatment of lumbar compartment syndrome the follow-up duration was less than 6 months, it is difficult to draw firm conclusions. To report a patient with lumbar paraspinal compartment syndrome who was treated conservatively over a follow-up period of 2 years. This is a case report of a 23-year-old male college student with lumbar paraspinal compartment syndrome who was treated conservatively. We report a case of a 23-year-old male college student with lumbar paraspinal compartment syndrome who was treated conservatively. We repeatedly checked his physical examination, laboratory tests, lumbar compartment pressures, and magnetic resonance imaging, and surgical teams were readily prepared to operate should the patient's condition worsen. To prevent complications of rhabdomyolysis, hydration and alkalization were performed. We followed him up to 2 years after discharge. Although the temporal changes on MRI up to the 1-year point, the patient continued to have no symptoms. Conservative therapy can be recommended if rhabdomyolysis is under control.

  14. A HOMOLOGOUS GENE-REPORTER SYSTEM FOR THE BASIDIOMYCETE SCHIZOPHYLLUM-COMMUNE BASED ON INTERNALLY DELETED HOMOLOGOUS GENES

    NARCIS (Netherlands)

    SCHUREN, FHJ; HARMSEN, MC; WESSELS, JGH

    Problems encountered in our attempts to achieve expression of heterologous genes, driven by ascomycetous regulatory sequences, in homobasidiomycetes led us to develop a gene-reporter system based on the expression of homologous genes in Schizophyllum commune. Internal deletions were made in the

  15. NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.

    Science.gov (United States)

    Kirby, J; Kavanagh, T A

    2002-11-01

    GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

  16. Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1.

    Science.gov (United States)

    Zhang, Ling; Liu, Meihong; Merling, Randall; Giam, Chou-Zen

    2006-08-01

    Potent activation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is mediated by the virus-encoded transactivator protein Tax and three imperfect 21-bp repeats in the viral long terminal repeats. Each 21-bp repeat contains a cAMP-responsive-element core flanked by 5' G-rich and 3' C-rich sequences. Tax alone does not bind DNA. Rather, it interacts with basic domain-leucine zipper transcription factors CREB and ATF-1 to form ternary complexes with the 21-bp repeats. In the context of the ternary complexes, Tax contacts the G/C-rich sequences and recruits transcriptional coactivators CREB-binding protein (CBP)/p300 to effect potent transcriptional activation. Using an easily transduced and chromosomally integrated reporter system derived from a self-inactivating lentivirus vector, we showed in a BRG1- and BRM1-deficient adrenal carcinoma cell line, SW-13, that Tax- and 21-bp repeat-mediated transactivation does not require BRG1 or BRM1 and is not enhanced by BRG1. With a similar reporter system, we further demonstrated that Tax- and tumor necrosis factor alpha-induced NF-kappaB activation occurs readily in SW-13 cells in the absence of BRG1 and BRM1. These results suggest that the assembly of stable multiprotein complexes containing Tax, CREB/ATF-1, and CBP/p300 on the 21-bp repeats is the principal mechanism employed by Tax to preclude nucleosome formation at the HTLV-1 enhancer/promoter. This most likely bypasses the need for BRG1-containing chromatin-remodeling complexes. Likewise, recruitment of CBP/p300 by NF-kappaB may be sufficient to disrupt histone-DNA interaction for the initiation of transcription.

  17. Children’s School-Breakfast Reports and School-Lunch Reports (in 24-hour Dietary Recalls): Conventional and Reporting-Error-Sensitive Measures Show Inconsistent Accuracy Results for Retention Interval and for Breakfast Location

    Science.gov (United States)

    Baxter, Suzanne Domel; Guinn, Caroline H.; Smith, Albert F.; Hitchcock, David B.; Royer, Julie A.; Puryear, Megan P.; Collins, Kathleen L.; Smith, Alyssa L.

    2017-01-01

    Validation-study data were analyzed to investigate retention interval (RI) and prompt effects on accuracy of fourth-grade children’s reports of school-breakfast and school-lunch (in 24-hour recalls), and accuracy of school-breakfast reports by breakfast location (classroom; cafeteria). Randomly-selected fourth-grade children at 10 schools in four districts were observed eating school-provided breakfast and lunch, and interviewed under one of eight conditions (two RIs [short (prior-24-hour recall obtained in afternoon); long (previous-day recall obtained in morning)] crossed with four prompts [forward (distant-to-recent), meal-name (breakfast, etc.), open (no instructions), reverse (recent-to-distant)]). Each condition had 60 children (half girls). Of 480 children, 355 and 409 reported meals satisfying criteria for reports of school-breakfast and school-lunch, respectively. For breakfast and lunch separately, a conventional measure—report rate—and reporting-error-sensitive measures—correspondence rate and inflation ratio—were calculated for energy per meal-reporting child. Correspondence rate and inflation ratio—but not report rate—showed better accuracy for school-breakfast and school-lunch reports with the short than long RI; this pattern was not found for some prompts for each sex. Correspondence rate and inflation ratio showed better school-breakfast report accuracy for the classroom than cafeteria location for each prompt, but report rate showed the opposite. For each RI, correspondence rate and inflation ratio showed better accuracy for lunch than breakfast, but report rate showed the opposite. When choosing RI and prompts for recalls, researchers and practitioners should select short RIs to maximize accuracy. Recommendations for prompt selections are less clear. As report rates distort validation-study accuracy conclusions, reporting-error-sensitive measures are recommended. PMID:26865356

  18. 3' Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

    Institute of Scientific and Technical Information of China (English)

    Jie Jing; Men Jing; Wang Chun-mei; Gao Xue-jun; Li Qing-zhang

    2012-01-01

    To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3' UTR was predicted by Target Scan 5.1 software, 3' UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3' UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3' UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.

  19. Necrobiotic granulomatous tattoo reaction: report of an unusual case showing features of both necrobiosis lipoidica and granuloma annulare patterns.

    Science.gov (United States)

    Wood, Andrew; Hamilton, Stuart A; Wallace, William A; Biswas, Asok

    2014-08-01

    The application of decorative tattoos to the skin is an ancient practice, which continues to be popular in the present day. Increasingly, a number of complications of tattoo application are recognized, including a diverse variety of cutaneous hypersensitivity reactions to tattoo pigments. Herein we describe an unusual case of a necrobiotic granulomatous tattoo reaction showing combined features of necrobiosis lipoidica and early granuloma annulare. The relevant literature is reviewed, and the practical significance of such unusual histological features as a part of the tattoo reaction is discussed.

  20. Bacillus subtilis from Soybean Food Shows Antimicrobial Activity for Multidrug-Resistant Acinetobacter baumannii by Affecting the adeS Gene.

    Science.gov (United States)

    Wang, Tieshan; Su, Jianrong

    2016-12-28

    Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii. Nineteen multidrug-resistant A. baumannii strains were clinifcally isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii. The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis. Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.

  1. Genetic variants in three genes and smoking show strong associations with susceptibility to exudative age-related macular degeneration in a Chinese population

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    cigarette smoking were also related to exudative AMD. After controlling for environmental risk factors, CFH and HTRA1 SNPs were independently associated with exudative AMD, with OR of 3.50 (1.45-8.45) for CT genotype in Y402H, 3.34 (1.33-8.36) for GG genotype in rs1410996 and 3.85 (1.58-9.42) for AA genotype in rs11200638 respectively. The interaction analysis between gene and environmental factors showed that smoking synergistically increased susceptibility of AMD for heterozygotes of rs1410996, with ORinteraction of 7.33 (Pinteraction=0.029). Conclusions In a Han Chinese population, CFH and HTRA1 polymorphisms appear to be independently and possibly additivelv hereditary contributors to exudative AMD. Y402H polymorphism conferred a significant but relatively lower contribution in Chinese than in Caucasians with a low frequency of risk allele. The gene-environment interaction may be a best way to encourage those with a high genetic risk to prevent AMD by avoiding modifiable factors until there is effective treatment for AMD.

  2. Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.

    Science.gov (United States)

    Marui, Junichiro; Yoshimi, Akira; Hagiwara, Daisuke; Fujii-Watanabe, Yoshimi; Oda, Ken; Koike, Hideaki; Tamano, Koichi; Ishii, Tomoko; Sano, Motoaki; Machida, Masayuki; Abe, Keietsu

    2010-08-01

    Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

  3. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  4. Seed dormancy release in Arabidopsis Cvi by dry after-ripening, low temperature, nitrate and light shows common quantitative patterns of gene expression directed by environmentally specific sensing.

    Science.gov (United States)

    Finch-Savage, William E; Cadman, Cassandra S C; Toorop, Peter E; Lynn, James R; Hilhorst, Henk W M

    2007-07-01

    The depth of seed dormancy can be influenced by a number of different environmental signals, but whether a common mechanism underlies this apparently similar response has yet to be investigated. Full-genome microarrays were used for a global transcript analysis of Arabidopsis thaliana Cape Verde Island accession seeds exposed to dry after-ripening (AR), or low temperature, nitrate and light when imbibed. Germination studies showed that the sensitivity of imbibed seeds to low temperature, nitrate and light was dependent upon the length of time spent AR following harvest. Seeds had an absolute requirement for light to complete dormancy release in all conditions, but this effect required an exposure to a prior dormancy relieving environment. Principal component analyses of the expression patterns observed grouped physiological states in a way that related to the depth of seed dormancy, rather than the type of environmental exposure. Furthermore, opposite changes in transcript abundance of genes in sets associated with dormancy, or dormancy relief through AR, were also related to the depth of dormancy and common to different environments. Besides these common quantitative changes, environment-specific gene expression patterns during dormancy relief are also described. For example, higher transcript abundance for genes linked to the process of nitrate accumulation, and nitrate reduction was associated with dormancy relief. The quantity of GA3ox1 transcripts increased during dormancy relief in all conditions, in particular when dormancy relief was completed by exposure to light. This contrasts with transcripts linked to abscisic acid (ABA) synthesis, which declined. The results are consistent with a role for the ABA/gibberellic acid balance in integrating dormancy-relieving environmental signals.

  5. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  6. Environmental Bacteriophages of the Emerging Enterobacterial Phytopathogen, Dickeya solani, Show Genomic Conservation and Capacity for Horizontal Gene Transfer between Their Bacterial Hosts

    Directory of Open Access Journals (Sweden)

    Andrew Day

    2017-08-01

    Full Text Available Dickeya solani is an economically important phytopathogen widespread in mainland Europe that can reduce potato crop yields by 25%. There are no effective environmentally-acceptable chemical systems available for diseases caused by Dickeya. Bacteriophages have been suggested for use in biocontrol of this pathogen in the field, and limited field trials have been conducted. To date only a small number of bacteriophages capable of infecting D. solani have been isolated and characterized, and so there is a need to expand the repertoire of phages that may have potential utility in phage therapy strategies. Here we describe 67 bacteriophages from environmental sources, the majority of which are members of the viral family Myoviridae. Full genomic sequencing of two isolates revealed a high degree of DNA identity with D. solani bacteriophages isolated in Europe in the past 5 years, suggesting a wide ecological distribution of this phage family. Transduction experiments showed that the majority of the new environmental bacteriophages are capable of facilitating efficient horizontal gene transfer. The possible risk of unintentional transfer of virulence or antibiotic resistance genes between hosts susceptible to transducing phages cautions against their environmental use for biocontrol, until specific phages are fully tested for transduction capabilities.

  7. Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

    Energy Technology Data Exchange (ETDEWEB)

    Fields, C.A.

    1994-09-01

    This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

  8. Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

    Science.gov (United States)

    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.

  9. Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model.

    Science.gov (United States)

    Yang, Feng-Yi; Chang, Wen-Yuan; Lin, Wei-Ting; Hwang, Jeng-Jong; Chien, Yi-Chun; Wang, Hsin-Ell; Tsai, Min-Lan

    2015-11-03

    The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2'-fluoro-2'-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P therapy in brain diseases.

  10. A candidate gene study of serotonergic pathway genes and pain relief during treatment with escitalopram in patients with neuropathic pain shows significant association to serotonin receptor2C (HTR2C)

    DEFF Research Database (Denmark)

    Brasch-Andersen, Charlotte; Møller, Malik U; Christiansen, Lene

    2011-01-01

    the association between polymorphisms in genes involved in the serotonergic pathway and the effect of escitalopram on peripheral neuropathic pain. METHODS: We genotyped 34 participants from a placebo-controlled trial of escitalopram in peripheral neuropathic pain for polymorphisms in five genes: the serotonin.......047), with 75% carrying the C allele being responders. The same tendency was seen in women. Similarly, carriership of the C allele at rs6318 was associated with better pain relief during treatment with escitalopram [odds ratio (OR) 15.5, p = 0.014)] Furthermore, there was a tendency of better relief...... with increasing number of short alleles for the 5-HTTLPR polymorphism of the serotonin transporter (OR 5.7, p = 0.057). None of the other polymorphisms showed a significant association with treatment response to escitalopram. CONCLUSION: This study indicates that variation in the HTR2C gene is associated...

  11. Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight.

    Science.gov (United States)

    Flachowsky, Henryk; Szankowski, Iris; Fischer, Thilo C; Richter, Klaus; Peil, Andreas; Höfer, Monika; Dörschel, Claudia; Schmoock, Sylvia; Gau, Achim E; Halbwirth, Heidrun; Hanke, Magda-Viola

    2010-02-01

    Transgenic apple plants (Malus x domestica cv. 'Holsteiner Cox') overexpressing the Leaf Colour (Lc) gene from maize (Zea mays) exhibit strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins). Greenhouse plants investigated in this study exhibit altered phenotypes with regard to growth habit and resistance traits. Lc-transgenic plants show reduced size, transversal gravitropism of lateral shoots, reduced trichome development, and frequently reduced shoot diameter and abnormal leaf development with fused leaves. Such phenotypes seem to be in accordance with a direct or an indirect effect on polar-auxin-transport in the transgenic plants. Furthermore, leaves often develop necrotic lesions resembling hypersensitive response lesions. In tests, higher resistance against fire blight (caused by the bacterium Erwinia amylovora) and against scab (caused by the fungus Venturia inaequalis) is observed. These phenotypes are discussed with respect to the underlying altered physiology of the Lc-transgenic plants. The results are expected to be considered in apple breeding strategies.

  12. A novel lamin A/C gene mutation causing spinal muscular atrophy phenotype with cardiac involvement: report of one case.

    Science.gov (United States)

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Kawamata, Jun; Shimohama, Shun

    2015-02-20

    Mutations of the lamin A/C gene have been associated with several diseases such as Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy and Charcot-Marie-Tooth disease, referred to as laminopathies. Only one report of spinal muscular atrophy and cardiomyopathy phenotype with lamin A/C gene mutations has been published. The concept that lamin A/C gene mutations cause spinal muscular atrophy has not been established. We report a man aged 65 years who presented with amyotrophy of lower limbs, arrhythmia and cardiac hypofunction. He showed gait disturbance since childhood, and his family showed similar symptoms. Neurological and electrophysiological findings suggested spinal muscular atrophy type 3. Gene analysis of lamin A/C gene showed a novel nonsense mutation p.Q353X (c.1057C > T). Further investigations revealed that he and his family members had cardiac diseases including atrioventricular block. We report the first Japanese case of spinal muscular atrophy phenotype associated with lamin A/C mutation. When a patient presents a spinal muscular atrophy phenotype and unexplained cardiac disease, especially when the family history is positive, gene analysis of lamin A/C gene should be considered.

  13. Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

    Science.gov (United States)

    Ansari, Israr-Ul H; Longacre, Melissa J; Stoker, Scott W; Kendrick, Mindy A; O'Neill, Lucas M; Zitur, Laura J; Fernandez, Luis A; Ntambi, James M; MacDonald, Michael J

    2017-03-15

    Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Genes of ACYL CARRIER PROTEIN Family Show Different Expression Profiles and Overexpression of ACYL CARRIER PROTEIN 5 Modulates Fatty Acid Composition and Enhances Salt Stress Tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jiexue Huang

    2017-06-01

    Full Text Available Acyl carrier proteins (ACPs are a group of small acidic proteins functioning as important cofactors in the de novo synthesis of fatty acids. In Arabidopsis, ACPs are encoded by a small gene family comprising five plastid members, AtACP1 to AtACP5, and three mitochondrial members. The biological functions and the transcriptional responses to abiotic stresses of most AtACPs have yet to be elucidated. The present study extends previous findings and provides new knowledge on the function of ACPs by examining the responses of AtACP-encoding genes to several abiotic stresses and, in particular, the role of AtACP5 in the adaptation to salt stress. Phylogenetic analysis showed that AtACP1, AtACP2, AtACP3, and AtACP5 can be classified into one group and separated from a group comprising AtACP4 and ACP homologs from related species. Quantitative RT-PCR analysis revealed that the expression of AtACP1, AtACP2, and AtACP3 was induced by drought. Both iron deficiency and nitrogen starvation resulted in down-regulation of AtACP4. The most pronounced response was observed for AtACP5, the expression of which was dramatically decreased by salt stress. Knock-out of AtACP5 showed increased sensitivity to NaCl stress, whereas transgenic lines overexpressing AtACP5 displayed increased salt tolerance relative to the wild-type. Overexpression of AtACP5 further led to an altered composition of fatty acids, mainly a decrease of oleic acid (C18:1 and an increase of palmitic acid (C16:0, and to a lower Na+/K+ ratio when compared to the salt stressed wild-type. The comprehensive transcriptional information on the small plastid AtACP gene family in response to various abiotic stresses and the further investigation of the AtACP5 indicate that AtACP5 might be critical for salt tolerance through alterations of the composition of fatty acids and, subsequently, the Na+/K+ ratio.

  15. Preliminary Report of Molecular Detection of Retinoblastoma Gene Mutations

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    To develop gene diagnosis for retinoblastoma predisposition, it is necessary to disclose the retinoblastoma gene mutations or deletions in detail. Genomic DNA from tumor and peripheral white blood cells in 33 patients with retinoblastoma was detected with 3.8kb probe derived from 3' end of retinoblastoma gene cDNA. The gene abnormalities, including deletion, partial deletion and rearrangement, were found in 18 patients. Further research will be aimed at microdeletions or mutations for those patients wti...

  16. Analysis of cosmid clones of nuclear DNA from Trypanosome brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

    NARCIS (Netherlands)

    D. Valerio (Dinko); T. de Lange; P. Borst (Piet); F.G. Grosveld (Frank); L.H.T. van der Ploeg

    1982-01-01

    textabstractTrypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expres

  17. A module-based analytical strategy to identify novel disease-associated genes shows an inhibitory role for interleukin 7 Receptor in allergic inflammation

    Directory of Open Access Journals (Sweden)

    Perkins Andy D

    2009-02-01

    Full Text Available Abstract Background The identification of novel genes by high-throughput studies of complex diseases is complicated by the large number of potential genes. However, since disease-associated genes tend to interact, one solution is to arrange them in modules based on co-expression data and known gene interactions. The hypothesis of this study was that such a module could be a found and validated in allergic disease and b used to find and validate one ore more novel disease-associated genes. Results To test these hypotheses integrated analysis of a large number of gene expression microarray experiments from different forms of allergy was performed. This led to the identification of an experimentally validated reference gene that was used to construct a module of co-expressed and interacting genes. This module was validated in an independent material, by replicating the expression changes in allergen-challenged CD4+ cells. Moreover, the changes were reversed following treatment with corticosteroids. The module contained several novel disease-associated genes, of which the one with the highest number of interactions with known disease genes, IL7R, was selected for further validation. The expression levels of IL7R in allergen challenged CD4+ cells decreased following challenge but increased after treatment. This suggested an inhibitory role, which was confirmed by functional studies. Conclusion We propose that a module-based analytical strategy is generally applicable to find novel genes in complex diseases.

  18. A module-based analytical strategy to identify novel disease-associated genes shows an inhibitory role for interleukin 7 Receptor in allergic inflammation.

    Science.gov (United States)

    Mobini, Reza; Andersson, Bengt A; Erjefält, Jonas; Hahn-Zoric, Mirjana; Langston, Michael A; Perkins, Andy D; Cardell, Lars Olaf; Benson, Mikael

    2009-02-12

    The identification of novel genes by high-throughput studies of complex diseases is complicated by the large number of potential genes. However, since disease-associated genes tend to interact, one solution is to arrange them in modules based on co-expression data and known gene interactions. The hypothesis of this study was that such a module could be a) found and validated in allergic disease and b) used to find and validate one ore more novel disease-associated genes. To test these hypotheses integrated analysis of a large number of gene expression microarray experiments from different forms of allergy was performed. This led to the identification of an experimentally validated reference gene that was used to construct a module of co-expressed and interacting genes. This module was validated in an independent material, by replicating the expression changes in allergen-challenged CD4+ cells. Moreover, the changes were reversed following treatment with corticosteroids. The module contained several novel disease-associated genes, of which the one with the highest number of interactions with known disease genes, IL7R, was selected for further validation. The expression levels of IL7R in allergen challenged CD4+ cells decreased following challenge but increased after treatment. This suggested an inhibitory role, which was confirmed by functional studies. We propose that a module-based analytical strategy is generally applicable to find novel genes in complex diseases.

  19. Analysis of S gene mutation of the hepatitis B virus in adult liver transplant recipients showing resistance to hepatitis B immunoglobulin therapy.

    Science.gov (United States)

    Park, G-C; Hwang, S; Ahn, C-S; Kim, K-H; Moon, D-B; Ha, T-Y; Song, G-W; Jung, D-H; Shin, Y W; Kim, S-H; Chang, K-H; Namgoong, J-M; Park, C-S; Park, H-W; Park, Y-H; Kang, S-H; Jung, B-H; Lee, S-G

    2013-10-01

    A considerable proportion of recipients of liver transplantations who are presented hepatitis B immunoglobulin (HBIG) monotherapy for hepatitis B virus (HBV) prophylaxis develop HBIG resistance. In this study, we investigated the mutation patterns in the major hydrophilic region (MHR) of amino acid sequences 100 to 160. Using the gene sequence analyzer for amino acid sequences 0 to 226 in the S/pre-S region we analyzed blood samples of 15 patients showing HBIG resistance after high-dose HBIG prophylaxis. Various mutations in the MHR were observed in 14/15 samples: Gly145Arg mutation in 8/13 Adr subtype and 1/2 Ayw subtype samples (60%). The next most common mutation was Gly165Trp in 8/13 Adr subtype but neither of 2 Ayw subtype samples (53.3%). Concurrent antiviral resistance was noted in 5 patients: lamivudine (n = 5), or entecavir (n = 3), but not adefovir, suggesting the occurrence of simultaneous, antiviral cross-resistances. Two patients underwent retransplantation due to the progression of HBV infection despite vigorous antiviral therapy. At diagnosis of HBV recurrence, the mean HBV DNA load was 6.5 × 10(6) copies/mL; 4 patients showed paradoxical coexistence of anti-HBs and HBsAg. Currently, 2 subjects show low-level HBV DNA replication in peripheral blood, although the other 12 had no DNA replication after prolonged antiviral therapy. This study suggested that various mutations in the "a" determinant were associated with HBIG resistance. Since treatment failure to rescue antiviral therapy was often associated with delayed detection of HBV recurrence rather than concurrent antiviral resistance, frequent HBV surveillance using more sensitive screening tests, such as HBeAg and HBV DNA polymerase chain reaction assay, seems to be mandatory. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Analysis of gene expression pattern and neuroanatomical correlates for SLC20A2 (PiT-2) shows a molecular network with potential impact in idiopathic basal ganglia calcification ("Fahr's disease").

    Science.gov (United States)

    da Silva, R J Galdino; Pereira, I C L; Oliveira, J R M

    2013-06-01

    Familial idiopathic basal ganglia calcification (FIBGC), also known as "Fahr's disease," is a neuropsychiatric disorder with motor and cognitive symptoms. It is characterized pathologically by bilateral calcification most commonly in the basal ganglia and also in other brain regions such as the thalamus and cerebellum. A recent report by Wang et al. (2012) discovered multiple families with FIBGC carrying mutations in the SLC20A2 gene, encoding the inorganic phosphate transporter PiT-2, which segregated in an autosomal dominant pattern. To understand further the role of SLC20A2 in FIBGC brain pathology, here we described the gene expression pattern across the whole brain for SLC20A2, using the Allen Institute Human Brain Atlas database. Microarray analysis provided evidence that the neuroanatomical pattern of expression for SLC20A2 is highest in the regions most commonly affected in FIBGC. Neuroanatomical regions that demonstrated high correlation or anti-correlation with SLC20A2 expression also showed a molecular network with potential to explain the limited neuroanatomical distribution of calcifications in IBGC. Lastly, these co-expression networks suggest additional further candidate genes for FIBGC.

  1. Genomic organization of the CC chemokine mip-3alpha/CCL20/larc/exodus/SCYA20, showing gene structure, splice variants, and chromosome localization.

    Science.gov (United States)

    Nelson, R T; Boyd, J; Gladue, R P; Paradis, T; Thomas, R; Cunningham, A C; Lira, P; Brissette, W H; Hayes, L; Hames, L M; Neote, K S; McColl, S R

    2001-04-01

    We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.

  2. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  3. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  4. Diagnostic accuracy studies of fine-needle aspiration show wide variation in reporting of study population characteristics: implications for external validity.

    Science.gov (United States)

    Schmidt, Robert L; Narra, Krishna K; Witt, Benjamin L; Factor, Rachel E

    2014-01-01

    Study comparisons rest on the assessment of applicability or external validity. Population characteristics are an important component of external validity and, although there has been a heightened awareness of deficiencies in reporting in diagnostic test accuracy (DTA) studies, the reporting of populations in DTA studies has not been investigated. To assess the quality of reporting of population descriptions in DTA studies for fine-needle aspiration cytology (FNAC). Literature survey of common population parameters and usage patterns in FNAC DTA studies. We randomly selected 20 FNAC DTA studies in 4 categories (salivary glands, lung, thyroid, and pancreas) and determined the frequency of parameter usage. Studies showed considerable variability in reporting patterns. On average, studies reported 2 to 4 parameters to describe study populations. Age, sex, and lesion size were most frequently reported. Sixteen percent of studies did not provide any population description. Population parameters were used to describe the sample population more frequently than to describe the selection process (P = .001). There were significant differences in the number of parameters specified by anatomic site (P = .001). Only 21% of studies provided a flow diagram. Thirty-three percent of studies mentioned the target population. Studies show considerable variability in the description of sample populations and the population selection process. Studies often fail to provide flow diagrams or to provide a clear statement of the research problem. There is considerable opportunity for studies to improve both descriptions of sample populations and the process used to select them.

  5. Deletion of a single-copy DAAM1 gene in congenital heart defect: a case report

    Directory of Open Access Journals (Sweden)

    Bao Bihui

    2012-08-01

    Full Text Available Abstract Background With an increasing incidence of congenital heart defects (CHDs in recent years, genotype-phenotype correlation and array-based methods have contributed to the genome-wide analysis and understanding of genetic variations in the CHD population. Here, we report a copy number deletion of chromosomal 14q23.1 in a female fetus with complex congenital heart defects. This is the first description of DAAM1 gene deletion associated with congenital heart anomalies. Case Presentation Compared with the control population, one CHD fetus showed a unique copy number deletion of 14q23.1, a region that harbored DAAM1 and KIAA0666 genes. Conclusions Results suggest that the copy number deletion on chromosome 14q23.1 may be critical for cardiogenesis. However, the exact relationship and mechanism of how DAAM1 and KIAA0666 deletion contributes to the onset of CHD is yet to be determined.

  6. The tyrosinase-positive oculocutaneous albinism gene shows locus homogeneity on chromosome 15q11-q13 and evidence of multiple mutations in southern African negroids

    Energy Technology Data Exchange (ETDEWEB)

    Kedda, M.A.; Stevens, G.; Manga, P.; Viljoen, C.; Jenkins, T.; Ramsay, M. (South African Institute for Medical Research, Johannesburg (South Africa) Univ. of Witwatersrand, Johannesburg (South Africa))

    1994-06-01

    Tyrosinase-positive oculocutaneous albinism (ty-pos OCA) is an autosomal recessive disorder of the melanin pigmentary system. South African ty-pos OCA individuals occur with two distinct phenotypes, with or without darkly pigmented patches (ephelides, or dendritic freckles) on exposed areas of the skin. These phenotypes are concordant within families, suggesting that there may be more than one mutation at the ty-pos OCA locus. Linkage studies carried out in 41 families have shown linkage between markers in the Prader-Willi/Angelman syndrome (PWS/AS) region on chromosome 15q11-q13 and ty-pos OCA. Analysis showed no obligatory crossovers between the alleles at the D15S12 locus and ty-pos OCA, suggesting that the D15S12 locus is very close to or part of the disease locus, which is postulated to be the human homologue, P, of the mouse pink-eyed dilution gene, p. Unlike caucasoid [open quotes]ty-pos OCA[close quotes] individuals, negroid ty-pos OCA individuals do not show any evidence of locus heterogeneity. Studies of allelic association between the polymorphic alleles detected at the D15S12 locus and ephelus status suggest that there was a single major mutation giving rise to ty-pos OCA without ephelides. There may, however, be two major mutations causing ty-pos OCA with ephelides, one associated with D15S12 allele 1 and the other associated with D15S12 allele 2. The two loci, GABRA5 and D15S24, flanking D15S12, are both hypervariable, and many different haplotypes were observed with the alleles at the three loci on both ty-pos OCA-associated chromosomes and [open quotes]normal[close quotes] chromosomes. No haplotype showed statistically significant association with ty-pos OCA, and thus none could be used to predict the origins of the ty-pos OCA mutations. On the basis of the D15S12 results, there is evidence for multiple ty-pos OCA mutations in southern African negroids. 31 refs., 1 fig., 3 tabs.

  7. Transgenic barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil.

    Science.gov (United States)

    Delhaize, Emmanuel; Taylor, Phillip; Hocking, Peter J; Simpson, Richard J; Ryan, Peter R; Richardson, Alan E

    2009-06-01

    Barley (Hordeum vulgare L.), genetically modified with the Al(3+) resistance gene of wheat (TaALMT1), was compared with a non-transformed sibling line when grown on an acidic and highly phosphate-fixing ferrosol supplied with a range of phosphorus concentrations. In short-term pot trials (26 days), transgenic barley expressing TaALMT1 (GP-ALMT1) was more efficient than a non-transformed sibling line (GP) at taking up phosphorus on acid soil, but the genotypes did not differ when the soil was limed. Differences in phosphorus uptake efficiency on acid soil could be attributed not only to the differential effects of aluminium toxicity on root growth between the genotypes, but also to differences in phosphorus uptake per unit root length. Although GP-ALMT1 out-performed GP on acid soil, it was still not as efficient at taking up phosphorus as plants grown on limed soil. GP-ALMT1 plants grown in acid soil possessed substantially smaller rhizosheaths than those grown in limed soil, suggesting that root hairs were shorter. This is a probable reason for the lower phosphorus uptake efficiency. When grown to maturity in large pots, GP-ALMT1 plants produced more than twice the grain as GP plants grown on acid soil and 80% of the grain produced by limed controls. Expression of TaALMT1 in barley was not associated with a penalty in either total shoot or grain production in the absence of Al(3+), with both genotypes showing equivalent yields in limed soil. These findings demonstrate that an important crop species can be genetically engineered to successfully increase grain production on an acid soil.

  8. Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Shaffer Benjamin

    2008-06-01

    Full Text Available Abstract Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12 p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using

  9. MNSs genotyping by MALDI-TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles.

    Science.gov (United States)

    Meyer, Stefan; Vollmert, Caren; Trost, Nadine; Sigurdardottir, Sonja; Portmann, Claudia; Gottschalk, Jochen; Ries, Judith; Markovic, Alexander; Infanti, Laura; Buser, Andreas; Amar El Dusouqui, Soraya; Rigal, Emmanuel; Castelli, Damiano; Weingand, Bettina; Maier, Andreas; Mauvais, Simon M; Sarraj, Amira; Braisch, Monica C; Thierbach, Jutta; Hustinx, Hein; Frey, Beat M; Gassner, Christoph

    2016-08-01

    Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs. © 2016 John Wiley & Sons Ltd.

  10. Inflammatory bowel disease gene discovery. CRADA final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-09-09

    The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.

  11. [Intraductal Papillary Mucinous Neoplasm (IPMN) of the Pancreas Showing High Accumulation of 18F-Fluorodeoxyglucose (FDG)--A Case Report].

    Science.gov (United States)

    Kubota, Masaru; Nishimura, Masashige; Nakatsuka, Rie; Miyazaki, Susumu; Danno, Katsuki; Motoori, Masaaki; Matsuda, Chu; Fujitani, Kazumasa; Iwase, Kazuhiro

    2015-11-01

    The diagnosis of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas includes radiographic (CT, MRI) and endoscopic evaluation (ERCP, EUS). The treatment strategy is outlined in the 2012 International Consensus Guidelines (ICG). Herein, we report a case initially not indicated for surgery. Four months after the initial diagnosis, the cystic lesion transformed into a solid mass-like lesion visible on CT. FDG-PET showed abnormal FDG uptake at the same location. Surgical resection was performed immediately, and the tumor was diagnosed as IPMN with inflammation. FDG-PET showed a false-positive diagnosis for the malignancy in this case of IPMN.

  12. Recombinant hybrid infectious hematopoietic necrosis virus (IHNV) carrying viral haemorrhagic septicaemia virus (VHSV) G or NV genes show different virulence properities

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Biacchesi, S.; Stegmann, Anders

    Viral haemorrhagic septicaemia virus (VHSV) is the economically most important viral disease in European rainbow trout farming. The virus was introduced to fresh water farms in the 1950ies from a reservoir of VHSV in the marine environment. Isolates from wild marine fish and fresh water farms....... By a reverse genetics approach using the related novirrhabdovirus infectious hematopoietic necrosis virus (IHNV) as basis, four hybrid IHNV-VHSV variants were generated. These chimeric variants included substitution of the IHNV glyco(G) or nonstrutrual (Nv) protein with the corresponding G or Nv-protein from...... or fresh water VHSV. Recombinant IHNV gained higher virulence following substitution of the homologous G gene with the VHSV G gene, while the opposite was the case following substitution of the Nv gene. These findings suggest that higher virulence of VHSV compared to IHNV might be related to the G protein...

  13. Gene expression profiles of vascular smooth muscle show differential expression of mitogen-activated protein kinase pathways during captopril therapy of heart failure.

    Science.gov (United States)

    Chen, Frank C; Brozovich, Frank V

    2008-01-01

    Congestive heart failure (CHF) is characterized by increased vascular tone and an impairment in nitric-oxide-mediated vasodilatation. We have demonstrated that the blunted response to nitric oxide is due, in part, to a reduction in the leucine-zipper-positive isoform of the myosin-targeting subunit (MYPT1) of myosin light-chain phosphatase. Additionally, we have shown that angiotensin-converting enzyme inhibition, but not afterload reduction with prazosin, preserves leucine-zipper-positive MYPT1 isoform expression in vascular smooth muscle cells and normalizes the sensitivity to cGMP-mediated vasodilatation. We therefore hypothesized that in CHF, growth regulators and cytokines downstream of the angiotensin II receptor are involved in modulating gene expression in vascular tissue. Rats were divided into control and captopril-treated groups following left coronary artery ligation. Gene expression profiles in the aorta and portal vein at baseline and 2 and 4 weeks after myocardial infarction (MI) were analyzed using microarray technology and quantitative real-time PCR. After MI, microarray analysis revealed differential mRNA expression of 21 genes in the aorta of captopril-treated rats 2 and 4 weeks after surgery when compared to gene expression profiles at baseline and without captopril therapy. Real-time PCR demonstrated that captopril suppressed the expression of protein kinases in the angiotensin-II-mediated mitogen-activated protein kinase signaling pathway, including Taok1 and Raf1. These data suggest that in CHF, captopril therapy modulates gene expression in vascular smooth muscle, and some of the beneficial effects of ACE inhibition may be due to differential gene expression in the vasculature.

  14. Molecular and Genetic Characterization of HIV-1 Tat Exon-1 Gene from Cameroon Shows Conserved Tat HLA-Binding Epitopes: Functional Implications

    Science.gov (United States)

    Teto, Georges; Fonsah, Julius Y.; Tagny, Claude T.; Mbanya, Dora; Nchindap, Emilienne; Kenmogne, Leopoldine; Fokam, Joseph; Njamnshi, Dora M.; Kouanfack, Charles; Njamnshi, Alfred K.; Kanmogne, Georgette D.

    2016-01-01

    HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA. PMID:27438849

  15. Giant Cell Tumor with Secondary Aneurysmal Bone Cyst Shows Heterogeneous Metabolic Pattern on (18)F-FDG PET/CT: A Case Report.

    Science.gov (United States)

    Park, Hee Jeong; Kwon, Seong Young; Cho, Sang-Geon; Kim, Jahae; Song, Ho-Chun; Kim, Sung Sun; Yoon, Yeon Hong; Park, Jin Gyoon

    2016-12-01

    Giant cell tumor (GCT) is a generally benign bone tumor accounting for approximately 5 % of all primary bone neoplasms. Cystic components in GCTs that indicate secondary aneurysmal bone cysts (ABCs) are reported in 14 % of GCTs. Although both of them have been described separately in previous reports that may show considerable fluorodeoxyglucose (FDG) uptake despite their benign nature, the findings of GCT with secondary ABC on (18)F-FDG positron emission tomography/computed tomography (PET/CT) have not been well-known. We report a case of GCT with secondary ABC in a 26-year-old woman. (18)F-FDG PET/CT revealed a heterogeneous hypermetabolic lesion in the left proximal femur with the maximum standardized uptake value of 4.7. The solid components of the tumor showed higher FDG uptake than the cystic components. These observations suggest that the ABC components in GCTs show heterogeneous metabolic patterns on (18)F-FDG PET/CT.

  16. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    NARCIS (Netherlands)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was

  17. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    NARCIS (Netherlands)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was

  18. The plant mitochondrial mat-r gene/nad1 gene complex. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Wolstenholme, D.R.

    1994-06-01

    The authors have completed sequencing the segments (totalling 19 kb, both complementary strands) of the maize mtDNA molecule that encode the entire NADH dehydrogenase subunit (nadl) gene. They have identified nucleotides in mature transcripts of the nadl gene that are edited and have generated clones of cDNAs of entire mature (fully spliced) nadl transcripts. They have examined the relative rates of splicing in transcripts of the four nadl gene group II introns and begun examining nadl intron cDNAs to determine the extent and distribution of RNA edits in introns, in order to evaluate the possibility that intron excision and exon splicing might be editing independent.

  19. Construction and preliminary evaluation of an Aspergillus flavus reporter gene construct as a potential tool for screening aflatoxin resistance.

    Science.gov (United States)

    Brown, Robert L; Brown-Jenco, Carmen S; Bhatnagar, Deepak; Payne, Gary A

    2003-10-01

    Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available. The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed. A reporter construct containing the Aspergillus flavus beta-tubulin gene promoter fused to Escherichia coli beta-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels. Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture. In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction. Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain. Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation.

  20. Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: LXL730724@hotmail.com; Liu Ying; He Yong; Wu Tao; Zhang Binqing; Gao Zairong; An Rui [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: zhyx1229@163.com

    2010-04-15

    Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with {sup 131}I-2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil ({sup 131}I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models. Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with {sup 131}I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1x10{sup 8} pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5x10{sup 8}, 1x10{sup 8}, 5x10{sup 7} and 1x10{sup 7} pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models. Results: From the ARG images, rats injected with Ad5-tk showed significant {sup 131}I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of {sup 131}I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5x10{sup 7} pfu, and the optimal Ad titer was 1x10{sup 8} pfu

  1. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    OpenAIRE

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Ameri...

  2. Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

    Directory of Open Access Journals (Sweden)

    Muh. Alias L. Rajamuddin

    2016-02-01

    Full Text Available Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP, caulifl ower mosaic virus (pCaMV-GFP,medaka β-actin (pmBA-GFP and Japanese fl ounder keratin (pJfKer-GFP promoters were introduced by electroporationmethod. Electroporation was performed using a gene pulser (BIORAD with voltage of 300 V, pulse length of 0.5 ms,pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expressionlevel using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus(34.10%±1.49 expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activitywith CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25. mBA promoter drove GFP expressionat medium level and similar number of fi lament callus (8.85%±2.31 expressing GFP with CaMV, while JfKer promoterhad lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26 and GFP expression level. PCRanalysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressingfi lament callus in the same size (about 0.6 kb with positive control of plasmid. Thus, CMV and CaMV promoters wasan appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combiningthis achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K.alvarezii can be feasible.

  3. Huntington’s disease blood and brain show a common gene expression pattern and share an immune signature with Alzheimer’s disease

    Science.gov (United States)

    Hensman Moss, Davina J.; Flower, Michael D.; Lo, Kitty K.; Miller, James R. C.; van Ommen, Gert-Jan B.; ’t Hoen, Peter A. C.; Stone, Timothy C.; Guinee, Amelia; Langbehn, Douglas R.; Jones, Lesley; Plagnol, Vincent; van Roon-Mom, Willeke M. C.; Holmans, Peter; Tabrizi, Sarah J.

    2017-01-01

    There is widespread transcriptional dysregulation in Huntington’s disease (HD) brain, but analysis is inevitably limited by advanced disease and postmortem changes. However, mutant HTT is ubiquitously expressed and acts systemically, meaning blood, which is readily available and contains cells that are dysfunctional in HD, could act as a surrogate for brain tissue. We conducted an RNA-Seq transcriptomic analysis using whole blood from two HD cohorts, and performed gene set enrichment analysis using public databases and weighted correlation network analysis modules from HD and control brain datasets. We identified dysregulated gene sets in blood that replicated in the independent cohorts, correlated with disease severity, corresponded to the most significantly dysregulated modules in the HD caudate, the most prominently affected brain region, and significantly overlapped with the transcriptional signature of HD myeloid cells. High-throughput sequencing technologies and use of gene sets likely surmounted the limitations of previously inconsistent HD blood expression studies. Our results suggest transcription is disrupted in peripheral cells in HD through mechanisms that parallel those in brain. Immune upregulation in HD overlapped with Alzheimer’s disease, suggesting a common pathogenic mechanism involving macrophage phagocytosis and microglial synaptic pruning, and raises the potential for shared therapeutic approaches. PMID:28322270

  4. Comparison of in vitro hormone activities of selected phthalates using reporter gene assays.

    Science.gov (United States)

    Shen, Ouxi; Du, Guizhen; Sun, Hong; Wu, Wei; Jiang, Yi; Song, Ling; Wang, Xinru

    2009-12-01

    Phthalates are widely used in the plastic industry and food packaging, imparting softness and flexibility to normally rigid plastic medical devices and children's toys. Even though phthalates display low general toxicity, there is increasing concern on the effects of endocrine system induced by some of phthalate compounds. The hormone activity of dibutyl phthalate (DBP), mono-n-butyl phthalate (MBP) and di-2-ethylhexyl phthalate (DEHP) were assessed using the luciferase reporter gene assays. The results showed that DBP, MBP and DEHP, not only exhibited potent antiandrogenic activity, with IC(50) value of 1.05x10(-6), 1.22x10(-7)M and exceeding 1x10(-4)M respectively, but also showed the androgenic activity with EC(50) value of 6.17x10(-6), 1.13x10(-5)M and exceeding 1x10(-4)M. We also found that all the three related chemicals possessed thyroid receptor (TR) antagonist activity with IC(50) of 1.31x10(-5), 2.77x10(-6)M and exceeding 1x10(-4)M respectively, and none showed TR agonist activity. These results indicate that TR might be the targets of industrial chemicals. In the ER mediate reporter gene assay, three chemicals showed no agonistic activity except for DBP, which appeared weakly estrogenic at the concentration of 1.0x10(-4)M. Together, the findings demonstrate that the three phthalates could simultaneously disrupt the function of two or more hormonal receptors. Therefore, these phthalates should be considered in risk assessments for human health.

  5. Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

    OpenAIRE

    Muh. Alias L. Rajamuddin; Alimuddin A; Utut Widyastuti; Irvan Faizal

    2016-01-01

    Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select asuitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Greenfl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycusalvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP),medaka β-actin (pmBA-GFP) and Japanese fl...

  6. In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, C.-H. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Liu, R.-S. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); National Yang-Ming University Medical School and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Wang, H.-E. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Hwang, J.-J. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Deng, W.-P. [Institute of Biomedical Material, Taipei Medical University, Taipei 112, Taiwan (China); Chen, J.-C. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Chen, F.-D. [Department of Medical Radiation Technology and Institute of Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China) and Institute of Radiological Sciences, Central Taiwan University of Science and Technology Taichung 112, Taiwan (China)]. E-mail: d49220009@ym.edu.tw

    2006-07-15

    The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [{sup 131}I] FIAU and [{sup 3}H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by

  7. Increase of Expression Levels of Reporter Gene in Transgenic Tobaccos by Matrix Attachment Regions

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The matrix attachment region (MAR) located downstream of Plastocyanin gene was isolated from the genome of pea. To study the effect of MARs on foreign gene expression in transgenic plants, T-DNA vector was constructed in which MARs flanked bothβ-glucuronidase(GUS) gene and selectable marker neomycin phosphotransferase (NPT-II) gene. The plant expression vectors were transferred into leaf discs via Agrobacterium-mediated transformation procedure. The result of GUS measurement showed that pea MAR could increase transgene expression level. The mean expression levels of GUS gene expression in population containing MARs could be increased twofold when compared with that of population without MARs.

  8. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity.

    Science.gov (United States)

    Legler, Juliette; Zeinstra, Laura M; Schuitemaker, Femke; Lanser, Peter H; Bogerd, Jan; Brouwer, Abraham; Vethaak, A Dick; De Voogt, Pim; Murk, Albertinka J; Van der Burg, Bart

    2002-10-15

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.

  9. Expression patterns of developmental regulatory genes show comparable divisions in the telencephalon of Xenopus and mouse: insights into the evolution of the forebrain.

    Science.gov (United States)

    Medina, Loreta; Brox, Aurora; Legaz, Isabel; García-López, Margarita; Puelles, Luis

    2005-09-15

    In this study, we review data on the existence of comparable divisions and subdivisions in the telencephalon of different groups of tetrapods based on expression of some developmental regulatory genes, having a particular focus in the comparison of the anuran amphibian Xenopus and the mouse. The available data on Xenopus, mouse, chick and turtle indicate that apparently all tetrapod groups possess the same molecularly distinct divisions and subdivisions in the telencephalon. This basic organization was likely present in the telencephalon of stem tetrapods. Each division/subdivision is characterized by expression of a unique combination of developmental regulatory genes, and appears to represent a self-regulated and topologically constant histogenetic brain compartment that gives rise to specific groups of cells. This interpretation has an important consequence for searching homologies, since a basic condition for cell groups in different vertebrates to be considered homologous is that they originate in the same compartment. However, evolution may allow individual cell groups derived from comparable (field homologous) subdivisions to be either similar or dissimilar across the vertebrate groups, giving rise to several possible scenarios of evolution, which include both the evolutionary conservation of similar (homologous) cells or the production of novel cell groups. Finally, available data in the lamprey, a jawless fish, suggest that not all telencephalic subdivisions were present at the origin of vertebrates, raising important questions about their evolution.

  10. Carcinoma showing thymus-like elements of the thyroid gland: report of three cases including one case with breast cancer history.

    Science.gov (United States)

    Zhang, Guanjun; Liu, Xi; Huang, Wei; Li, Xiaofeng; Johnstone, Marianne; Deng, Yuan; Ke, Yongqiang; Nunes, Quentin M; Wang, Hongyan; Wang, Yili; Zhang, Xuebin

    2015-01-01

    Carcinoma showing thymus-like elements (CASTLE) is a rare malignant tumor of the thyroid or adjacent neck soft tissues, whose histogenesis is still debated. It may resemble other primary or metastatic poorly differentiated tumors histologically and the differential diagnosis is crucial for CASTLE has a better prognosis. However, CASTLE as a second primary tumor has not been reported in the literature. We report three cases of thyroid CASTLE, including a unique tumor following breast-conserving surgery for early-stage breast invasive carcinoma. There were two female and one male. All three tumors were located in the right lobe of the thyroid, and one tumor showed extension into the surrounding soft tissue. Histologically, all tumors showed expansive growth and consisted of cords, nests or sheets of epithelial cells divided into irregularly shaped lobules by fibrous connective tissue with lymphoplasmacytic infiltration. Focal squamous differentiation resembling Hassall's corpuscles were observed. All cases stained positively for CD5, CD117, high molecular weight cytokeratin, cytokeratin, P63, carcinoembryonic antigen and epithelial membrane antigen. Positive staining for Bcl-2 in two cases and chromogranin A in one case was noted. Ki-67 expression ranged from 15 to 25%. Thyroid transcription factor and CD3 were negative. There was no evidence of recurrent or metastatic disease at following surgery. These features demonstrated CASTLE may arise from branchial pouch remnants, the thyroid solid cell nests. CASTLE is a rare entity, awareness of its occurrence as a second primary tumor is important to avoid overtreatment because it is associated with a favorable prognosis.

  11. Genetic analysis of the regulation of TCH gene expression, Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Braam, Janet

    2008-10-28

    investigation of CML24 function and regulation led to the finding that CML24 has a critical role in nitric oxide regulation. Distinct tilling mutant alleles demonstrated that CML24 can act as a switch in the response to day length perception. Because of potential redundancy with the related CML23 gene, CML23 T-DNA insertion mutants were identified and characterized. Together, CML23 and CML24 impact the autonomous regulatory pathway of the transition to flowering. Nitric oxide levels are elevated in cml23/cml24 double mutants. Therefore, CML23 and CML24 are potential calcium sensors regulate nitric oxide accumulation. In collaboration with Drs. McCann and Carpita, fourier transform infrared spectroscopy (FTIR) was used to assess, verify and classify wall architectural changes that occur as a result of single XTH insertion mutations. Thirty-four homozygous mutant lines of Arabidopsis representing 21 members of the xyloglucan endotransglucosylase/hydrolase gene family provided a set of mutants to characterize. Kohonen networks classified cell wall architectures of xth mutant lines and previously characterized cell wall mutants. The xth mutants were found to have chemical changes in their cell walls not detectable as phenotypic growth and development changes, consistent with the existence of feed-back loops that modify wall composition in response to a life-long deficiency of a cell wall enzyme. To gain insight into the potential physiological relevance of the distinct members of the XTH family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data revealed that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs showed XTH::GUS expression and most, if not all, are found to express

  12. Allelic variants at codon 146 in the PRNP gene show significant differences in the risk for natural scrapie in Cypriot goats.

    Science.gov (United States)

    Ortiz-Pelaez, A; Georgiadou, S; Simmons, M M; Windl, O; Dawson, M; Arnold, M E; Neocleous, P; Papasavva-Stylianou, P

    2015-04-01

    Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

  13. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    Science.gov (United States)

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  14. Genetic variation and recombination of RdRp and HSP 70h genes of Citrus tristeza virus isolates from orange trees showing symptoms of citrus sudden death disease

    Directory of Open Access Journals (Sweden)

    Pappas Georgios J

    2008-01-01

    Full Text Available Abstract Background Citrus sudden death (CSD, a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the disease's distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV. CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms. Results A total of 286 RNA-dependent-RNA polymerase (RdRp and 284 heat shock protein 70 homolog (HSP70h gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD. Conclusion We suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence of a single CTV variant within these populations that triggers the disease.

  15. The soybean mycorrhiza-inducible phosphate transporter gene, GmPT7, also shows localized expression at the tips of vein endings of senescent leaves.

    Science.gov (United States)

    Inoue, Yuki; Kobae, Yoshihiro; Omoto, Eiji; Tanaka, Aiko; Banba, Mari; Takai, Shoko; Tamura, Yosuke; Hirose, Aya; Komatsu, Kunihiko; Otagaki, Shungo; Matsumoto, Shogo; Taniguchi, Mitsutaka; Masuta, Chikara; Ishimoto, Masao; Hata, Shingo

    2014-12-01

    GmPT7 was originally identified as an arbuscular mycorrhiza-inducible gene of soybean that encodes a member of subfamily I in the PHOSPHATE TRANSPORTER 1 family. In the present study, we established conditions under which a number of dwarf soybean plants complete their life cycles in a growth chamber. Using this system, we grew transgenic soybean with a GmPT7 promoter-β-glucuronidase fusion gene and evaluated GmPT7 expression in detail. GmPT7 was highly expressed in mature, but not in collapsed, arbuscule-containing cortical cells, suggesting its importance in the absorption of fungus-derived phosphate and/or arbuscule development. GmPT7 was also expressed in the columella cells of root caps and in the lateral root primordia of non-mycorrhizal roots. The expression of GmPT7 occurred only in the late stage of phosphorus translocation from leaves to seeds, after water evaporation from the leaves ceased, and later than the expression of GmUPS1-2, GmNRT1.7a and GmNRT1.7b, which are possibly involved in nitrogen export. GmPT7 expression was localized in a pair of tracheid elements at the tips of vein endings of senescent leaves. Transmission electron microscopy revealed that the tip tracheid elements in yellow leaves were still viable and had intact plasma membranes. Thus, we think that GmPT7 on the plasma membranes transports phosphate from the apoplast into the tip elements. GmPT7 knockdown resulted in no significant effects, the function of GmPT7 remaining to be clarified. We propose a working model in which phosphate incorporated in vein endings moves to seeds via xylem to phloem transfer.

  16. Novel Pd(II)-salen complexes showing high in vitro anti-proliferative effects against human hepatoma cancer by modulating specific regulatory genes

    NARCIS (Netherlands)

    Azam, M.; Hussain, Z.; Warad, I.; Al-Resayes, S.I.; Khan, M.S.; Shakir, M.; Trzesowska-Kruszynska, A.; Kruszynski, R.

    2012-01-01

    We have reported the synthesis of a novel salen ligand and its mononuclear Pd-salen complexes derived from 2-{[2-hydroxy-3-{[(E)-(2-hydroxyphenyl)methylidene]amino}propyl)imino]methyl}phen ol. The newly synthesized and isolated Pd(II) complexes have been identified and fully characterized by various

  17. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    NARCIS (Netherlands)

    Yang, X.; Kraan, P.M. van der; Dolder, J. van den; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2007-01-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and

  18. Undifferentiated (Anaplastic Carcinoma of the Pancreas with Osteoclast-Like Giant Cells Showing Various Degree of Pancreas Duct Involvement. A Case Report and Literature Review

    Directory of Open Access Journals (Sweden)

    Vlad Maksymov

    2011-03-01

    Full Text Available Context Undifferentiated (anaplastic carcinoma of the pancreas with osteoclast-like giant cells is exceedingly rare. The prognosis of undifferentiated carcinoma is worse than that of poorly differentiated ductal adenocarcinoma of the pancreas; however, undifferentiated carcinoma with osteoclast-like giant cells might have a more favorable prognosis. Case report We report the case of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells, showing an intraductal growth pattern with various degree of pancreas duct involvement in the different areas. As a result, we were able to demonstrate the entire spectrum of changes, ranging from the early, minimal intraluminal growth to the partial or complete occlusion of the branches of the main pancreatic duct, and finally invasion and formation of the large necrotic/degenerated cysts. Conclusions Our findings support the epithelial origin of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In early stages, the affected pancreatic duct epithelium was intermingled with nonepithelial component and had an immunoprofile distinctive from the epithelial lining of the uninvolved (normal pancreatic ducts. Distinctive immunoprofile (CK 5/6, p63 and p53 positive of the epithelial component and p63 and p53 positivity of the nonepithelial component should be explained and further investigated in the similar cases. Our findings support prior assertions that undifferentiated carcinoma of the pancreas with osteoclast-like giant cells may develop from carcinoma in situ within the main pancreatic duct or its branches

  19. Wheat TILLING mutants show that the vernalization gene VRN1 down-regulates the flowering repressor VRN2 in leaves but is not essential for flowering.

    Directory of Open Access Journals (Sweden)

    Andrew Chen

    Full Text Available Most of the natural variation in wheat vernalization response is determined by allelic differences in the MADS-box transcription factor VERNALIZATION1 (VRN1. Extended exposures to low temperatures during the winter (vernalization induce VRN1 expression and promote the transition of the apical meristem to the reproductive phase. In contrast to its Arabidopsis homolog (APETALA1, which is mainly expressed in the apical meristem, VRN1 is also expressed at high levels in the leaves, but its function in this tissue is not well understood. Using tetraploid wheat lines with truncation mutations in the two homoeologous copies of VRN1 (henceforth vrn1-null mutants, we demonstrate that a central role of VRN1 in the leaves is to maintain low transcript levels of the VRN2 flowering repressor after vernalization. Transcript levels of VRN2 were gradually down-regulated during vernalization in both mutant and wild-type genotypes, but were up-regulated after vernalization only in the vrn1-null mutants. The up-regulation of VRN2 delayed flowering by repressing the transcription of FT, a flowering-integrator gene that encodes a mobile protein that is transported from the leaves to the apical meristem to induce flowering. The role of VRN2 in the delayed flowering of the vrn1-null mutant was confirmed using double vrn1-vrn2-null mutants, which flowered two months earlier than the vrn1-null mutants. Both mutants produced normal flowers and seeds demonstrating that VRN1 is not essential for wheat flowering, which contradicts current flowering models. This result does not diminish the importance of VRN1 in the seasonal regulation of wheat flowering. The up-regulation of VRN1 during winter is required to maintain low transcript levels of VRN2, accelerate the induction of FT in the leaves, and regulate a timely flowering in the spring. Our results also demonstrate the existence of redundant wheat flowering genes that may provide new targets for engineering wheat

  20. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    Science.gov (United States)

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-03

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

  1. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    Science.gov (United States)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  2. ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor, Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

    Directory of Open Access Journals (Sweden)

    Rebecca Louise Harris

    2012-01-01

    Full Text Available Background. The asialoglycoprotein receptor (ASGPR is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2, encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR, expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver.

  3. First report of the blaVIM gene in environmental isolates of Buttiauxella sp.

    Science.gov (United States)

    Pitondo-Silva, André; Martins, Vinicius Vicente; Stehling, Eliana Guedes

    2015-04-01

    Several works have demonstrated the presence of metallo-β-lactamases (MBLs) in clinical bacteria. However, in environmental isolates, few works have reported on these enzymes. In this study, we report for the first time two environmental isolates of Buttiauxella sp. recovered from chrysanthemum plantations in Brazil containing blaVIM gene and producing MBLs.

  4. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

    Science.gov (United States)

    Kirov, Julia V; Adkisson, Michael; Nava, A J; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K; Lloyd, K C Kent; de Jong, Pieter; West, David B

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

  5. A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

    Directory of Open Access Journals (Sweden)

    Ted W Simon

    Full Text Available A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR by 2,3,7,8-tetrachlorodibenzodioxin (TCDD and subsequent binding the activated AHR to xenobiotic response elements (XREs on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT. In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism's ability to respond on a phenotypic level to various stimuli within an inconstant environment.

  6. Biological Education of IVFRU and FIAU for HSV1-TK Reporter Gene Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Su Hee; Kim, Eun Jung; Lee, Eun Ah; Lee, Jong Chan; Choi, Tae Hyun; Lee, Kyo Chul; An, Gwang Il; Cheon, Gi Jeong [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    The Herpes Simplex Virus Type1-thymidine kinase (HSV1-TK) system is a useful gene therapy monitoring method. HSV1-TK is one of the most widely used effector gene systems used for imaging gene expression, in association with its use as a reporter gene. It has resulted the development of a number of radiolabeled HSV1-TK substrates for the non-invasive detection of HSV1-TK expression. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. Prodrug activation or sucide gene therapy has been shown to be successful in potentiating the therapeutic index by sensitizing genetically modified tumor cells to various prodrugs or enhancing the action of commonly used chemotherapeutic agents. The most studied prodrug activation approaches involve transfection of tumors with HSV1-TK gene. (Z)-5-(2-iodovinyl)-2'-fluoro- 2'-deoxyuridine (IVFRU) possesses a 2'-fluoro substituent in the ribose configuration, is considered to protect IVFRU from enzyme mediated degradation in vivo. It is obviously potential substrates for HSV1-TK imaging. 2'-Fiuoro-2'-deoxy-1-{beta}-D-arabinofuranosyl- 5-iodo-uridine (FIAU), an anticancer drug widely used in clinical practice, is an analogue of thymidine. In a series of studies using adenovirus vector for gene transfer described the appropriate combination of exogenously introduced HSV1-TK as a 'marker/reporter gene' and radiolabelled FIAU as a 'marker substrate/reporter probe' for monitoring gene therapy and gene expression.

  7. Prenatal Diagnosis of 45,X/46,XX Mosaicism with Presence of SRY Gene. A Case Report

    Directory of Open Access Journals (Sweden)

    Pedro Alí Díaz-Véliz Jiménez

    2013-10-01

    Full Text Available The most common karyotype of the Turner syndrome is 45,X, although it may occur as mosaic 45,X/46,XX. In the Provincial Medical Genetics Center, a 42-year-old pregnant woman underwent an amniocentesis which led to the detection of mosaic Turner Syndrome (45,X/46,XX. A male was diagnosed through ultrasound and a sample of amniotic fluid was sent to the National Medical Genetics Center to confirm it. A study of the SRY gene was completed and the result was positive. The patient decided to terminate the pregnancy. The pathology report showed a fetal corpse of 25, 3 weeks of gestation, with penis and morphologically normal scrotal sacs, presenting alterations by cysts and hypoplasia of the prostate and seminal vesicles as well as testicular absence. In the literature, cases of XX males are considered uncommon while those combining a mosaic 45, X/46, XX with a SRY gene are less addressed; and therein lies the importance of this case. This article presents the results of a prenatal diagnosis of this rare chromosomal aberration.

  8. Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Taher uz Zaman

    2014-11-01

    Conclusions: This is probably the first reported outbreak of multidrug/carbapenem-resistant Klebsiella infection involving the OXA-48 gene from Saudi Arabia. Although the presence of ESBLs such as OXA, CTX-M, TEM, and SHV are predictable reasons for resistance, variations in the Omp-36 gene might also have precipitated this phenomenon. Disruption of the Omp-36 sequence by large insertional elements, the insertion of two amino acids in a very crucial part of this protein, and the presence of a premature stop codon in one isolate might have rendered this protein incomplete and non-functional. The study also demonstrated that more than one type of clone was responsible for this reported apparent outbreak and that ST29, a clone not reported from this region before, was the major clone responsible.

  9. Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

    DEFF Research Database (Denmark)

    Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

    ). But evidence of specific siRNA inhibition in living fish is still needed. Using the small interfering RNAs (siRNAs), messenger RNA (mRNA) can be targeted resulting in degradation of targeted transcript or translational repression. Reporter genes such as luciferase and green fluorescence protein (GFP) can......RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011...... be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...

  10. Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

    DEFF Research Database (Denmark)

    Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

    2011-01-01

    ). But evidence of specific siRNA inhibition in living fish is still needed. Using the small interfering RNAs (siRNAs), messenger RNA (mRNA) can be targeted resulting in degradation of targeted transcript or translational repression. Reporter genes such as luciferase and green fluorescence protein (GFP) can......RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011...... be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...

  11. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

    1999-01-01

    cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV)(2)-luciferase vector using the new nonliposomal transfection reagent FuGene, Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold...... antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were......-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription, To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast...

  12. Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung [School of Midicine, Chonnam National University, Gwangju (Korea, Republic of)

    2004-07-01

    The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10{sup 6}) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways.

  13. Genetics and molecular biology of methanogen genes. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Konisky, J.

    1997-10-07

    Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 C for Methanococcus voltage, 70 C for Methanococcus thermolithotrophicus, 80 C for Methanococcus igneus and 80--90 C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor, Ap{sub 5}A [P{sup 1}, P{sup 5}-di(adenosine-5{prime}) pentaphosphate], a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular weight (approximately 23--25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N-terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases including an enzyme isolated from the Archeon, Sulfolobus acidocaldarius. If verified as a nucleotide binding domain, the methanogen sequence would represent a novel nucleotide binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.

  14. Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

    Directory of Open Access Journals (Sweden)

    Orozco Javier I

    2011-10-01

    Full Text Available Abstract Introduction Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. Case presentation A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. Conclusion In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.

  15. The capsule biosynthesis locus of Haemophilus influenzae shows conspicuous similarity to the corresponding locus in Haemophilus sputorum and may have been recruited from this species by horizontal gene transfer.

    Science.gov (United States)

    Nielsen, Signe M; de Gier, Camilla; Dimopoulou, Chrysoula; Gupta, Vikas; Hansen, Lars H; Nørskov-Lauritsen, Niels

    2015-06-01

    The newly described species Haemophilus sputorum has been cultured from the upper respiratory tract of humans and appears to have little pathogenic potential. The species encodes a capsular biosynthesis locus of approximately 12  kb composed of three distinct regions. Region I and III genes, involved in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H. sputorum. Three ORFs are present in region II of the sequenced strain of H. sputorum, of which a putative phosphotransferase showed homology with corresponding genes from H. influenzae serotype c and f. Phylogenetic analysis of housekeeping genes from 24 Pasteurellaceae species showed that H. sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may have been recruited relatively recently from the commensal species H. sputorum by horizontal gene transfer.

  16. Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.

    Science.gov (United States)

    Xu, Wenxin; Zhou, You; Chollet, Raymond

    2003-05-01

    Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.

  17. New imaging probes to track cell fate: reporter genes in stem cell research.

    Science.gov (United States)

    Jurgielewicz, Piotr; Harmsen, Stefan; Wei, Elizabeth; Bachmann, Michael H; Ting, Richard; Aras, Omer

    2017-07-03

    Cell fate is a concept used to describe the differentiation and development of a cell in its organismal context over time. It is important in the field of regenerative medicine, where stem cell therapy holds much promise but is limited by our ability to assess its efficacy, which is mainly due to the inability to monitor what happens to the cells upon engraftment to the damaged tissue. Currently, several imaging modalities can be used to track cells in the clinical setting; however, they do not satisfy many of the criteria necessary to accurately assess several aspects of cell fate. In recent years, reporter genes have become a popular option for tracking transplanted cells, via various imaging modalities in small mammalian animal models. This review article examines the reporter gene strategies used in imaging modalities such as MRI, SPECT/PET, Optoacoustic and Bioluminescence Imaging. Strengths and limitations of the use of reporter genes in each modality are discussed.

  18. Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter.

    Science.gov (United States)

    Sakanaka, Mikiyasu; Tamai, Saki; Hirayama, Yosuke; Onodera, Ai; Koguchi, Hiroka; Kano, Yasunobu; Yokota, Atsushi; Fukiya, Satoru

    2014-11-01

    Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia coli can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum α-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. coli. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruEKFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. coli. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. coli. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research.

  19. A systemic lupus erythematosus gene expression array in disease diagnosis and classification: a preliminary report.

    Science.gov (United States)

    Juang, Y-T; Peoples, C; Kafri, R; Kyttaris, V C; Sunahori, K; Kis-Toth, K; Fitzgerald, L; Ergin, S; Finnell, M; Tsokos, G C

    2011-03-01

    Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease diagnosed on the presence of a constellation of clinical and laboratory findings. At the pathogenetic level, multiple factors using diverse biochemical and molecular pathways have been recognized. Succinct recognition and classification of clinical disease subsets, as well as the availability of disease biomarkers, remains largely unsolved. Based on information produced by the present authors' and other laboratories, a lupus gene expression array consisting of 30 genes, previously claimed to contribute to aberrant function of T cells, was developed. An additional eight genes were included as controls. Peripheral blood was obtained from 10 patients (19 samples) with SLE and six patients with rheumatoid arthritis (RA) as well as 19 healthy controls. T cell mRNA was subjected to reverse transcription and PCR, and the gene expression levels were measured. Conventional statistical analysis was performed along with principal component analysis (PCA) to capture the contribution of all genes to disease diagnosis and clinical parameters. The lupus gene expression array faithfully informed on the expression levels of genes. The recorded changes in expression reflect those reported in the literature by using a relatively small (5 ml) amount of peripheral blood. PCA of gene expression levels placed SLE samples apart from normal and RA samples regardless of disease activity. Individual principal components tended to define specific disease manifestations such as arthritis and proteinuria. Thus, a lupus gene expression array based on genes previously claimed to contribute to immune pathogenesis of SLE may define the disease, and principal components of the expression of 30 genes may define patients with specific disease manifestations.

  20. Reporter gene imaging of immune responses to cancer: progress and challenges.

    Science.gov (United States)

    Dubey, Purnima

    2012-01-01

    Immune responses to cancer are dynamic processes which take place through the concerted activity of innate and adaptive cell populations. In order to fully understand the efficacy of immune therapies for cancer, it is critical to understand how the treatment modulates the function of each cell type involved in the anti-tumor immune response. Molecular imaging is a versatile method for longitudinal studies of cellular localization and function. The development of reporter genes for tracking cell movement and function was a powerful addition to the immunologist's toolbox. This review will highlight the advances and challenges in the use of reporter gene imaging to track immune cell localization and function in cancer.

  1. Optimization of reporter gene assay: several factors influencing detection of promoter activity

    Institute of Scientific and Technical Information of China (English)

    XUE Li-xiang; WENG Mo; ZHANG Zong-yu; TONG Tan-jun

    2007-01-01

    Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.Methods Human embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination,such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA.Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

  2. Prenatal Diagnosis in a Family of TNFRSF11A (RANK Gene Mutation Detection: A Case Report

    Directory of Open Access Journals (Sweden)

    Mutlu Karkucak

    2014-08-01

    Full Text Available Autosomal recessive osteoporosis (ARO is a severe disease causing death usually at infancy or childhood. RANKL coded by TNFSF11 gene and RANK coded by TNFRSF11A gene are important proteins for osteoclast maturation and it is indicated that mutation on these genes plays an important role for ARO development. It is reported in this article that c.508 A→G homozygote mutation (pArg170Gly is observed in TNFRSF11A gene of 2 children of consanguineous couple. Mutation analysis performed on CVS material during the next pregnancy revealed heterozygous mutation in the fetus. The pregnancy was continued to term and a healthy boy was delivered. Prenatal mutation analysis is important for diseases with known mutations to relieve parental anxiety and provide genetic counselling for the family.

  3. Identification of epigenetically downregulated Tmem70 and Ube2e2 in rat liver after 28-day treatment with hepatocarcinogenic thioacetamide showing gene product downregulation in hepatocellular preneoplastic and neoplastic lesions produced by tumor promotion.

    Science.gov (United States)

    Mizukami, Sayaka; Yafune, Atsunori; Watanabe, Yousuke; Nakajima, Kota; Jin, Meilan; Yoshida, Toshinori; Shibutani, Makoto

    2017-01-15

    The present study identified genes showing promoter region hypermethylation by CpG island microarrays in the liver of rats treated with hepatocarcinogen thioacetamide (TAA) for 28days. Among 47 hypermethylated genes, Hist1h2aa, Tmem70, Ube2e2, and Slk were confirmed to show hypermethylation by methylation-specific quantitative polymerase chain reaction (PCR) and pyrosequencing analyses as well as downregulation of transcript levels by real-time reverse transcription-PCR analysis in the livers of rats treated with TAA. All gene products of the 4 selected genes showed decreased immunoreactivity forming negative liver cell foci in a subpopulation of glutathione S-transferase placental form (GST-P)(+) foci in TAA-promoted rat livers in a two-stage hepatocarcinogenesis model. Among them, TMEM70 and UBE2E2 showed increased incidences of negative foci in GST-P(+) foci by promotion of all examined TAA, β-naphthoflavone, piperonyl butoxide, fenbendazole and phenobarbital, while HIST1H2AA and SLK did not respond to all promotive treatments. In the late stage of tumor promotion by TAA, the incidence of GST-P(+) proliferative lesions with downregulation of TMEM70 or UBE2E2 was higher in adenomas and carcinomas than liver cell foci. TMEM70 plays a role in mitochondrial oxidative phosphorylation, and UBE2E2 participates in the stabilization of cell cycle regulatory proteins. Therefore, our results indicate that aberrant epigenetic gene downregulation suggestive of a metabolic shift of cellular respiration from oxidative phosphorylation to glycolysis and aberrant cell cycle regulation facilitating cell proliferation from as early as 28days after hepatocarcinogen treatment contribute to tumor development.

  4. Oxygen Sensitivity of Reporter Genes: Implications for Preclinical Imaging of Tumor Hypoxia

    Directory of Open Access Journals (Sweden)

    Ivana Cecic

    2007-07-01

    Full Text Available Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ. Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing β-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-β-galactoside (DDAOG. Generation of signal in in vivo tumor models expressing bioluminescent or β-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5, 6-dimethylxanthenone-4-acetic acid (DMXAA. Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas β-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining β-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

  5. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    NARCIS (Netherlands)

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

    2002-01-01

    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-

  6. A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

    DEFF Research Database (Denmark)

    Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

    2010-01-01

    of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching...

  7. Multiple insulin degrading enzyme variants alter in vitro reporter gene expression.

    Directory of Open Access Journals (Sweden)

    Olivia Belbin

    Full Text Available The insulin degrading enzyme (IDE variant, v311 (rs6583817, is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ, decreased risk for Alzheimer's disease (AD and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957, for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2 successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04 but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5' to the promoter (1.18 fold-change, p = 0.006 and 3' to the reporter gene (1.29 fold change, p = 0.003, an effect contributed to by four variants (v10, v315, v154 and v311. Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.

  8. WUS and STM-based reporter genes for studying meristem development in poplar

    Science.gov (United States)

    We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5’ flanking regions of close homologs were used to drive expression o...

  9. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

    NARCIS (Netherlands)

    Legler, J.; Dennekamp, M.; Vethaak, A.D.; Brouwer, A.; Koeman, J.H.; Burg, van der B.; Murk, A.J.

    2002-01-01

    Sediments may be the ultimate sink for persistent (xeno-) estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-

  10. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    Science.gov (United States)

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  11. Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Sonja Schallenberg

    Full Text Available CD4(+CD25(+ regulatory T (Treg cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4(+CD8(+ double-positive (DP and CD4(+CD8(? single-positive stages of thymic development, as well as in postthymic CD4(+ T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3(+ Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3(+ DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4(+ T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3(+ Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3(+ Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

  12. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  13. Adults with developmental dyslexia show selective impairments in time-based and self-initiated prospective memory: Self-report and clinical evidence.

    Science.gov (United States)

    Smith-Spark, James H; Zięcik, Adam P; Sterling, Christopher

    2017-03-01

    Prospective memory (PM; memory for delayed intentions) would seem to be impaired in dyslexia but evidence is currently limited in scope. There is a need, therefore, firstly, to explore PM under controlled conditions using a broader range of PM tasks than used previously and, secondly, to determine whether objectively measured and self-reported PM problems can be found in the same individuals with dyslexia. The responses of 30 adults with dyslexia were compared with those of 30 IQ-matched adults without dyslexia on a self-report and a clinical measure of PM. Dyslexia-related deficits were shown on the clinical measure overall and, more particularly, when PM responses had to be made to cues based on time rather than environmental events. Adults with dyslexia were also more likely to forget to carry out an intention under naturalistic conditions 24h later. On the self-report questionnaire, the group with dyslexia reported significantly more frequent problems with PM overall, despite using more techniques to aid their memory. In particular, problems were identified with longer-term PM tasks and PM which had to be self-initiated. Dyslexia-related PM deficits were found under both laboratory and everyday conditions in the same participants; the first time that this has been demonstrated. These findings support previous experimental research which has highlighted dyslexia-related deficits in PM when the enacting of intentions is based on time cues and/or has to be self-initiated rather than being in prompted by environmental events. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

  15. Recurrent anencephaly: a case report and examination of the VANGL1 and FOXN1 genes.

    Science.gov (United States)

    Sergi, Consolato; Gekas, Jean; Kamnasaran, Deepak

    2013-07-01

    We report a new and rare case of recurrent anencephaly in a family with no other apparent abnormalities. The karyotypes of the family and all affected subjects were normal. Thorough mutational analyses of VANGL1 of chromosome 1p13.1 and FOXN1 of chromosome 17q11-q12, genes that are associated with phenotypes of the anencephaly spectrum, unfortunately did not disclose any DNA variations in an affected fetus of this family. The etiology of recurrent anencephaly in this family is therefore due to mutations in genes yet to be discovered, perhaps of the planar cell polarity pathway, or to possible environmental gestational factors during development.

  16. Molecular characterization of a maize regulatory gene. Progress report, July 1989--March 1990

    Energy Technology Data Exchange (ETDEWEB)

    Wessler, S.

    1990-12-31

    This progress report contains information concerning the characterization of the Maize regulatory gene. The findings of this research program have immediate significance. Firstly, it provides support for the notion that R proteins, produced by the regulatory gene, are functionally equivalent. Secondly, the success of these experiments provides a simple transient assay for either natural or constructed R protein mutations. The relative ease of this assay coupled with overnight results are important prerequisites to the proposed experiments involving a structure-function analysis of the R protein.

  17. Identification of a male meiosis-specific gene, Tcte2, which is differentially spliced in species that form sterile hybrids with laboratory mice and deleted in t chromosomes showing meiotic drive.

    Science.gov (United States)

    Braidotti, G; Barlow, D P

    1997-06-01

    Tcte2 (t complex testes expressed 2) is a male meiosis-specific gene that maps to band 3.3 of mouse chromosome 17. Two distinct male fertility defects, hybrid sterility and transmission ratio distortion, have previously been mapped to this region. Hybrid sterility arises in crosses between different mouse species and the F1 generation males have defects in the first meiotic division and are sterile. Transmission ratio distortion is shown by males heterozygous for the t haplotype form of chromosome 17 and is a type of meiotic drive in which male gametes function unequally at fertilization. The Tcte2 gene expresses a coding mRNA and a number of putative non-ORF transcripts in meiosis I. A deletion of the 5' part of the locus abolishes Tcte2 expression on the t haplotype form of chromosome 17. Additionally, the series of putative non-ORF RNAs at the Tcte2 locus are differentially spliced in species that show hybrid sterility when crossed to laboratory mice. The identification of polymorphisms in t haplotypes and in different mouse species allows alleles of Tcte2 to be proposed as candidates for loci which contribute to both meiotic drive and hybrid sterility phenotypes. While theoretical considerations have previously been used to propose that speciation and meiotic drive involve alleles of the same genes, Tcte2 is the first cloned candidate gene to support this link at a molecular level.

  18. Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay.

    Science.gov (United States)

    Johansen, Audny; Collet, Bertrand; Sandaker, Elin; Secombes, Christopher J; Jørgensen, Jorunn B

    2004-02-01

    We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.

  19. An assessment of recently published gene expression data analyses: reporting experimental design and statistical factors

    Directory of Open Access Journals (Sweden)

    Azuaje Francisco

    2006-06-01

    Full Text Available Abstract Background The analysis of large-scale gene expression data is a fundamental approach to functional genomics and the identification of potential drug targets. Results derived from such studies cannot be trusted unless they are adequately designed and reported. The purpose of this study is to assess current practices on the reporting of experimental design and statistical analyses in gene expression-based studies. Methods We reviewed hundreds of MEDLINE-indexed papers involving gene expression data analysis, which were published between 2003 and 2005. These papers were examined on the basis of their reporting of several factors, such as sample size, statistical power and software availability. Results Among the examined papers, we concentrated on 293 papers consisting of applications and new methodologies. These papers did not report approaches to sample size and statistical power estimation. Explicit statements on data transformation and descriptions of the normalisation techniques applied prior to data analyses (e.g. classification were not reported in 57 (37.5% and 104 (68.4% of the methodology papers respectively. With regard to papers presenting biomedical-relevant applications, 41(29.1 % of these papers did not report on data normalisation and 83 (58.9% did not describe the normalisation technique applied. Clustering-based analysis, the t-test and ANOVA represent the most widely applied techniques in microarray data analysis. But remarkably, only 5 (3.5% of the application papers included statements or references to assumption about variance homogeneity for the application of the t-test and ANOVA. There is still a need to promote the reporting of software packages applied or their availability. Conclusion Recently-published gene expression data analysis studies may lack key information required for properly assessing their design quality and potential impact. There is a need for more rigorous reporting of important experimental

  20. Case report of novel CACNA1A gene mutation causing episodic ataxia type 2

    Directory of Open Access Journals (Sweden)

    David Alan Isaacs

    2017-05-01

    Full Text Available Background: Episodic ataxia type 2 (OMIM 108500 is an autosomal dominant channelopathy characterized by paroxysms of ataxia, vertigo, nausea, and other neurologic symptoms. More than 50 mutations of the CACNA1A gene have been discovered in families with episodic ataxia type 2, although 30%–50% of all patients with typical episodic ataxia type 2 phenotype have no detectable mutation of the CACNA1A gene. Case: A 46-year-old Caucasian man, with a long history of bouts of imbalance, vertigo, and nausea, presented to our hospital with 2 weeks of ataxia and headache. Subsequent evaluation revealed a novel mutation in the CACNA1A gene: c.1364 G > A Arg455Gln. Acetazolamide was initiated with symptomatic improvement. Conclusion: This case report expands the list of known CACNA1A mutations associated with episodic ataxia type 2.

  1. Case report of novel CACNA1A gene mutation causing episodic ataxia type 2.

    Science.gov (United States)

    Isaacs, David Alan; Bradshaw, Michael J; Brown, Kelly; Hedera, Peter

    2017-01-01

    Episodic ataxia type 2 (OMIM 108500) is an autosomal dominant channelopathy characterized by paroxysms of ataxia, vertigo, nausea, and other neurologic symptoms. More than 50 mutations of the CACNA1A gene have been discovered in families with episodic ataxia type 2, although 30%-50% of all patients with typical episodic ataxia type 2 phenotype have no detectable mutation of the CACNA1A gene. A 46-year-old Caucasian man, with a long history of bouts of imbalance, vertigo, and nausea, presented to our hospital with 2 weeks of ataxia and headache. Subsequent evaluation revealed a novel mutation in the CACNA1A gene: c.1364 G > A Arg455Gln. Acetazolamide was initiated with symptomatic improvement. This case report expands the list of known CACNA1A mutations associated with episodic ataxia type 2.

  2. Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter.

    Science.gov (United States)

    Cui, Zhong-Li; Zhang, Xiao-Zhou; Zhang, Zhong-Hui; Li, Shun-Peng

    2004-07-01

    A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.

  3. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae

    Science.gov (United States)

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

    Abstract Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon‐optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half‐lives of 40 and 5 min, respectively. The commercial substrate Nano‐Glo® is compatible with detection of yNluc bioluminescence in bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  4. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation.

    Directory of Open Access Journals (Sweden)

    Alexander R Mendenhall

    Full Text Available In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to

  5. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;

    2011-01-01

    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-induced firefly luciferase activity to be normalized...

  6. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;

    2011-01-01

    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...

  7. A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

    DEFF Research Database (Denmark)

    Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

    2010-01-01

    Mechanical forces are translated into biochemical signals and contribute to cell differentiation and phenotype maintenance. Mesenchymal stem cells and their tissue-specific offspring, as osteoblasts and chondrocytes, cells of cardiovascular tissues and lung cells are sensitive to mechanical loading...... but molecules and mechanisms involved have to be unraveled. It is well established that cellular mechanotransduction is mediated e.g. by activation of the transcription factor SP1 and by kinase signaling cascades resulting in the activation of the AP1 complex. To investigate cellular mechanisms involved...... in mechanotransduction and to analyze substances, which modulate cellular mechanosensitivity reporter gene constructs, which can be transfected into cells of interest might be helpful. Suitable small-scale bioreactor systems and mechanosensitive reporter gene constructs are lacking. To analyze the molecular mechanisms...

  8. Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports

    Science.gov (United States)

    Lakraj, Amanda Amrita; Miller, Geoffrey; Vortmeyer, Alexander O.; Khokhar, Babar; Nowak, Richard J.; DiCapua, Daniel B.

    2013-01-01

    Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia. Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC). Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy. Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature. PMID:23483815

  9. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    Energy Technology Data Exchange (ETDEWEB)

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  10. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

    Directory of Open Access Journals (Sweden)

    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  11. Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Ya-Ju Hsieh

    2014-01-01

    Full Text Available Multimodality imaging using noncytotoxic triple fusion (TF reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl, monomeric red fluorescence protein (mrfp, and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39 were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET. The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

  12. Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

    2009-02-15

    expression by polymerase chain reaction in adenovirus vector-infected cardiac myocytes was significantly higher than that in pDC316-tk/lipoplex-transducted ones (semiquantitative analysis, 3.11{+-}0.14 versus 1.60{+-}0.05, P<.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rates were 81.70{+-}0.40% in Ad5-tk and 22.06{+-}0.32% in liposome (P<.01). Conclusions: Both adenovirus and liposome could transfer reporter gene into cardiac myocytes successfully, and the expressed exogenous protein could form functional enzymes efficiently. However, the adenovirus vector acted more efficiently than did liposome, with a higher uptake rate of the reporter probe. Thus, adenovirus is competent for gene transfer in cardiac reporter gene imaging.

  13. Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

    Science.gov (United States)

    Fatahi-Bafghi, Mehdi; Heidarieh, Parvin; Rasouli-Nasab, Masoumeh; Habibnia, Shadi; Hashemi-Shahraki, Abdorazagh; Eshraghi, Seyyed Saeed

    2016-10-01

    Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

  14. Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signalling Pathway

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    Yueming Kelly Sun

    2016-12-01

    Full Text Available Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signalling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2. KAI2 is a homologue of DWARF 14 (D14, the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2. We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signalling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analogue. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signalling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signalling pathway.

  15. A cross-sectional study of self-reported chemical-related sensitivity is associated with gene variants of drug-metabolizing enzymes

    Directory of Open Access Journals (Sweden)

    Fabig Nathalie

    2007-02-01

    Full Text Available Abstract Background N-acetyltransferases (NAT and glutathione S-transferases (GST are involved in the metabolism of several ubiquitous chemical substances leading to the activation and detoxification of carcinogenic heterocyclic and aromatic amines. Since polymorphisms within these genes are described to influence the metabolism of ubiquitous chemicals, we conducted the present study to determine if individuals with self-reported chemical-related sensitivity differed from controls without self-reported chemical-related sensitivity with regard to the distribution of genotype frequencies of NAT2, GSTM1, GSTT1, and GSTP1 polymorphisms. Methods Out of 800 subjects who answered a questionnaire of ten items with regard to their severity of chemical sensitivity 521 unrelated individuals agreed to participate in the study. Subsequently, genetic variants of the NAT2, GSTM1, GSTT1, and GSTP1 genes were analyzed. Results The results show significant differences between individuals with and without self-reported chemical-related sensitivity with regard to the distribution of NAT2, GSTM1, and GSTT1 gene variants. Cases with self-reported chemical-related sensitivity were significantly more frequently NAT2 slow acetylators (controlled OR = 1.81, 95% CI = 1.27–2.59, P = 0.001. GSTM1 and GSTT1 genes were significantly more often homozygously deleted in those individuals reporting sensitivity to chemicals compared to controls (GSTM1: controlled OR 2.08, 95% CI = 1.46–2.96, P = 0.0001; GSTT1: controlled OR = 2.80, 95% CI = 1.65–4.75, P = 0.0001. Effects for GSTP1 gene variants were observed in conjunction with GSTM1, GSTT1 and NAT2 gene. Conclusion The results from our study population show that individuals being slow acetylators and/or harbouring a homozygous GSTM1 and/or GSTT1 deletion reported chemical-related hypersensitivity more frequently.

  16. First report of a de novo germline mutation in the MLH1 gene

    Institute of Scientific and Technical Information of China (English)

    Rein P Stulp; Yvonne J Vos; Bart Mol; Arend Karrenbeld; Monique de Raad; Huub JC van der Mijle; Rolf H Sijmons

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC)is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven,or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient's parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  17. Development of a facile method for high throughput screening with reporter gene assays.

    Science.gov (United States)

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  18. A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.

    Science.gov (United States)

    Ogg, M S; Williams, J M; Tarbit, M; Goldfarb, P S; Gray, T J; Gibson, G G

    1999-03-01

    1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.

  19. The capsule biosynthesis locus of Haemophilus influenzae show conspicuous similarity to the corresponding locus in Haemophilus sputorum and may have been recruited from this species by horizontal gene transfer

    DEFF Research Database (Denmark)

    Nielsen, Signe Maria; de Gier, Camilla; Dimopoulou, Chrysoula

    2015-01-01

    in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H....... sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may relatively recently have been recruited...

  20. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

    Science.gov (United States)

    Fischer, Martin A; Güllert, Simon; Neulinger, Sven C; Streit, Wolfgang R; Schmitz, Ruth A

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  1. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  2. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    Energy Technology Data Exchange (ETDEWEB)

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  3. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    Energy Technology Data Exchange (ETDEWEB)

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  4. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report.

    Science.gov (United States)

    Lee, Sin Hang

    2016-01-01

    Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain.

  5. Methylene tetrahydrofolate reductase gene mutation together with anticardiolipin antibody during pregnancy: a case report

    Directory of Open Access Journals (Sweden)

    Egle Couto

    Full Text Available CONTEXT: High plasmatic homocysteine levels have been associated with arterial and venous thrombosis. The C677T methylene tetrahydrofolate reductase (MTHFR gene mutation is one of the known causes for high homocysteine levels in plasma. Anticardiolipin antibody (ACA is also associated with thrombosis and, along with other clinical complications such as recurrent abortion and stillbirth, is part of the antiphospholipid syndrome. DESIGN: Case report. CASE REPORT: A 19-year-old woman with two gestations and one parity (G2P1 had exhibited deep venous thrombosis in her previous puerperal period. Investigation of thrombophilic factors revealed ACA-IgM and heterozygous C677T mutation in the MTHFR gene. Lupus anticoagulant, protein C, protein S and antithrombin III deficiencies, and Leiden factor V and the G20210A mutation in the prothrombin gene, were not detected. The patient received 55,000 IU of subcutaneous heparin daily, from the 15th to the 36th week of pregnancy, when vaginal delivery took place. There were no clinical complications during the puerperal period and she was discharged three days after delivery, while still using oral anticoagulants.

  6. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    Science.gov (United States)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

  7. Highly efficient production of the staphylococcal nuclease reporter in Lactobacillus bulgaricus governed by the promoter of the hlbA gene.

    Science.gov (United States)

    Chouayekh, Hichem; Serror, Pascale; Boudebbouze, Samira; Maguin, Emmanuelle

    2009-04-01

    Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) genome sequence analysis revealed the presence of two genes that encode histone-like HU proteins (hlbA and hlbB) showing extensive similarity to other bacterial homologues. These genes were found to be extremely conserved among several L. bulgaricus strains. The hlbA gene was shown to be constitutively transcribed from a unique promoter (phlbA) during normal growth, whereas hlbB did not seem to be expressed under usual laboratory conditions. Using a reporter cassette in which the staphylococcal nuclease was fused at its N-terminus to the lactococcal signal peptide Usp45 (SP Usp45), we have demonstrated that phlbA promotes high expression of the reporter in L. bulgaricus, which correlated with an abundant secretion of the mature nuclease in the supernatant fraction. Quantification of the exported enzyme reveals a secretion level approximately threefold higher when the expression of the reporter was under the control of phlbA compared with the lactococcal usp45 promoter. Together, these results indicate that phlbA is suitable for gene expression in L. bulgaricus, that SP Usp45 is functionally recognized and processed by the L. bulgaricus secretion machinery and that the nuclease reporter gene can be used for the identification of exported products in this bacterium.

  8. Showing Value (Editorial

    Directory of Open Access Journals (Sweden)

    Denise Koufogiannakis

    2009-06-01

    Full Text Available When Su Cleyle and I first decided to start Evidence Based Library and Information Practice, one of the things we agreed upon immediately was that the journal be open access. We knew that a major obstacle to librarians using the research literature was that they did not have access to the research literature. Although Su and I are both academic librarians who can access a wide variety of library and information literature from our institutions, we belong to a profession where not everyone has equal access to the research in our field. Without such access to our own body of literature, how can we ever hope for practitioners to use research evidence in their decision making? It would have been contradictory to the principles of evidence based library and information practice to do otherwise.One of the specific groups we thought could use such an open access venue for discovering research literature was school librarians. School librarians are often isolated and lacking access to the research literature that may help them prove to stakeholders the importance of their libraries and their role within schools. Certainly, school libraries have been in decline and the use of evidence to show value is needed. As Ken Haycock noted in his 2003 report, The Crisis in Canada’s School Libraries: The Case for Reform and Reinvestment, “Across the country, teacher-librarians are losing their jobs or being reassigned. Collections are becoming depleted owing to budget cuts. Some principals believe that in the age of the Internet and the classroom workstation, the school library is an artifact” (9. Within this context, school librarians are looking to our research literature for evidence of the impact that school library programs have on learning outcomes and student success. They are integrating that evidence into their practice, and reflecting upon what can be improved locally. They are focusing on students and showing the impact of school libraries and

  9. First report of multiresistance gene cfr in Enterococcus species casseliflavus and gallinarum of swine origin.

    Science.gov (United States)

    Liu, Yang; Wang, Yang; Dai, Lei; Wu, Congming; Shen, Jianzhong

    2014-06-01

    The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr in Enterococcus species of swine origin. Twenty-five cfr-carrying Enterococcus isolates were collected from swine in Beijing, Guangzhou, and Shandong, China. The isolates consist of 24 Enterococcus casseliflavus and one Enterococcus gallinarum isolate, and exhibited six SmaI PFGE patterns. The cfr gene was located on plasmids in all isolates except E. casseliflavus En83, in which cfr was located on the chromosomal DNA. The cfr gene environments in most of these isolates contain DNA sequences similar to pEF-01, which was first found in Enterococcus. However, inverse PCR analysis suggested that the cfr-carrying circular forms might be different from pEF-01. The circular forms in Eg51 and its transconjugant, and En23, En10, and En94 are similar to the circular form in pEF-01, except for the truncated IS1216, which is replaced by a transposase of the IS256 family in En24. The cfr circular form could not be detected in either En77 or En83, and the same cfr-carrying segments of ∼ 10 kb had only 3500bp of sequence similar to pEF-01. This is the first report of cfr gene in E. casseliflavus and E. gallinarum. The potential dissemination of the multidrug resistance gene amongst different bacterial species, especially in enterococci of human and animal origins, is concerning and should be closely monitored.

  10. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Konrad E. Mueller

    2016-01-01

    Full Text Available Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM. We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.

  11. User-centered design of multi-gene sequencing panel reports for clinicians.

    Science.gov (United States)

    Cutting, Elizabeth; Banchero, Meghan; Beitelshees, Amber L; Cimino, James J; Fiol, Guilherme Del; Gurses, Ayse P; Hoffman, Mark A; Jeng, Linda Jo Bone; Kawamoto, Kensaku; Kelemen, Mark; Pincus, Harold Alan; Shuldiner, Alan R; Williams, Marc S; Pollin, Toni I; Overby, Casey Lynnette

    2016-10-01

    The objective of this study was to develop a high-fidelity prototype for delivering multi-gene sequencing panel (GS) reports to clinicians that simulates the user experience of a final application. The delivery and use of GS reports can occur within complex and high-paced healthcare environments. We employ a user-centered software design approach in a focus group setting in order to facilitate gathering rich contextual information from a diverse group of stakeholders potentially impacted by the delivery of GS reports relevant to two precision medicine programs at the University of Maryland Medical Center. Responses from focus group sessions were transcribed, coded and analyzed by two team members. Notification mechanisms and information resources preferred by participants from our first phase of focus groups were incorporated into scenarios and the design of a software prototype for delivering GS reports. The goal of our second phase of focus group, to gain input on the prototype software design, was accomplished through conducting task walkthroughs with GS reporting scenarios. Preferences for notification, content and consultation from genetics specialists appeared to depend upon familiarity with scenarios for ordering and delivering GS reports. Despite familiarity with some aspects of the scenarios we proposed, many of our participants agreed that they would likely seek consultation from a genetics specialist after viewing the test reports. In addition, participants offered design and content recommendations. Findings illustrated a need to support customized notification approaches, user-specific information, and access to genetics specialists with GS reports. These design principles can be incorporated into software applications that deliver GS reports. Our user-centered approach to conduct this assessment and the specific input we received from clinicians may also be relevant to others working on similar projects.

  12. Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes.

    Science.gov (United States)

    Jindal, Sunita; Longchar, Bendangchuchang; Singh, Alka; Gupta, Vikrant

    2015-01-01

    Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants.

  13. The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

    Directory of Open Access Journals (Sweden)

    Dashuai Mu

    Full Text Available Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3 was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%. To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.

  14. Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

    NARCIS (Netherlands)

    Legler, J.; Zeinstra, L.M.; Schuitemaker, F.; Lanser, P.H.; Bogerd, J.; Brouwer, A.; Vethaak, A.D.; Voogt, de P.; Murk, A.J.; Burg, van der B.

    2002-01-01

    Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably trans

  15. A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2012-08-01

    Full Text Available Abstract Background Transcription activator-like effectors (TALEs are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP applications. Conclusion We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

  16. Search for major genes with progeny test data to accelerate the development of genetically superior loblolly pine. Technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-02-15

    This report details the progress of the three tasks of this project. The tasks are: (1) develop genetic models and analytical methods; (2) molecular confirmation of major gene segregation; and (3) develop strategies for marker-assisted breeding.

  17. p53 Promoter-based Reporter Gene in vitro Assays for Quick Assessment of Agents with Genotoxic Potential

    Institute of Scientific and Technical Information of China (English)

    Huaixing LI; Ke SHI; Ruiwen CHEN; Yan HE; Dan WU; Shuhan SUN

    2007-01-01

    The p53 promoter-based green fluorescent protein (GFP) and luciferase reporter gene assays have been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or 5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cells exposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of the cells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reporter gene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.

  18. single nucleotide Polymorphisms rs2227284, rs2243283 and rs2243288 in the Il-4 Gene show no association with susceptibility to Chronic Hepatitis B in a Chinese Han Population

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Objective To investigate the relationship between single nucleotide polymorphisms (SNPs) of the interleukin-4 (IL-4) gene and outcome of hepatitis B virus (HBV) infection in a Chinese Han population. Methods Total of 501 patients with chronic hepatitis B virus (HBV) infection and 301 controls with self-limiting HBV infection were studied. Three tag SNPs in the IL-4 gene (rs2227284G/T, rs2243283C/G and rs2243288A/G) were genotyped by the Multiplex snapshot technique. The genotype and allele frequencies were calculated and analyzed. Results The three SNPs showed no significant genotype/allele associations with chronic HBV infection. Overall allele P values were:rs2227284, P=0.655, odds ratio (OR) [95%conifdence interval (CI)]=1.070 (0.793-1.445);rs2243283, P=0.849, OR (95%CI)=0.976 (0.758-1.257);rs2243288, P=0.659, OR (95%CI) = 1.060 (0.818-1.375). Overall genotype P values were: rs2227284, P = 0.771; rs2243283, P = 0.571;rs2243288, P=0.902. There were no statistically signiifcant differences between patients with chronic HBV infection and controls. Haplotypes generated by these three SNPs also had no signiifcant differences between the two groups. Conclusions The three tag SNPs of IL-4 were not associated with the outcome of HBV infection in the Han Chinese population.

  19. Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?

    Science.gov (United States)

    Di Fruscio, Giuseppina; Garofalo, Arcomaria; Mutarelli, Margherita; Savarese, Marco; Nigro, Vincenzo

    2016-01-01

    Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.

  20. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  1. Sporadic Cerebral Cavernous Malformations: Report of Further Mutations of CCM Genes in 40 Italian Patients

    Directory of Open Access Journals (Sweden)

    Rosalia D’Angelo

    2013-01-01

    Full Text Available Cerebral cavernous malformations (CCMs are vascular lesions characterized by abnormally enlarged capillary cavities, affecting the central nervous system. CCMs can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (K-Rev interaction trapped 1 (KRIT1, CCM2 (MGC4607, and CCM3 (PDCD10. CCMs occur as a single or multiple malformations that can lead to seizures, focal neurological deficits, hemorrhagic stroke, and headache. However, patients are frequently asymptomatic. In our previous mutation screening, performed in a cohort of 95 Italian patients, both sporadic and familial, we have identified several mutations in CCM genes, three of which in three distinct sporadic patients. In this study, representing further molecular screening of the three CCM genes, in a south Italian cohort of CCM patients enrolled by us in the last three years, we report the identification of other four new mutations in 40 sporadic patients with either single or multiple CCM.

  2. Sporadic Cerebral Cavernous Malformations: Report of Further Mutations of CCM Genes in 40 Italian Patients

    Science.gov (United States)

    D'Angelo, Rosalia; Alafaci, Concetta; Scimone, Concetta; Ruggeri, Alessia; Salpietro, Francesco Maria; Bramanti, Placido; Tomasello, Francesco; Sidoti, Antonina

    2013-01-01

    Cerebral cavernous malformations (CCMs) are vascular lesions characterized by abnormally enlarged capillary cavities, affecting the central nervous system. CCMs can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1 (K-Rev interaction trapped 1 (KRIT1)), CCM2 (MGC4607), and CCM3 (PDCD10). CCMs occur as a single or multiple malformations that can lead to seizures, focal neurological deficits, hemorrhagic stroke, and headache. However, patients are frequently asymptomatic. In our previous mutation screening, performed in a cohort of 95 Italian patients, both sporadic and familial, we have identified several mutations in CCM genes, three of which in three distinct sporadic patients. In this study, representing further molecular screening of the three CCM genes, in a south Italian cohort of CCM patients enrolled by us in the last three years, we report the identification of other four new mutations in 40 sporadic patients with either single or multiple CCM. PMID:24058906

  3. Brief Report: Aggression and Stereotypic Behavior in Males with Fragile X Syndrome-- Moderating Secondary Genes in a "Single Gene" Disorder

    Science.gov (United States)

    Hessl, David; Tassone, Flora; Cordeiro, Lisa; Koldewyn, Kami; McCormick, Carolyn; Green, Cherie; Wegelin, Jacob; Yuhas, Jennifer; Hagerman, Randi J.

    2008-01-01

    Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine…

  4. Brief Report: Aggression and Stereotypic Behavior in Males with Fragile X Syndrome-- Moderating Secondary Genes in a "Single Gene" Disorder

    Science.gov (United States)

    Hessl, David; Tassone, Flora; Cordeiro, Lisa; Koldewyn, Kami; McCormick, Carolyn; Green, Cherie; Wegelin, Jacob; Yuhas, Jennifer; Hagerman, Randi J.

    2008-01-01

    Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine…

  5. Familial adult spinal muscular atrophy associated with the VAPB gene: report of 42 cases in Brazil

    Directory of Open Access Journals (Sweden)

    Victor Kosac

    2013-10-01

    Full Text Available Familial spinal muscular atrophy (FSMA associated with the vesicle-associated membrane protein-associated protein B (VAPB gene is a rare autosomal dominant disease with late onset and slow progression. We studied 10 of 42 patients from 5 families by taking clinical histories and performing physical exams, electrophysiological studies, and genetic tests. All patients presented late onset disease with slow progression characterized by fasciculations, proximal weakness, amyotrophy, and hypoactive deep tendon reflex, except two who exhibited brisk reflex. Two patients showed tongue fasciculations and respiratory insufficiency. Electrophysiological studies revealed patterns of lower motor neuron disease, and genetic testing identified a P56S mutation of the VAPB gene. Although it is a rare motor neuron disease, FSMA with this mutation might be much more prevalent in Brazil than expected, and many cases may be undiagnosed. Genetic exams should be performed whenever it is suspected in Brazil.

  6. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    Energy Technology Data Exchange (ETDEWEB)

    Plotan, Monika; Elliott, Christopher T. [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom); Scippo, Marie Louise [Department of Food Sciences, University of Liege, 4000 Liege (Belgium); Muller, Marc [Molecular Biology and Genetic Engineering GIGA-R, University of Liege, 4000 Liege (Belgium); Antignac, Jean-Philippe [LABERCA, ENVN, USC INRA 2013, BP 50707, 44 307, Nantes (France); Malone, Edward [The State Laboratory, Young' s Cross, Celbridge, Co. Kildare (Ireland); Bovee, Toine F.H. [RIKILT Institute of Food Safety, P.O. Box 230, AE Wageningen 6700 (Netherlands); Mitchell, Samuel [Agri-Food and Biosciences Institute, Belfast BT9 5PX (United Kingdom); Connolly, Lisa, E-mail: l.connolly@qub.ac.uk [Institute of Agri-Food and Land Use, School of Biological Sciences, Queen' s University Belfast, Belfast BT95AG, Northern Ireland (United Kingdom)

    2011-08-26

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC{sub 50} of 0.01 ng mL{sup -1} and 0.16 ng mL{sup -1} respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally

  7. Gene polymorphisms in association with self-reported stroke in US adults

    Directory of Open Access Journals (Sweden)

    Amy Z Fan

    2010-03-01

    Full Text Available Amy Z Fan1, Jing Fang1, Ajay Yesupriya2, Man-huei Chang2, Greta Kilmer1, Meaghan House3, Donald Hayes1, Renée M Ned2, Nicole F Dowling2, Ali H Mokdad1 1National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 2Office of Public Health Genomics, Coordinating Center for Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA; 3School of Public Health, Emory University, Atlanta, GA, USAPurpose: Epidemiologic studies suggest that several gene variants increase the risk of stroke, and population-based studies help provide further evidence. We identified polymorphisms associated with the prevalence of self-reported stroke in US populations using a representative sample.Methods: Our sample comprised US adults in the Third National Health and Nutrition Examination (NHANES III DNA bank. We examined nine candidate gene variants within ACE, F2, F5, ITGA2, MTHFR, and NOS3 for associations with self-reported stroke. We used multivariate regression and Cox proportional hazards models to test the association between these variants and history of stroke.Results: In regression models, the rs4646994 variant of ACE (I/I and I/D genotypes was associated with higher prevalence adjusted prevalence odds ratio [APOR] = 2.66 [1.28, 5.55] and 2.23 [1.30, 3.85], respectively compared with the D/D genotype. The heterozygous genotype of MTHFR rs1801131 (A/C was associated with lower prevalence of stroke (APOR = 0.48 [0.25, 0.92] compared with A/A and C/C genotypes. For rs2070744 of NOS3, both the C/T genotype (APOR = 1.91 [1.12, 3.27] and C/C genotype (APOR = 3.31 [1.66, 6.60] were associated with higher prevalence of stroke compared with the T/T genotype.Conclusion: Our findings suggest an association between the prevalence of self-reported stroke and polymorphisms in ACE, MTHFR, and NOS3 in a population-based sample. Keywords: stroke, gene, polymorphisms, NHANES III, gene

  8. Genetic heritability of ischemic stroke and the contribution of previously reported candidate gene and genomewide associations.

    Science.gov (United States)

    Bevan, Steve; Traylor, Matthew; Adib-Samii, Poneh; Malik, Rainer; Paul, Nicola L M; Jackson, Caroline; Farrall, Martin; Rothwell, Peter M; Sudlow, Cathie; Dichgans, Martin; Markus, Hugh S

    2012-12-01

    The contribution of genetics to stroke risk, and whether this differs for different stroke subtypes, remainsuncertain. Genomewide complex trait analysis allows heritability to be assessed from genomewide association study (GWAS) data. Previous candidate gene studies have identified many associations with stoke but whether these are important requires replication in large independent data sets. GWAS data sets provide a powerful resource to perform replication studies. We applied genomewide complex trait analysis to a GWAS data set of 3752 ischemic strokes and 5972 controls and determined heritability for all ischemic stroke and the most common subtypes: large-vessel disease, small-vessel disease, and cardioembolic stroke. By systematic review we identified previous candidate gene and GWAS associations with stroke and previous GWAS associations with related cardiovascular phenotypes (myocardial infarction, atrial fibrillation, and carotid intima-media thickness). Fifty associations were identified. For all ischemic stroke, heritability was 37.9%. Heritability varied markedly by stroke subtype being 40.3% for large-vessel disease and 32.6% for cardioembolic but lower for small-vessel disease (16.1%). No previously reported candidate gene was significant after rigorous correction for multiple testing. In contrast, 3 loci from related cardiovascular GWAS studies were significant: PHACTR1 in large-vessel disease (P=2.63e(-6)), PITX2 in cardioembolic stroke (P=4.78e(-8)), and ZFHX3 in cardioembolic stroke (P=5.50e(-7)). There is substantial heritability for ischemic stroke, but this varies for different stroke subtypes. Previous candidate gene associations contribute little to this heritability, but GWAS studies in related cardiovascular phenotypes are identifying robust associations. The heritability data, and data from GWAS, suggest detecting additional associations will depend on careful stroke subtyping.

  9. Mutations of SCN4A gene cause different diseases: 2 case reports and literature review.

    Science.gov (United States)

    Liu, Xiao-li; Huang, Xiao-jun; Luan, Xing-hua; Zhou, Hai-yan; Wang, Tian; Wang, Jing-yi; Chen, Sheng-di; Tang, Hui-dong; Cao, Li

    2015-01-01

    SCN4A encodes the Nav1.4 channel and mutations in SCN4A lead to different ionic channelopathies. In this study, one sporadic individual of periodic paralysis, one paramyotonia family and 200 normal healthy controls are enrolled. Genomic DNA was extracted from peripheral blood leukocytes, followed by polymerase chain reaction and DNA sequencing of candidate genes, including SCN4A and CACNA1S. As a result, heterozygous mutations c.2024G>A (R675Q) and c.1333G>A (V445M) of gene SCN4A were identified in the hypokalemic periodic paralysis patient and the paramyotonia congenita family respectively. Both mutations were not detected in healthy controls. Compared with reported cases, patients with mutation R675Q usually do not present hypokalemic periodic paralysis but hyperkalemic or normokalemic periodic paralysis. The mutation V445M was first reported in Chinese patients with nondystrophic myotonias. In addition, we carried out literature review by summarizing clinical features of the 2 mutations and establish the genotype-phenotype correlations to provide guidance for diagnosis.

  10. Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil.

    Science.gov (United States)

    Camargo, I L B C; Barth, A L; Pilger, K; Seligman, B G S; Machado, A R L; Darini, A L C

    2004-11-01

    In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 microg/ml. This isolate was sent to a reference laboratory (Laboratorio Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

  11. Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

    Directory of Open Access Journals (Sweden)

    I.L.B.C. Camargo

    2004-11-01

    Full Text Available In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

  12. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  13. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    Directory of Open Access Journals (Sweden)

    Huifen Xu

    2015-01-01

    Full Text Available Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene was confirmed to have a high tolerance for mutagenesis by this new assay.

  14. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  15. The Health Show

    OpenAIRE

    Swann, David

    2011-01-01

    Dr David Swann interviewed on The Health Show, Series 1, Episode 5, 2011 for BBC World about the award-winning 21st Century Nursing Bag. BBC World News reaches 241million people every week, available in 296 million homes, 1.8 million hotel rooms and has the highest average viewership on a weekday of any international news channel. The Health Show is a new 26-part series for BBC World News covering the most important news stories from around the world.

  16. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli.

    Science.gov (United States)

    Paproski, Robert J; Li, Yan; Barber, Quinn; Lewis, John D; Campbell, Robert E; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene

  17. Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-11-01

    A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

  18. Combination therapy and evaluation of therapeutic effect in hepatocellular carcinoma cell using triple reporter genes; containing for NIS, HSV1-sr39tk and GFP

    Energy Technology Data Exchange (ETDEWEB)

    Lee, You La; Lee, Yong Jin; Ahn, Sohn Joo; Ahn, Byeong Cheol; Lee, Sang Woo; Yoo, Jeong Soo; Lee, Jae Tae [Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To identify therapeutic effect after combine Sodium Iodine Symporter (NIS) and Mutant Herpes-simplex virus type 1 sr39tk (HSV1-sr39tk) expression in hepatocellular carcinoma cell, we transfected triple gene and investigated the properties of these gene ability in hepatocellular carcinoma cell line. After making vector with gene encoding a fusion protein comprised of HSV1-sr39tk and green florescence protein (GFP), to make triple reporter genes NIS gene was further fused to the vector using IRES vector. The vector expressing triple reporter gene was transfected to the Huh-7 cell line using liposome. Functions of hNIS and HSV1-sr39tk expression were confirmed by radio iodine uptake with and without perchlorate and [3H]-penciclovir (3-H PCV) uptake, respectively. To evaluate therapeutic effect in vitro, GCV and I-131 was treated in Huh-7/NTG cell and dual therapy performed. An animal imaging acquired using Optix and microPET in vivo. I-125 uptake was increased up to 100-fold compare to that of non-transfected cells. The transfected cell accumulated H-3 PCV up to 53 times higher at 2 hour than that of non-transfected cells. With fluorescence microscopy, green fluorescence was detected in the transfected cell. In cytotoxic studies, the cell viability of Huh-7/NTG cell was decreased to 41 % of control cell at 10ug/ml GCV concentrations. The survival rate of the Huh-7/NTG cell treated with I-131 decreased up to 16%. In I-131 and GCV dual therapy, Huh-7/NTG cell survival rate decreased up to 4%. In animal studies, Huh-7/NTG tumors showed higher uptake of 18F-FHBG and I-124 than Huh-7 tumors. GFP signal is also higher in Huh-7/NTG tumor than control. We successfully constructed a vector with delivery two therapeutic genes and one reporter gene and transfected the vector to a Huh-7 cell. The hepatocellular carcinoma cell transfected with the vector can be treated with GCV and I-131. The effect of dual gene therapy could be easily assessed by the optical reporter gene imaging.

  19. A Fashion Show

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    <正>Story: The yearly fashion show day.The children take turns to walk on the stage and show the class their favorite clothes.Now it’s Joe’s and Phoebe’s turn.Joe walks on the stage and says,“My shorts are blue.Do you like my blue shorts?”On the other side of the stage, Phoebe is wearing her favorite pink skirt.“My skirt is pink.Do you like my pink skirt?”asks

  20. On not showing scalps

    DEFF Research Database (Denmark)

    Marselis, Randi Lorenz

    2016-01-01

    proposed by Janet Marstine, the editor of the Routledge Companion to Museum Ethics, I show how the museum succeeded in engaging users in questions of museum ethics. However, this specific debate on human remains in museums developed into an encounter between a global, museological discourse...

  1. Violence and TV Shows

    OpenAIRE

    ÖZTÜRK, Yrd. Doç. Dr. Şinasi

    2008-01-01

    This study aims to discuss theories on theviolent effects of TV shows on viewers, especiallyon children. Therefore, this study includes a briefdiscussion of definitions of violence, discussionof violence theories, main results of researcheson televised violence, measuring TV violence,perception of televised violence, individualdifferences and reactions to TV violence,aggressiveness and preferences for TV violence.

  2. Honored Teacher Shows Commitment.

    Science.gov (United States)

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  3. A Visionary Show

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Seduction. Distinction. Relax. Pulsation. These are the "style universes" on display at Première Vision, heralded as "The World’s Premiere Fabric Show." Started more than 35 years ago by 15 French weavers, Première Vision has expanded beyond its

  4. Honored Teacher Shows Commitment.

    Science.gov (United States)

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  5. MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

    2016-11-15

    Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

  6. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    Science.gov (United States)

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  7. Shanghai Shows Its Heart

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The city known as China’s economic powerhouse showed a more caring face as host of the Special Olympic Games Between October 2 and 11,the Special Olympics Summer Games were hosted in Shanghai,the first time the 40-year-old athletic com- petition for people with intellectual disabilities came to a developing country. This Special Olympics was also larger than all previous games in temps of the number of athletes.

  8. THE USE OF gusA REPORTER GENE TO MONITOR THE SURVIVAL OF INTRODUCED BACTERIA IN THE SOIL

    Directory of Open Access Journals (Sweden)

    Edi Husen

    2013-07-01

    Full Text Available An effective marker to monitor the survival of introduced bacteria in the soil is required for further evaluation of their beneficial effects on plant growth. This study tested the use of gusA gene as a marker to trace the fate of three Gram negative bacteria in the root, rhizosphere, and soil. The study was conducted at the laboratory and greenhouse of the National Institute of Molecular Biology and Biotechnology, Philippines from January to December 2001. Isolates TCaR 61 and TCeRe 60, and Azotobacter vinelandii Mac 259 were selected as test bacteria based on their ability to produce indole-3acetic acid and solubilize precipitated phosphate, which may promote plant growth in the field. These bacteria were marked with gusA reporter gene from Escherichia coli strain S17-1(λ-pir containing mTn5SSgusA21. The gusA (β-glucuronidase gene from the donor (E. coli was transferred to each bacterium (recipient through bacterial conjugation in mating procedures using tryptone-yeast agar followed by the selection of the transconjugants (bacteria receiving gusA in tryptone-yeast agar supplemented with double antibiotics and X-GlcA (5bromo-4chloro- 3indoxyl-β-D-glucuronic acid. The antibiotics used were rifampicin and either streptomycin or spectinomycin based on antibiotic profiles of the donor and recipients. The results showed that the insertion of gusA gene into bacterial genomes of the recipient did not impair its phenotypic traits; the growth rates of the transconjugants as well as their ability to produce indole-3acetic acid and solubilize precipitated phosphate in pure culture were similar to their wild types. All transconjugants colonized the roots of hot pepper (Capsicum annuum L. and survived in the rhizosphere and soil until the late of vegetative growth stage. The distinct blue staining of transconjugants as the expression of gusA gene in media containing X-GlcA coupled with their resistance to rifampicin and streptomycin or spectinomycin

  9. Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

    Science.gov (United States)

    Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C; Merk, Denis R; Lyons, Jennifer K; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N; Ray, Pritha; Patel, Manishkumar; Chang, Ya-Fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C; Dash, Rajesh; Yang, Phillip C; Brinton, Todd J; Yock, Paul G; McConnell, Michael V; Gambhir, Sanjiv S

    2016-09-01

    Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

  10. First report on interferon related developmental regulator-1 from Macrobrachium rosenbergii: bioinformatic analysis and gene expression.

    Science.gov (United States)

    Arockiaraj, Jesu; Easwvaran, Sarasvathi; Vanaraja, Puganeshwaran; Singh, Arun; Othman, Rofina Yasmin; Bhassu, Subha

    2012-05-01

    This study reports the first full length gene of interferon related developmental regulator-1 (designated as MrIRDR-1), identified from the transcriptome of Macrobrachium rosenbergii. The complete gene sequence of the MrIRDR-1 is 2459 base pair long with an open reading frame of 1308 base pairs and encoding a predicted protein of 436 amino acids with a calculated molecular mass of 48 kDa. The MrIRDR-1 protein contains a long interferon related developmental regulator super family domain between 30 and 330. The mRNA expressions of MrIRDR-1 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) infected M. rosenbergii were examined using qRT-PCR. The MrIRDR-1 is highly expressed in hepatopancreas along with all other tissues (walking leg, gills, muscle, haemocyte, pleopods, brain, stomach, intestine and eye stalk). After IHHNV infection, the expression is highly upregulated in hepatopancreas. This result indicates an important role of MrIRDR-1 in prawn defense system.

  11. Short report: polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia.

    Science.gov (United States)

    Huaman, Maria Cecilia; Yoshinaga, Kazumi; Suryanatha, Aan; Suarsana, Nyoman; Kanbara, Hiroji

    2004-07-01

    The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

  12. Involvement of the BDNF gene in loneliness in adolescence: a report of opposite gene effects in boys and girls.

    Directory of Open Access Journals (Sweden)

    Maaike Verhagen

    Full Text Available Previous research has shown that loneliness has a heritable component and that genes within the serotonin-, dopamine-, and oxytocin systems are related to loneliness in adolescence. In the present study, the relation between the BDNF Val66Met polymorphism and loneliness in adolescent boys and girls was examined in a longitudinal study spanning five annual waves (N = 305. Latent growth curve modeling (LGCM was used to examine the baseline level and the change in loneliness over time. The main finding was that the BDNF gene was not related to loneliness in the total sample. A BDNF by sex interaction was found, in that Met carrying girls had the highest levels of loneliness at baseline, whereas in boys the ValVal genotype was related to higher levels of loneliness. Our results underline the importance of sex-stratified analyses when examining effects of the BDNF genotype and the necessity of conducting gene studies to intermediate phenotypes of loneliness.

  13. Development of the 5-HT2CR-Tango System Combined with an EGFP Reporter Gene.

    Science.gov (United States)

    Watanabe, Yoshihisa; Tsujimura, Atsushi; Aoki, Miku; Taguchi, Katsutoshi; Tanaka, Masaki

    2016-02-01

    The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

  14. Not a "reality" show.

    Science.gov (United States)

    Wrong, Terence; Baumgart, Erica

    2013-01-01

    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show.

  15. A novel mutation in the ADA gene causing severe combined immunodeficiency in an Arab patient: a case report

    Directory of Open Access Journals (Sweden)

    Hellani Ali

    2009-04-01

    Full Text Available Abstract Introduction About 20% of the cases of human severe combined immunodeficiency are the result of the child being homozygous for defective genes encoding the enzyme adenosine deaminase. To our knowledge, the mutation pattern in Arab patients with severe combined immunodeficiency has never been reported previously. Case presentation A 14-month-old Arab boy had clinical features typical of severe combined immunodeficiency. His clinical picture and flow cytometric analysis raised the diagnosis of adenosine deaminase deficiency and prompted us to screen the adenosine deaminase gene for mutation(s. We detected a novel mutation in exon 9 of the adenosine deaminase gene (p.Arg282>Gln, which we believe is the cause of the severe combined immunodeficiency phenotype observed in our patient. Conclusion This is the first report of adenosine deaminase mutation in an Arab patient with severe combined immunodeficiency due to a novel pathogenic mutation in the adenosine deaminase gene.

  16. [Homozygous E387K (1159G>A) mutation of the CYP1B1 gene in a Roma boy affected with primary congenital glaucoma. Case report].

    Science.gov (United States)

    Vogt, Gábor; Kádasi, Ľudevit Lajos; Czeizel, Endre

    2014-08-17

    Primary congenital glaucoma was diagnosed in a son (born in 2009) of a healthy, non-consanguineous Roma couple. This couple terminated their next two pregnancies because of the 25% recurrence risk of this autosomal recessive ophthalmological abnormality. Molecular genetic analysis showed the homozygote E387K mutation of the CYP1B1 gene in the proband and the presence of this gene mutation in heterozygous form in both parents. This gene mutation is characteristic for Slovakian Roma population. There are two objectives of this case report. On one hand this finding indicates the genetic relationship of Slovakian and Hungarian Romas. On the other hand, the couple plans to have further pregnancies, and prenatal genetic test may help to assess the possible recurrence risk of this hereditary disease.

  17. Identification of a novel Penicillium chrysogenum rhamnogalacturonan rhamnohydrolase and the first report of a rhamnogalacturonan rhamnohydrolase gene.

    Science.gov (United States)

    Matsumoto, Shotaro; Yamada, Hiroyuki; Kunishige, Yuika; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

    2017-03-01

    Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of β-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.

  18. Phyllodes tumor showing intraductal growth.

    Science.gov (United States)

    Makidono, Akari; Tsunoda, Hiroko; Mori, Miki; Yagata, Hiroshi; Onoda, Yui; Kikuchi, Mari; Nozaki, Taiki; Saida, Yukihisa; Nakamura, Seigo; Suzuki, Koyu

    2013-07-01

    Phyllodes tumor of the breast is a rare fibroepithelial lesion and particularly uncommon in adolescent girls. It is thought to arise from the periductal rather than intralobular stroma. Usually, it is seen as a well-defined mass. Phyllodes tumor showing intraductal growth is extremely rare. Here we report a girl who has a phyllodes tumor with intraductal growth.

  19. Public medical shows.

    Science.gov (United States)

    Walusinski, Olivier

    2014-01-01

    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre.

  20. No effect of high fat diet-induced obesity on spontaneous reporter gene mutations in gpt delta mice.

    Science.gov (United States)

    Takasu, Shinji; Ishii, Yuji; Matsushita, Kohei; Kuroda, Ken; Kijima, Aki; Kodama, Yukio; Ogawa, Kumiko; Umemura, Takashi

    2014-01-01

    A large number of epidemiological studies have demonstrated that obesity is a risk factor for several human cancers. Several animal studies using rodents with diet-induced or genetic obesity have also demonstrated that obesity can promote tumor development. However, the effects of obesity on the early stages of carcinogenesis, and especially on the spontaneous occurrence of somatic gene mutations, remain unclear. To investigate the effects of obesity on the rate of spontaneous gene mutations, we performed reporter gene mutation assays in liver, kidney, and colon, organs in which obesity appears to be associated with cancer development on the basis of epidemiological or animal studies, in mice with high fat diet (HFD)-induced obesity. Six-week-old male and female C57BL/6 gpt delta mice were fed HFD or standard diet (STD) for 13 or 26 weeks. At the end of the experiments, reporter gene mutation assays of liver, kidney, and colon were performed. Final body weights and serum leptin levels of male and female mice fed HFD for 13 or 26 weeks were significantly increased compared with corresponding STD-fed groups. Reporter gene mutation assays of liver, kidney, and colon revealed that there were no significant differences in gpt or Spi- mutant frequencies between STD- and HFD-fed mice in either the 13-week or 26-week groups. These results indicate that HFD treatment and consequent obesity does not appear to influence the spontaneous occurrence of somatic gene mutations.

  1. The Great Cometary Show

    Science.gov (United States)

    2007-01-01

    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

  2. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

    Science.gov (United States)

    Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

    2014-10-01

    Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

  3. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  4. Associations between oxytocin receptor gene (OXTR) polymorphisms and self-reported aggressive behavior and anger: Interactions with alcohol consumption.

    Science.gov (United States)

    Johansson, Ada; Westberg, Lars; Sandnabba, Kenneth; Jern, Patrick; Salo, Benny; Santtila, Pekka

    2012-09-01

    Oxytocin has been implicated in the regulation of social as well as aggressive behaviors, and in a recent study we found that the effect of alcohol on aggressive behavior was moderated by the individual's genotype on an oxytocin receptor gene (OXTR) polymorphism (Johansson et al., 2012). In this study we wanted to deepen and expand the analysis by exploring associations between three (rs1488467, rs4564970, rs1042778) OXTR polymorphisms and aggressive behavior, trait anger as well as anger control in a population-based sample of Finnish men and women (N=3577) aged between 18 and 49 years (M=26.45 years, SD=5.02). A specific aim was to investigate if the polymorphisms would show interactive effects with alcohol consumption on aggressive behavior and trait anger, as well as to explore whether these polymorphisms affect differences in anger control between self-reported sober and intoxicated states. The results showed no main effects of the polymorphisms, however, three interactions between the polymorphisms and alcohol consumption were found. The effect of alcohol consumption on aggressive behavior was moderated by the genotype of the individual on the rs4564970 polymorphism, in line with previous results (Johansson et al., 2012). For trait anger, both the rs1488467 and the rs4564970 polymorphisms interacted with alcohol consumption. It appears that the region of the OXTR gene including both the rs4564970 and the rs1488467 polymorphisms may be involved in the regulation of the relationship between alcohol and aggressive behavior as well as between alcohol and the propensity to react to situations with elevated levels of anger.

  5. Familial migraine: Exclusion of the susceptibility gene from the reported locus of familial hemiplegic migraine on 19p

    Energy Technology Data Exchange (ETDEWEB)

    Hovatta, I.; Peltonen, L. [National Public Health Institute, Helsinki (Finland); Kallela, M.; Faerkkilae, M. [Helsinki Univ. Central Hospital (Finland)

    1994-10-01

    Genetic isolates are highly useful in analyses of the molecular background of complex diseases since the enrichment of a limited number of predisposing genes can be predicted in representative families or in specific geographical regions. It has been suggested that the pathophysiology and etiology of familial hemiplegic migraine (FHM) and typical migraine with aura are most probably the same. Recent assignment of FHM locus to chromosome 19p in two French families makes it now possible to test this hypothesis. We report here linkage data on four families with multiple cases of migraine disorder originating from the genetically isolated population of Finland. We were interested to discover whether the migraine in these families would also show linkage to the markers on 19p. We could exclude a region of 50 cM, flanking the reported FHM locus, as a site of migraine locus in our four families. It seems evident that locus heterogeneity exists between different diagnostic classes of migraine spectrum of diseases and also between different ethnic groups. 10 refs., 2 figs., 1 tab.

  6. A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet

    Directory of Open Access Journals (Sweden)

    Kloos Dorothee U

    2004-12-01

    Full Text Available Abstract Background Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. Results Suppression Subtractive Hybridization (SSH was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12 were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. Conclusions This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed.

  7. 'Obesity Gene' Doesn't Affect Ability to Lose Weight: Report

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_161090.html 'Obesity Gene' Doesn't Affect Ability to Lose Weight: ... 21, 2016 (HealthDay News) -- Having the so-called "obesity gene" doesn't affect people's ability to shed ...

  8. A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.

    Directory of Open Access Journals (Sweden)

    Baris Tursun

    Full Text Available Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination ("recombineering" represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5' and 3' cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.

  9. Developing in vitro reporter gene assays to assess the hormone receptor activities of chemicals frequently detected in drinking water.

    Science.gov (United States)

    Sun, Hong; Si, Chaozong; Bian, Qian; Chen, Xiaodong; Chen, Liansheng; Wang, Xinru

    2012-08-01

    The present study intended to develop receptor-mediated luciferase reporter gene assays to evaluate and compare the estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) activities of target chemicals. Di-2-ethylhexyl-phthalate (DEHP), chlorpyrifos (CPF), 2,4-dichlorophenoxyacetic acid (2,4-D) and bisphenol A (BPA) are some of the most common contaminants in drinking water and are frequently detected in China and worldwide. The chemicals were tested at concentrations of 0.1, 1, 10 and 100 times their maximum contaminant level in drinking water. The results showed that BPA possessed various activities on ER, AR and TR. DEHP and CPF could suppress 17β-estradiol or testosterone activity with different potencies, and DEHP possessed weaker anti-thyroid hormone activity. 2,4-D showed no agonist or antagonist activity against these hormone receptors, but it significantly enhanced the activity of testosterone through AR. Furthermore, the mixture of DEHP and CPF exhibited stronger ER and AR antagonist activities than each single component alone, but their combined effects were less than the expected effects based on the additive model. These results implied that the transcription activation mediated by hormone receptors was the potential endocrine-disrupting mechanism of the test chemicals. Our study also provided useful tools for evaluation of their endocrine disrupting activity.

  10. Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).

    Science.gov (United States)

    Schmid, Claudia; Arndt, Christian; Reifferscheid, Georg

    2003-02-05

    The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

  11. Versatile dual reporter gene systems for investigating stop codon readthrough in plants.

    Directory of Open Access Journals (Sweden)

    Nga T Lao

    Full Text Available BACKGROUND: Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA in an mRNA. However, many viruses (and some cellular mRNAs contain "stop" codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a "readthrough", or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. METHODOLOGY: We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. CONCLUSIONS: We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a 'leaky' UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV readthrough context.

  12. Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg

    2016-01-01

    Background: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major...... roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans...... results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi....

  13. Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression.

    Science.gov (United States)

    Wang, H G; Wang, X F; Jing, X Y; Li, Z; Zhang, Y; Lv, Z J

    2011-08-26

    Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.

  14. Imprinted genes and transpositions: epigenomic targets for low dose radiation effects. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Jirtle, Randy L.

    2012-10-11

    The overall hypothesis of this grant application is that low dose ionizing radiation (LDIR) elicits adaptive responses in part by causing heritable DNA methylation changes in the epigenome. This novel postulate was tested by determining if the level of DNA methylation at the Agouti viable yellow (A{sup vy}) metastable locus is altered, in a dose-dependent manner, by low dose radiation exposure (<10 cGy) during early gestation. This information is particularly important to ascertain given the increased use of CT scans in disease diagnosis, increased number of people predicted to live and work in space, and the present concern about radiological terrorism. We showed for the first time that LDIR significantly increased DNA methylation at the A{sup vy} locus in a sex-specific manner (p=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 cGy and 7.6 cGy with maximum effects at 1.4 cGy and 3.0 cGy (p<0.01). Offspring coat color was concomitantly shifted towards pseudoagouti (p<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring (p<0.05). Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic Avy mouse model epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful. Our findings not only have significant implications concerning the mechanism of hormesis, but they also emphasize the potential importance of this phenomenon in determining human risk at low radiation doses. Since the epigenetic regulation of genes varies markedly between species, the effect of LDIR on other epigenetically labile genes (e.g. imprinted genes) in

  15. An open label phase II study evaluating first-line EGFR tyrosine kinase inhibitor erlotinib in non-small cell lung cancer patients with tumors showing high EGFR gene copy number

    Science.gov (United States)

    Kowalczyk, Anna; Suszko-Kazarnowicz, Malgorzata; Duchnowska, Renata; Szczesna, Aleksandra; Ratajska, Magdalena; Sowa, Aleksander; Limon, Janusz; Biernat, Wojciech; Burzykowski, Tomasz; Jassem, Jacek; Dziadziuszko, Rafal

    2017-01-01

    Background First-line treatment with epidermal growth factor receptor (EGFR) inhibitors in NSCLC is effective in patients with activating EGFR mutations. The activity of erlotinib in patients harboring high EGFR gene copy number has been considered debatable. Patients and Methods A multicenter, open-label, single-arm phase II clinical trial was performed to test the efficacy of erlotinib in the first-line treatment of NSCLC patients harboring high EGFR gene copy number defined as =4 copies in =40% of cells. Findings Between December 2007 and April 2011, tumor samples from 149 subjects were screened for EGFR gene copy number by fluorescence in-situ hybridization (FISH), Out of 49 patients with positive EGFR FISH test, 45 were treated with erlotinib. Median PFS in the intent-to-treat population was 3.3 months (95%CI: 1.83.9 months), and median overall survival was 7.9 months (95% CI: 5.112.6 months). Toxicity profile of erlotinib was consistent with its known safety profile. The trial was stopped prematurely at 63% of originally planned sample size due to accumulating evidence that EGFR gene copy number should not be used to select NSCLC patients to first-line therapy with EGFR TKI. Data on erlotinib efficacy according to EGFR, KRAS and BRAF mutations are additionally presented. Interpretation This trial argues against using high gene copy number for selection of NSCLC patients to first-line therapy with EGFR TKIs. The study adds to the discussion on efficacy of other targeted agents in patients with target gene amplified tumors. PMID:27924059

  16. Asynchronous Bilateral Renal Infarction and Thrombophilia With Associated Gene Mutations in a 43-Year-Old Man: A Case Report.

    Science.gov (United States)

    Zhou, Xu-Jie; Liu, Li-Jun; Chen, Min; Zhou, Fu-De

    2016-04-01

    Renal infarction (RI) is frequently misdiagnosed or diagnosed late because of its rarity and nonspecific clinical presentation, which may result in irreversible damage to the renal parenchyma or increase the risk of other embolic events affecting additional organs. Multiple causal mechanisms and cases of idiopathic RI have been reported, but the causal factors are not clear in most cases.Here, we report the case of a patient with heterochronic bilateral RI caused by thrombophilia. Although he had several risk factors for hypercoagulation disorders, two gene mutations-MTHFR 677 C>T and PLG 1858G>A-were identified by genome sequencing of the entire exome. The findings suggest the possibility of a synergistic relationship between the two gene mutations.Thus, screening for gene mutations may provide additional clues for clarifying the cause of RI and thrombophilia.

  17. Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays.

    Science.gov (United States)

    Kojima, Hiroyuki; Sata, Fumihiro; Takeuchi, Shinji; Sueyoshi, Tatsuya; Nagai, Tadanori

    2011-02-27

    The nuclear receptor, pregnane X receptor (PXR), is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism. Recent studies have shown that PXR activation may affect energy metabolism as well as the endocrine and immune systems. In this study, we characterized and compared the agonistic activities of a variety of pesticides against human PXR (hPXR) and mouse PXR (mPXR). We tested the hPXR and mPXR agonistic activity of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 7 ureas, and 44 others) by reporter gene assays using COS-7 simian kidney cells. Of the 200 pesticides tested, 106 and 93 activated hPXR and mPXR, respectively, and a total of 111 had hPXR and/or mPXR agonistic activity with greater or lesser inter-species differences. Although all of the pyrethroids and most of the organochlorines and acid amides acted as PXR agonists, a wide range of pesticides with diverse structures also showed hPXR and/or mPXR agonistic activity. Among the 200 pesticides, pyributicarb, pretilachlor, piperophos and butamifos for hPXR, and phosalone, prochloraz, pendimethalin, and butamifos for mPXR, acted as particularly potent activators at low concentrations in the order of 10⁻⁸-10⁻⁷ M. In addition, we found that several organophosphorus oxon- and pyributicarb oxon-metabolites decreased PXR activation potency compared to their parent compounds. These results suggest that a large number of structurally diverse pesticides and their metabolites possess PXR-mediated transcriptional activity, and their ability to do so varies in a species-dependent manner in humans and mice. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  18. Pathogenic classification of LPL gene variants reported to be associated with LPL deficiency

    DEFF Research Database (Denmark)

    Rodrigues, Rute; Artieda, Marta; Tejedor, Diego

    2016-01-01

    BACKGROUND: Lipoprotein lipase (LPL) deficiency is a serious lipid disorder of severe hypertriglyceridemia (SHTG) with chylomicronemia. A large number of variants in the LPL gene have been reported but their influence on LPL activity and SHTG has not been completely analyzed. Gaining insight...

  19. Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants

    NARCIS (Netherlands)

    Mirabella, R.; Franken, C.; Krogt, van der G.N.M.; Bisseling, T.; Geurts, R.

    2004-01-01

    Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this

  20. Lenz-Majewski syndrome: Report of a case with novel mutation in PTDSS1 gene.

    Science.gov (United States)

    Tamhankar, Parag M; Vasudevan, Lakshmi; Bansal, Vandana; Menon, Shyla R; Gawde, Harshavardhan M; D'Souza, Aruna; Babu, Shiny; Kondurkar, Shweta; Adhia, Rashmi; Das, Dhanjit Kumar

    2015-08-01

    Lenz-Majewski syndrome (LMS) is an extremely rare syndrome characterized by osteosclerosis, intellectual disability, characteristic facies and distinct craniofacial, dental, cutaneous and distal - limb anomalies. Recently, mutations in PTDSS1 gene have been identified as causative in six unrelated individuals. We report the seventh mutation proven case of LMS and provide a concise review of all known patients till date.

  1. Establishing the lysine-rich protein CEST reporter gene as a CEST MR imaging detector for oncolytic virotherapy

    NARCIS (Netherlands)

    C.T. Farrar (Christian T.); J.S. Buhrman (Jason); G. Liu (Guanshu); A. Kleijn (Anne); M.L.M. Lamfers (Martine); M.T. McMahon (Michael T.); A.A. Gilad (Assaf A.); G. Fulci (Giulia)

    2015-01-01

    textabstractPurpose: To (a) evaluate whether the lysine-rich protein (LRP) magnetic resonance (MR) imaging reporter gene can be engineered into G47Δ, a herpes simplex-derived oncolytic virus that is currently being tested in clinical trials, without disrupting its therapeutic effectiveness and (b) e

  2. Lineage switch in relapse of acute leukemia with rearrangement of MLL gene (KMT2A. literature review and case reports

    Directory of Open Access Journals (Sweden)

    E. A. Zerkalenkova

    2016-01-01

    Full Text Available Lineage switch is a rare phenomenon in which a transition from lymphoid to myeloid was observed in relapse of acute leukemia, or vice versa. This paper presents the four clinical case reports of acute lymphoblastic leukemia with MLL gene rearrangement (KMT2A with myeloid phenotype in relapse.

  3. Stable reporter cell lines for peroxisome proliferator-activated receptor y (PPARy)-mediated modulation of gene expression

    NARCIS (Netherlands)

    Gijsbers, L.; Man, H.Y.; Kloet, S.K.; Haan, de L.H.J.; Keijer, J.; Rietjens, I.; Burg, van der B.J.; Aarts, J.M.M.J.G.

    2011-01-01

    Activation of peroxisome proliferator-activated receptor ¿ (PPAR¿) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement

  4. Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants

    NARCIS (Netherlands)

    Mirabella, R.; Franken, C.; Krogt, van der G.N.M.; Bisseling, T.; Geurts, R.

    2004-01-01

    Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this proble

  5. Using of green fluorescent reporter gene (GFP) to monitor the fate of Fusarium moniliforme mycoparasitized by Trichoderma viride

    Institute of Scientific and Technical Information of China (English)

    ZHU Ting-heng; WANG Wei-xia; WANG Chang-chun; YANG Rui-qin; CAI Xin-zhong

    2004-01-01

    @@ Fusarium moniliforme Sheld. is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus.

  6. LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo

    Science.gov (United States)

    Bozcal, Elif; Dagdeviren, Melih; Uzel, Atac

    2017-01-01

    It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice. PMID:28235077

  7. Tokyo Motor Show 2003; Tokyo Motor Show 2003

    Energy Technology Data Exchange (ETDEWEB)

    Joly, E.

    2004-01-01

    The text which follows present the different techniques exposed during the 37. Tokyo Motor Show. The report points out the great tendencies of developments of the Japanese automobile industry. The hybrid electric-powered vehicles or those equipped with fuel cells have been highlighted by the Japanese manufacturers which allow considerable budgets in the research of less polluting vehicles. The exposed models, although being all different according to the manufacturer, use always a hybrid system: fuel cell/battery. The manufacturers have stressed too on the intelligent systems for navigation and safety as well as on the design and comfort. (O.M.)

  8. Pine Gene Discovery Project - Final Report - 08/31/1997 - 02/28/2001

    Energy Technology Data Exchange (ETDEWEB)

    Whetten, R. W.; Sederoff, R. R.; Kinlaw, C.; Retzel, E.

    2001-04-30

    Integration of pines into the large scope of plant biology research depends on study of pines in parallel with study of annual plants, and on availability of research materials from pine to plant biologists interested in comparing pine with annual plant systems. The objectives of the Pine Gene Discovery Project were to obtain 10,000 partial DNA sequences of genes expressed in loblolly pine, to determine which of those pine genes were similar to known genes from other organisms, and to make the DNA sequences and isolated pine genes available to plant researchers to stimulate integration of pines into the wider scope of plant biology research. Those objectives have been completed, and the results are available to the public. Requests for pine genes have been received from a number of laboratories that would otherwise not have included pine in their research, indicating that progress is being made toward the goal of integrating pine research into the larger molecular biology research community.

  9. An evolutionary conserved region (ECR in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex

    Directory of Open Access Journals (Sweden)

    Haddley Kate

    2011-05-01

    Full Text Available Abstract Background Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs, in which the degree of conservation can be comparable with exonic regions suggesting functional significance. Results We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1 supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 in vitro. Conclusion Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a a strong enhancer that functions in neurons and b a transcription factor that may modulate the function of that enhancer.

  10. Construction of a multiplex promoter reporter platform to monitor Staphylococcus aureus virulence gene expression and the identification of usnic acid as a potent suppressor of psm gene expression

    Directory of Open Access Journals (Sweden)

    Peng GAO

    2016-08-01

    Full Text Available As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  11. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

    Science.gov (United States)

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

  12. [Influence of genetic mutations on clinical presentation of subretinal neovascularization. Report 2: The impact of HTRA and VEGF genes polymorphism].

    Science.gov (United States)

    Budzinskaia, M V; Pogoda, T V; Strelkova, I D; Chikun, E A; Shchegoleva, I V; Kazarian, É É; Galoian, N S

    2011-01-01

    A detailed analysis of influence of HTRA (serine peptidase) and VEGF (vascular endothelial growth factor) genes mutations is presented. The presence of one gene copy with allele of A- polymorphism rs1120638 of HTRA1 gen, T- polymorphism rs10490924 and de11443in54 of ARMS2 gene increases the risk of CNV in patients with AMD. The feature of clinical presentation in patients with CNV associated with (-625) A mutation of promoter region of HTRA1 gene in two chromosomes was fulminant course of the disease from exudative to scarring processes with fibrous tissue formation not just with sub-, but also intra- and preretinal localization. Genetic screening showed that combination of studied mutations (402H, (-625) A and (-251) A in both gene copies of CFH, HTRA and IL-8) results in the most severe and rapidly progressing form of the disease. Two new mutations were revealed in promoter region of VEGF gene: G > A replacement in position of (-72) nucleotide from transcription start and G > A replacement in 5'-nontranslated region of the 1st gene exon in position of (+31) nucleotide from transcription start.

  13. Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks.

    Science.gov (United States)

    de Andrade, Caroline Pinto; de Oliveira, Eduardo Conceição; Leal, Juliano Souza; de Almeida, Laura Lopes; de Castro, Luiza Amaral; da Silva, Sergio Ceroni; Driemeier, David

    2015-08-01

    Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protein that leads to deposition and aggregation in the host's central nervous system. Occurrence of the natural disease can be influenced by host genetic factors, such as a single nucleotide polymorphism of the prion protein gene. This study reports three scrapie-affected Dorper flocks located on three different farms in Brazil. The objective of this study was to analyze these three flocks using scrapie diagnostics, combining histology, immunohistochemistry, genotyping, and western blot assays. For immunohistochemistry, 192 sheep were selected and 308 sheep blood samples were taken for genotyping. A total of 22 sheep were scrapie positive by immunohistochemistry. Of these, four presented clinical signs and had scrapie immunoreactivity at the obex in western blot assays. The sheep without clinical signs were positive in lymphoid organs, such as the third eyelid and rectal mucosa. The major genotypes found on the flocks were ARQ/ARQ, ARQ/ARR, and ARQ/VRQ for codons 136, 154, and 171. Most of the sheep were considered to be at moderate to high risk, based on risk groups for developing scrapie. Some blood samples were sequenced, and polymorphisms were identified in other codons, such as 127, 142, and 143. Our data demonstrate the importance of preclinical scrapie diagnosis in Brazilian sheep, as most of the affected sheep showed no clinical signs, and emphasize the relevance of genotyping other Dorper sheep to determine the genotypic profile of the breed.

  14. Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters.

    Directory of Open Access Journals (Sweden)

    Sunil Laxman

    Full Text Available BACKGROUND: During continuous growth in specific chemostat cultures, budding yeast undergo robust oscillations in oxygen consumption that are accompanied by highly periodic changes in transcript abundance of a majority of genes, in a phenomenon called the Yeast Metabolic Cycle (YMC. This study uses fluorescent reporters of genes specific to different YMC phases in order to visualize this phenomenon and understand the temporal regulation of gene expression at the level of individual cells within the cycling population. METHODOLOGY: Fluorescent gene expression reporters for different phases of the YMC were constructed and stably integrated into the yeast genome. Subsequently, these reporter-expressing yeast were used to visualize YMC dynamics at the individual cell level in cultures grown in a chemostat or in a microfluidics platform under varying glucose concentrations, using fluorescence microscopy and quantitative Western blots. CONCLUSIONS: The behavior of single cells within a metabolic cycling population was visualized using phase-specific fluorescent reporters. The reporters largely recapitulated genome-specified mRNA expression profiles. A significant fraction of the cell population appeared to exhibit basal expression of the reporters, supporting the hypothesis that there are at least two distinct subpopulations of cells within the cycling population. Although approximately half of the cycling population initiated cell division in each permissive window of the YMC, metabolic synchrony of the population was maintained. Using a microfluidics platform we observed that low glucose concentrations appear to be necessary for metabolic cycling. Lastly, we propose that there is a temporal window in the oxidative growth phase of the YMC where the cycling population segregates into at least two subpopulations, one which will enter the cell cycle and one which does not.

  15. Immunoglobulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Honjo, T. (Kyoto Univ. (Japan)); Alt, F.W. (Columbia Univ., Dobbs Ferry, NY (USA). Hudson Labs.); Rabbitts, T.H. (Medical Research Council, Cambridge (UK))

    1989-01-01

    This book reports on the structure, function, and expression of the genes encoding antibodies in normal and neoplastic cells. Topics covered are: B Cells; Organization and rearrangement of immunoglobin genes; Immunoglobin genes in disease; Immunoglobin gene expression; and Immunoglobin-related genes.

  16. Report on emerging technologies for translational bioinformatics: a symposium on gene expression profiling for archival tissues

    Directory of Open Access Journals (Sweden)

    Waldron Levi

    2012-03-01

    Full Text Available Abstract Background With over 20 million formalin-fixed, paraffin-embedded (FFPE tissue samples archived each year in the United States alone, archival tissues remain a vast and under-utilized resource in the genomic study of cancer. Technologies have recently been introduced for whole-transcriptome amplification and microarray analysis of degraded mRNA fragments from FFPE samples, and studies of these platforms have only recently begun to enter the published literature. Results The Emerging Technologies for Translational Bioinformatics symposium on gene expression profiling for archival tissues featured presentations of two large-scale FFPE expression profiling studies (each involving over 1,000 samples, overviews of several smaller studies, and representatives from three leading companies in the field (Illumina, Affymetrix, and NuGEN. The meeting highlighted challenges in the analysis of expression data from archival tissues and strategies being developed to overcome them. In particular, speakers reported higher rates of clinical sample failure (from 10% to 70% than are typical for fresh-frozen tissues, as well as more frequent probe failure for individual samples. The symposium program is available at http://www.hsph.harvard.edu/ffpe. Conclusions Multiple solutions now exist for whole-genome expression profiling of FFPE tissues, including both microarray- and sequencing-based platforms. Several studies have reported their successful application, but substantial challenges and risks still exist. Symposium speakers presented novel methodology for analysis of FFPE expression data and suggestions for improving data recovery and quality assessment in pre-analytical stages. Research presentations emphasized the need for careful study design, including the use of pilot studies, replication, and randomization of samples among batches, as well as careful attention to data quality control. Regardless of any limitations in quantitave transcriptomics for

  17. Trypsinization severely perturbs radioiodide transport via membrane Na/I symporter proteolysis: implications for reporter gene imaging

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Kyung-Ho [Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710 (Korea, Republic of)], E-mail: jkhope.jung@sbri.co.kr; Paik, Jin-Young [Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710 (Korea, Republic of); Lee, You La; Lee, Yong Jin; Lee, Jaetae [Department of Nuclear Medicine and Pharmacology, School of Medicine, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Lee, Kyung-Han [Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710 (Korea, Republic of)], E-mail: khnm.lee@samsung.com

    2009-11-15

    Introduction: Cell preparation procedures injurious to Na/I symporters (NIS) could deter their usefulness for reporter gene assays and in vivo cell imaging. In this study, we investigated the effects of cell collection by trypsinization on radioiodide transport and in vivo cell imaging results. Methods: The influence of trypsinization procedures on {sup 125}I transport was evaluated using Huh-7/NIS hepatoma cells. The effects of graded concentrations of trypsin and EDTA were assessed on Huh-7/NIS and A431/NIS lung cancer cells. Trypsin-induced NIS proteolysis was investigated by immunoblots of plasma membrane prepared from adenovirus-infected mouse liver tissue. {sup 99m}Tc-O{sub 4}{sup -} scintigraphy was performed in Balb/C nude mice at 1 and 4 h following administration of Huh-7/NIS cells collected with and without trypsin. Results: {sup 125}I Transport ability of Huh-7/NIS cells was severely impaired within minutes of standard trypsinization and further deteriorated up to 24 h after termination of treatment. This perturbation was caused by trypsin, which dose- and time-dependently induced substantial reductions of {sup 125}I uptake in Huh-7/NIS and A431/NIS cells. Immunoblot analysis revealed significant dose- and time-dependent losses of membrane NIS protein by trypsin. NIS proteolysis was completely blocked by soybean trypsin inhibitor, and partial protection was offered by the substrates iodide and perchlorate. On {sup 99m}Tc-O{sub 4}{sup -} scintigraphy of mice, cells prepared by trypsinization were poorly visualized, whereas those collected with a nonenzymatic method showed significantly better uptake and contrast. Conclusion: Trypsinization leads to serious perturbations in iodide accumulating capacity through tryptic degradation of membrane NIS protein. Hence, NIS-based reporter assays and in vivo cell imaging studies may benefit from better-optimized cell cultivation and harvesting procedures.

  18. Unintentional miRNA ablation is a risk factor in gene knockout studies: a short report.

    Directory of Open Access Journals (Sweden)

    Ivan Osokine

    2008-02-01

    Full Text Available One of the most powerful techniques for studying the function of a gene is to disrupt the expression of that gene using genetic engineering strategies such as targeted recombination or viral integration of gene trap cassettes. The tremendous utility of these tools was recognized this year with the awarding of the Nobel Prize in Physiology or Medicine to Capecchi, Evans, and Smithies for their pioneering work in targeted recombination mutagenesis in mammals. Another noteworthy discovery made nearly a decade ago was the identification of a novel class of non-coding genes called microRNAs. MicroRNAs are among the largest known classes of regulatory elements with more than 1000 predicted to exist in the mouse genome. Over 50% of known microRNAs are located within introns of coding genes. Given that currently about half of the genes in mouse have been knocked out, we investigated the possibility that intronic microRNAs may have been coincidentally deleted or disrupted in some of these mouse models. We searched published murine knockout studies and gene trap embryonic stem cell line databases for cases where a microRNA was located within or near the manipulated genomic loci, finding almost 200 cases where microRNA expression may have been disrupted along with another gene. Our results draw attention to the need for careful planning in future knockout studies to minimize the unintentional disruption of microRNAs. These data also raise the possibility that many knockout studies may need to be reexamined to determine if loss of a microRNA contributes to the phenotypic consequences attributed to loss of a protein-encoding gene.

  19. Concomitant homozygosity for the prothrombin gene variant with mild deficiency of antithrombin III in a patient with multiple hepatic infarctions: a case report

    Directory of Open Access Journals (Sweden)

    Macheta M

    2010-04-01

    Full Text Available Abstract Introduction Hereditary causes of visceral thrombosis or thrombosis should be sought among young patients. We present a case of a young man presenting with multiple hepatic infarctions resulting in portal hypertension due to homozygosity of the prothrombin gene mutation not previously described in literature. Case presentation A 42-year-old Caucasian man with a previous history of idiopathic deep vein thrombosis 11 years earlier presented with vague abdominal pains and mildly abnormal liver function tests. An ultrasound and computed tomography scan showed evidence of hepatic infarction and portal hypertension (splenic varices. A thrombophilia screen confirmed a homozygous mutation for the prothrombin gene mutation, with mildly reduced levels of anti-thrombin III (AT III. Subsequent testing of his father and brother revealed heterozygosity for the same gene mutation. Conclusion Hepatic infarction is unusual due to the rich dual arterial and venous blood supply to the liver. In the absence of an arterial or haemodynamic insult causing hepatic infarction, a thrombophilia should be considered. To our knowledge, this is the first reported case of a hepatic infarction due to homozygosity of the prothrombin gene mutation. It is unclear whether homozygotes have a higher risk of thrombosis than heterozygotes. In someone presenting with a first thrombosis with this mutation, the case for life-long anticoagulation is unclear, but it may be necessary to prevent a second and more severe second thrombotic event, as occurred in this case.

  20. In vitro reporter gene assays for assessment of PPAR- and Nrf2-mediated health effects of tomato and its bioactive constituents

    OpenAIRE

    Gijsbers, L.

    2013-01-01

    The consumption of food products with health-promoting properties, such as for example margarines with plant sterols, fruit juice enriched with calcium and cereals with (soluble) fibre, has increased rapidly during the last years. The present thesis provides proof-of-principle that reporter gene assays are effective tools to investigate the effects of functional foods and food compounds on gene expression pathways. In order to test fruits and vegetables for selected functions in reporter gene...

  1. Genome-wide gene expression profiling and a forward genetic screen show that differential expression of the sodium ion transporter Ena21 contributes to the differential tolerance of Candida albicans and Candida dubliniensis to osmotic stress.

    LENUS (Irish Health Repository)

    Enjalbert, Brice

    2009-04-01

    Candida albicans is more pathogenic than Candida dubliniensis. However, this disparity in virulence is surprising given the high level of sequence conservation and the wide range of phenotypic traits shared by these two species. Increased sensitivity to environmental stresses has been suggested to be a possible contributory factor to the lower virulence of C. dubliniensis. In this study, we investigated, in the first comparison of C. albicans and C. dubliniensis by transcriptional profiling, global gene expression in each species when grown under conditions in which the two species exhibit differential stress tolerance. The profiles revealed similar core responses to stresses in both species, but differences in the amplitude of the general transcriptional responses to thermal, salt and oxidative stress. Differences in the regulation of specific stress genes were observed between the two species. In particular, ENA21, encoding a sodium ion transporter, was strongly induced in C. albicans but not in C. dubliniensis. In addition, ENA21 was identified in a forward genetic screen for C. albicans genomic sequences that increase salt tolerance in C. dubliniensis. Introduction of a single copy of CaENA21 was subsequently shown to be sufficient to confer salt tolerance upon C. dubliniensis.

  2. Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Michelmore, R.

    1994-09-01

    The goal of this project was to develop a transposon mutagenesis system for lettuce and to clone and characterize disease resistance genes by transposon tagging. The majority of studies were conducted with the Ac/Ds System. Researchers made and tested several constructs as well as utilized constructions shown to be functional in other plant species. Researchers demonstrated movement of Ac and DS in lettuce; however, they transposed at much lower frequencies in lettuce than in other plant species. Therefore, further manipulation of the system, particularly for flower specific expression of transposase, is required before a routine transposon system is available for lettuce. Populations of lettuce were generated and screened to test for the stability of resistance genes and several spontaneous mutations were isolated. Researchers also identified a resistance gene mutant in plants transformed with a Ds element and chimeric transposase gene. This is currently being characterized in detail.

  3. Bardet-Biedl syndrome in Denmark-report of 13 novel sequence variations in six genes

    DEFF Research Database (Denmark)

    Hjortshøj, Tina Duelund; Grønskov, Karen; Philp, Alisdair R

    2010-01-01

    Bardet-Biedl syndrome (BBS) is an autosomal recessive disease characterized by retinal dystrophy, polydactyly, obesity, learning disabilities, renal involvement, and male hypogenitalism. BBS is genetically heterogeneous with mutations of 14 genes, accounting for approximately 70% of cases. Triall...

  4. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    Energy Technology Data Exchange (ETDEWEB)

    Okita, T.W.

    1999-04-01

    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  5. Polymorphisms in stromal genes and susceptibility to serous epithelial ovarian cancer: a report from the Ovarian Cancer Association Consortium

    DEFF Research Database (Denmark)

    Amankwah, Ernest K; Wang, Qinggang; Schildkraut, Joellen M

    2011-01-01

    Alterations in stromal tissue components can inhibit or promote epithelial tumorigenesis. Decorin (DCN) and lumican (LUM) show reduced stromal expression in serous epithelial ovarian cancer (sEOC). We hypothesized that common variants in these genes associate with risk. Associations with sEOC among...

  6. Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Gabriel Rinaldi

    Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

  7. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae. Progress report, March 1993

    Energy Technology Data Exchange (ETDEWEB)

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the {beta}-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate {beta}-carboxy-cis,cis-muconate. {beta}-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for {beta}-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to {beta}-carboxy-cis,cis-muconate.

  8. Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Dudaladava Volha

    2006-01-01

    Full Text Available Abstract Global analysis of gene expression by DNA microarrays is nowadays a widely used tool, especially relevant for cancer research. It helps the understanding of complex biology of cancer tissue, allows identification of novel molecular markers, reveals previously unknown molecular subtypes of cancer that differ by clinical features like drug susceptibility or general prognosis. Our aim was to compare gene expression profiles in breast cancer that develop against a background of inherited predisposing mutations versus sporadic breast cancer. In this preliminary study we analysed seven hereditary, BRCA1 mutation-linked breast cancer tissues and seven sporadic cases that were carefully matched by histopathology and ER status. Additionally, we analysed 6 samples of normal breast tissue. We found that while the difference in gene expression profiles between tumour tissue and normal breast can be easily recognized by unsupervised algorithms, the difference between those two types of tumours is more discrete. However, by supervised methods of data analysis, we were able to select a set of genes that may differentiate between hereditary and sporadic tumours. The most significant difference concerns genes that code for proteins engaged in regulation of transcription, cellular metabolism, signalling, proliferation and cell death. Microarray results for chosen genes (TOB1, SEPHS2 were validated by real-time RT-PCR.

  9. Infection of Atlantic salmon with Moritella viscosus compared to a mechanical tissue injury model in rainbow trout show similar expression patterns of cytokine genes and may be related to triggering of the same signaling pathways

    DEFF Research Database (Denmark)

    Physical damage of tissue and multiple kinds of infections are found to cause inflammatory reactions in mammals. Regardless of the difference between non-pathogenic induced tissue damage and a bacterial infection, many of the same pathways and genes are triggered. To determine if the same...... was sampled from infected fish at 4, 7 and 14 days post infection. Samples were obtained from site of lesions and from locations without clinical signs of disease and lesions. To compare the inflammatory reactions from infected fish relative to sterile, mechanical tissue damage, rainbow trout (Oncorhynchus...... mykiss) were subjected to controlled tissue disruption applying sterile needles to skin and muscle tissue to one side of the fish. Samples were taken 4, 8, 24 hours and 7 days post injury from both the injured side and non injured site, internal control. From both studies, the samples were subject...

  10. In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

    Directory of Open Access Journals (Sweden)

    Hornsey Mark A

    2006-05-01

    Full Text Available Abstract Background Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. Results The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8 and mesenchymal (smooth muscle actin markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase, goblet cells (Periodic Acid Schiff positive, enteroendocrine cells (chromogranin A and Paneth cells (lysozyme. Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. Conclusion We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up

  11. Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker

    Directory of Open Access Journals (Sweden)

    Ning Zhangyong

    2011-12-01

    Full Text Available Abstract Background Reporter genes are often used as a selectable marker for generation of recombinant viruses in order to investigate the mechanism of pathogenesis and to obtain candidate vaccine viruses. Routine selection of the recombinant parapoxvirus is time-consuming and labor intensive. Therefore, developing a novel method for selection is critical. Results In this study, we developed a rapid method to generate recombinant Orf viruses (ORFV based on the enhanced green fluorescent protein (EGFP reporter gene as a selectable marker. The coding sequence of EGFP gene was amplified from pEGFP-N1 vector and subcloned into the pZIPPY-neo/gus plasmid under the control of the early-late vaccinia virus (VACV VV7.5 promoter and flanked by two multiple cloning sites (MCS to generate a novel transfer vector pSPV-EGFP. Using the pSPV-EGFP, two recombination cassettes pSPV-113LF-EGFP-113RF and pSPV-116LF-EGFP-116RF were constructed by cloning the flanking regions of the ORFV113 and ORFV116 and inserted into two MCS flanking the EGFP gene. Using this novel system, two single gene deletion mutants OV-IA82Δ113 and OV-IA82Δ116 were successfully generated. Conclusions This approach shortens the time needed to generate recombinant ORFVs (rORFVs. Thus, the pSPV-EGFP vector provides a direct, fast, and convenient way to manipulate the recombinant viruses, indicating that it is highly suited for its designed purpose.

  12. Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992

    Energy Technology Data Exchange (ETDEWEB)

    Michelmore, R.

    1994-06-01

    Our goal is to clone genes in lettuce determining resistance to downy mildew. One approach involves the mobilization of transposons into resistance genes to mutate and tag the target gene. Because transposons have yet to be isolated and characterized from lettuce, the majority of our experiments have involved Ac from corn as this is increasingly the best characterized transposon. Over the past several years, various labs have contributed to a detailed understanding of the biology of Ac in corn and heterologous plant species. We have collaborated closely with several of these labs, exchanged materials and incorporated their advances into our analysis of transposition in lettuce. The original proposal described the development of a transposon mutagenesis system for lettuce and its subsequent use to tag disease resistance genes. The development phase involved characterization and manipulation of Ac transposition, identification of suitable whole plant selectable markers for the construction of chimeric non-autonomous elements, and investigation of the stability of resistance genes. Investigation of Ac transposition in lettuce has received the majority of our attention. Initially, we made a simple construct with wildtype Ac and introduced it into lettuce. No transposition was observed; although other labs demonstrated that the same construct was functional in tomato. We then focused on assaying for Ac transposition with constructs of increasing sophistication that had been demonstrated by others to be functional in other species. The latest constructs for transposon mutagenesis clearly demonstrated transposition in lettuce. This allowed us to generate seed stocks that we will start to screen for insertional inactivation of resistance genes this year.

  13. A novel SNP in 3' UTR of INS gene: A case report of neonatal diabetes mellitus.

    Science.gov (United States)

    Bogari, Neda M; Rayes, Husni H; Mostafa, Fakri; Abdel-Latif, Azza M; Ramadan, Abeer; Al-Allaf, Faisal A; Taher, Mohiuddin M; Fawzy, Ahmed

    2015-09-01

    Neonatal diabetes mellitus (NDM) is a rare condition with a prevalence of 1 in 300,000 live births. We have found 3 known SNPs in 5'UTR and a novel SNP in 3' UTR in the INS gene. These SNPs were present in 9-month-old girl from Saudi Arabia and also present in the father and mother. The novel SNP we found is not present in 1000 Genome project or other databases. Further, the newly identified 3' UTR mutation in the INS gene may abolish the polyadenylation signal and result in severe RNA instability.

  14. First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis.

    Science.gov (United States)

    Chen, Lei; Zhang, Honghai; Liu, Guangshuai; Sha, Weilai

    2016-05-01

    The aim of this study was to investigate the bacterial community in the distal gut of dholes (Cuon alpinus) based on the analysis of bacterial 16S rRNA gene sequences. Fecal samples were collected from five healthy unrelated dholes captured from Qilian Mountain in Gansu province of China. The diversity of the fecal bacteria community was investigated by constructing a polymerase chain reaction (PCR)-amplified 16S rRNA gene clone library. Bacterial 16S rRNA gene was amplified by using universal bacterial primers 27F and 1492R. A total of 275 chimera-free near full length 16S rRNA gene sequences were collected, and 78 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified according to the 97 % sequence similarity. Forty-two OTUs (53.8 %) showed less than 98 % sequence similarity to 16S rRNA gene sequences reported previously. Phylogenetic analysis demonstrated that dhole bacterial community comprised five different phyla, with the majority of sequences being classified within the phylum Bacteroidetes (64.7 %), followed by Firmicutes (29.8 %), Fusobacteria (4.7 %),Proteobacteria (0.4 %), and Actinobacteria (0.4 %). The only order Bacteroidales in phylum Bacteroidetes was the most abundant bacterial group in the intestinal bacterial community of dholes. Firmicutes and Bacteroidetes were the two most diverse bacterial phyla with 46.2 and 44.9 % of OTUs contained, respectively. Bacteroidales and Clostridiales were the two most diverse bacterial orders that contained 44.9 and 39.7 % of OTUs, respectively.

  15. A Variant Form of the Human Deleted in Malignant Brain Tumor 1 (DMBT1) Gene Shows Increased Expression in Inflammatory Bowel Diseases and Interacts with Dimeric Trefoil Factor 3 (TFF3)

    DEFF Research Database (Denmark)

    Madsen, Jens; Sorensen, Grith Lykke; Nielsen, Ole Stig;

    2013-01-01

    The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human......-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue...

  16. First report of a de novo germline mutation in the MLH1 gene

    NARCIS (Netherlands)

    Stulp, Rein P; Vos, Yvonne J; Mol, Bart; Karrenbeld, Arend; de Raad, Monique; van der Mijle, Huub J C; Sijmons, Rolf H

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC) is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast m

  17. Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene

    Science.gov (United States)

    An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were tr...

  18. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    Science.gov (United States)

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

  19. Evidence of associations between cytokine genes and subjective reports of sleep disturbance in oncology patients and their family caregivers.

    Directory of Open Access Journals (Sweden)

    Christine Miaskowski

    Full Text Available The purposes of this study were to identify distinct latent classes of individuals based on subjective reports of sleep disturbance; to examine differences in demographic, clinical, and symptom characteristics between the latent classes; and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. Among 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their FCs, growth mixture modeling (GMM was used to identify latent classes of individuals based on General Sleep Disturbance Scale (GSDS obtained prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs and haplotypes in candidate cytokine genes were interrogated for differences between the two latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on GSDS group membership. Two latent classes were identified: lower sleep disturbance (88.5% and higher sleep disturbance (11.5%. Participants who were younger and had a lower Karnofsky Performance status score were more likely to be in the higher sleep disturbance class. Variation in two cytokine genes (i.e., IL6, NFKB predicted latent class membership. Evidence was found for latent classes with distinct sleep disturbance trajectories. Unique genetic markers in cytokine genes may partially explain the interindividual heterogeneity characterizing these trajectories.

  20. Molecular advances in reporter genes: the need to witness the function of stem cells in failing heart in vivo.

    Science.gov (United States)

    Agostini, Silvia; Recchia, Fabio A; Lionetti, Vincenzo

    2012-06-01

    Stem cells possess the ability to terminally differentiate in cell phenotypes belonging to several different lineages. Over the last decade, transplant of adult stem cells into the injuried myocardium has been widely studied as a revolutionary approach to promote the non-pharmacological improvement or replacement of the lost function. In spite of the tantalizing perspectives and controversial results, several questions about the viability and biology of transplanted stem cells in the beating heart still remain unanswered, mostly because of the current technological limitations. Recent advances in bio- and nano-technology are allowing the development of molecular probes for imaging thus providing a better understanding of stem cells physiology and fate in vivo. Reporter gene based molecular imaging is a high-throughput and sensitive tool used to unscramble over time the mechanisms underlying cell-induced myocardial repair in vivo. To date, the employed reporter genes have been exogenous (proteins which are expressed after gene engineering), or endogenous (detected by tracer substrates). This review will highlight current and outstanding experimental investigations, which are developing new probes to monitor the fate of stem cells transplanted in failing myocardium in vivo.

  1. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika

    2009-06-24

    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  2. Identification of the first in Poland CACNA1A gene mutation in familial hemiplegic migraine. Case report.

    Science.gov (United States)

    Kierdaszuk, Biruta; Dziewulska, Dorota; Pronicka, Ewa; Trubicka, Joanna; Rokicki, Dariusz; Piekutowska-Abramczuk, Dorota; Kaliszewska, Magdalena; Tonska, Katarzyna; Bartnik, Ewa; Ploski, Rafal; Kaminska, Anna M

    Migraine is a common neurological disorder characterized by a particular phenotype, complex pathophysiology and a heterogeneous genetic background. Among several heritable forms, familial hemiplegic migraine is the best described one. In the majority of cases it is caused by mutations in one of three different genes. Clinical symptoms of a 47 year old proband (and independently described in his 20 year old son) as well as differential diagnosis are discussed in the presented report. The most characteristic were recurrent attacks of blurred vision, paresthesias and hemiparesis often accompanied by speech disturbances and followed by severe headache with vomiting. Advanced morphological and genetic procedures were required to exclude MELAS, CADASIL and Call-Fleming syndrome. Finally, the definite diagnosis was possible after the application of the whole exome sequencing technique. It confirmed, for the first time in the Polish population, a heterozygous T666M mutation (c.1997C>T; p.Thr666Met) in the CACNA1A gene in the proband, the proband's son and in several other family members. The presented report provides clinical and genetic insight into familial hemiplegic migraine 1 resulting from a mutation in the CACNA1A gene. Copyright © 2017 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging

    Directory of Open Access Journals (Sweden)

    Vladimir Ponomarev

    2003-05-01

    Full Text Available To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSVi-tk/GFP. Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS, the addition of a nuclear export signal (NES, a combination of both mutation types, and a truncation of the first 135 by of the native hsvl-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improved metabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU. The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and γ-camera.

  4. Majewski osteodysplastic primordial dwarfism type II (MOPD II) syndrome previously diagnosed as Seckel syndrome: report of a novel mutation of the PCNT gene.

    Science.gov (United States)

    Piane, Maria; Della Monica, Matteo; Piatelli, Gianluca; Lulli, Patrizia; Lonardo, Fortunato; Chessa, Luciana; Scarano, Gioacchino

    2009-11-01

    We report on a 3-year-old boy with prenatal onset of proportionate dwarfism, postnatal severe microcephaly, high forehead with receded hairline, sparse scalp hair, beaked nose, mild retrognathia and hypotonia diagnosed at birth as Seckel syndrome. At age 3 years, he became paralyzed due to a cerebrovascular malformation. Based on the clinical and radiological features showing evidence of skeletal dysplasia, the diagnosis was revised to Majewski osteodysplastic primordial dwarfism type II (MOPD II) syndrome. Western blot analysis of the patient's lymphoblastoid cell line lysate showed the absence of the protein pericentrin. Subsequent molecular analysis identified a novel homozygous single base insertion (c.1527_1528insA) in exon 10 of the PCNT gene, which leads to a frameshift (Treo510fs) and to premature protein truncation. PCNT mutations must be considered diagnostic of MOPD II syndrome. A possible role of pericentrin in the development of cerebral vessels is suggested. Copyright 2009 Wiley-Liss, Inc.

  5. The second report of a new hypomyelinating disease due to a defect in the VPS11 gene discloses a massive lysosomal involvement.

    Science.gov (United States)

    Hörtnagel, Konstanze; Krägeloh-Mann, Inge; Bornemann, Antje; Döcker, Miriam; Biskup, Saskia; Mayrhofer, Heidi; Battke, Florian; du Bois, Gabriele; Harzer, Klaus

    2016-11-01

    Vesicular protein sorting-associated proteins (VPS, including VPS11) are indispensable in the endocytic network, in particular the endosome-lysosome biogenesis. Exome sequencing revealed the homozygous variant p.Leu387_ Gly395del in the VPS11 gene in two siblings. On immunoblotting, the mutant VPS11 protein showed a distinctly reduced immunostaining intensity. The children presented with primary and severe developmental delay associated with myoclonic seizures, spastic tetraplegia, trunk and neck hypotonia, blindness, hearing loss, and microcephaly. Neuro-imaging showed severe hypomyelination affecting cerebral and cerebellar white matter and corpus callosum, in the absence of a peripheral neuropathy. Electron microscopy of a skin biopsy revealed clusters of membranous cytoplasmic bodies in dermal unmyelinated nerve axons, and numbers of vacuoles in eccrine sweat glands, similar to what is seen in a classic lysosomal storage disease (LSD). Bone marrow cytology showed a high number of storage macrophages with a micro-vacuolated cytoplasm. Biochemically, changes in urinary glycosphingolipids were reminiscent of those in prosaposin deficiency (another LSD). The clinical and neuro-imaged features in our patients were almost identical to those in some recently reported patients with another variant in the VPS11 gene, p.Cys846Gly; underlining the presumed pathogenic potential of VPS11 defects. A new feature was the morphological evidence for lysosomal storage in VPS11 deficiency: This newly characterised disease can be viewed as belonging to the complex field of LSD.

  6. Orengedoku-to augmentation in cases showing partial response to yokukan-san treatment: a case report and literature review of the evidence for use of these Kampo herbal formulae

    Directory of Open Access Journals (Sweden)

    Okamoto H

    2013-01-01

    Full Text Available Hideki Okamoto,1 Atsushi Chino,1 Yoshiro Hirasaki,1 Keigo Ueda,1 Masaomi Iyo,2 Takao Namiki11Department of Japanese-Oriental (Kampo Medicine, Chiba University Graduate School of Medicine, Chiba City, Japan; 2Department of Psychiatry, Chiba University Graduate School of Medicine, Chiba City, Japan Background: Yokukan-san, a Japanese traditional herbal (Kampo prescription, has recently gathered increasing attention due to accumulating reports showing its remarkable efficacy in treating a wide variety of diseases refractory to conventional medicine as well as the behavioral and psychological symptoms of dementia. As yokukan-san has become broadly integrated with conventional medicine, augmentation therapy with other Kampo prescriptions has become necessary when the yokukan-san has been only partially efficacious. In this paper, we report three cases in which the addition of orengedoku-to, another Kampo formula, to yokukan-san was remarkably effective.Cases: Case 1 was an 85-year-old man with Alzheimer-type dementia who had become aggressive during the past 2 years. Three milligrams of aripiprazole completely suppressed his problematic behaviors but had to be stopped because of extrapyramidal symptoms. In the second case, a 44-year-old man with methamphetamine-induced psychosis had suffered from serious tardive dystonia for 2 years. No conventional approach had improved his tardive dystonia. The third case was a 29-year-old engineer who often failed to resist aggressive impulses and was diagnosed with intermittent explosive disorder. He was prescribed 5 mg of olanzapine, which did not suppress his extraordinary anger and caused somnolence even though the dose was low.Interventions and outcomes: Yokukan-san was complementarily added to the patients' regular medication and exerted a definitive but partial effect in all cases. The addition of orengedoku-to to yokukan-san exerted the same efficacy as aripiprazole in controlling aggressiveness in Case 1

  7. Familial Hemophagocytic Lymphohistiocytosis with A665 G Perforin Gene Mutation: A Case Report

    Directory of Open Access Journals (Sweden)

    Idil Yenicesu

    2012-09-01

    Full Text Available Familial hemophagocytic lymphohistiocytosis (FHL is a genetically heterogeneous disease. Presentation of the disease such as primarily fever, hepatosplenomegaly, and cytopenia, which are the results of functional degradation in cytotoxic T-lymphocytes and natural killer cells, activation of macrophages and T-lymphocytes, over production of proinflammatory cytokines, and hemophagocytosis. In all, 5 genetic loci have been identified in FHL, and all known affected genes encode critical components of the granule exocytosis pathway, which is essential for the release of cytotoxic granules and proteases that are necessary for targeted cell death. Herein we present an FHL patient with a severe clinical course and a very rare perforin gene mutation. The patient was homozygous for A665G mutation. However, the child died in a short period of time. Prenatal diagnosis was performed in the family and the fetus was found to be heterozygous for the mutation.

  8. In vivo Monitoring of microRNA Biogenesis Using Reporter Gene Imaging

    OpenAIRE

    2013-01-01

    MicroRNAs are small noncoding RNAs regulating gene expression, through base paring with their target mRNAs, which have been actively investigated as key regulators in a wide range of biological processes. Conventional methods such as Northern blot are generally time-consuming, non-repeatable, and cannot be applied in vivo due to the requirement for cell fixation. Therefore, a noninvasive imaging system is required for the monitoring of microRNA biogenesis to understand the versatile functions...

  9. A variant form of the human deleted in malignant brain tumor 1 (DMBT1 gene shows increased expression in inflammatory bowel diseases and interacts with dimeric trefoil factor 3 (TFF3.

    Directory of Open Access Journals (Sweden)

    Jens Madsen

    Full Text Available The protein deleted in malignant brain tumors (DMBT1 and the trefoil factor (TFF proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340. Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6 reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340 positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (p<0.0001 and Crohn's disease (p = 0.006 compared to normal colon tissue. Immunohistochemical analysis of trefoil factor TFF1, 2 and 3 showed that TFF1 and 3 localized to goblet cells in both normal colon tissue and in tissue from patients with ulcerative colitis or Crohn's disease. No staining for TFF2 was seen in goblet cells in normal colon tissue whereas the majority of tissue sections in ulcerative colitis and Crohn's disease showed sparse and scattered TFF2 positive goblet cells. DMBT1 and TFF proteins did therefore not co-localize in the same cells but localized in adjacent cells in the colon. The interaction between DMBT1(gp340 and trefoil TFFs proteins was investigated using an ELISA assay. DMBT1(gp340 bound to solid-phase bound recombinant dimeric TFF3 in a calcium dependent manner (p<0.0001 but did not bind to recombinant forms of monomeric TFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.

  10. [Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Guerinot, M.L.

    1992-06-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  11. Single-gene dual-color reporter cell line to analyze RNA synthesis in vivo.

    Science.gov (United States)

    Palangat, Murali; Larson, Daniel R

    2016-07-01

    RNA synthesis occurs through the multi-step process of transcription which consists of initiation, elongation, termination, and cleavage of the nascent RNA. In recent years, post-initiation events have attracted considerable attention as regulatory steps in gene expression. In particular, changes in elongation rate have been proposed to alter RNA fate either through changes in RNA secondary structure or recruitment of trans-acting factors, but systematic approaches for perturbing and measuring elongation rate are currently lacking. Here, we describe a system for precisely measuring elongation dynamics for single nascent transcripts at a single gene locus in human cell lines. The system is based on observing the production of fluorescently labeled RNA stem loops which flank a region of interest. The region of interest can be altered using flp recombinases, thus allowing one to study the effects of cis-acting sequences on transcription rate. The dual-color RNAs which are made during this process are exported and translated, thus enabling visualization of each step in gene expression.

  12. A variant form of the human deleted in malignant brain tumor 1 (DMBT1) gene shows increased expression in inflammatory bowel diseases and interacts with dimeric trefoil factor 3 (TFF3).

    Science.gov (United States)

    Madsen, Jens; Sorensen, Grith Lykke; Nielsen, Ole; Tornøe, Ida; Thim, Lars; Fenger, Claus; Mollenhauer, Jan; Holmskov, Uffe

    2013-01-01

    The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340). Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6) reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (pTFF3 in a calcium dependent manner (pTFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.

  13. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  14. Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs

    Directory of Open Access Journals (Sweden)

    Danae Campos-Melo

    2014-09-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs.

  15. Molecular Screening of "MECP2" Gene in a Cohort of Lebanese Patients Suspected with Rett Syndrome: Report on a Mild Case with a Novel Indel Mutation

    Science.gov (United States)

    Corbani, S.; Chouery, E.; Fayyad, J.; Fawaz, A.; El Tourjuman, O.; Badens, C.; Lacoste, C.; Delague, V.; Megarbane, A.

    2012-01-01

    Background: Rett syndrome (RTT), an X-linked, dominant, neurodevelopment disorder represents 10% of female subjects with profound intellectual disability. Mutations in the "MECP2" gene are responsible for up to 95% of the classical RTT cases, and nearly 500 different mutations distributed throughout the gene have been reported. Methods: We report…

  16. Molecular Screening of "MECP2" Gene in a Cohort of Lebanese Patients Suspected with Rett Syndrome: Report on a Mild Case with a Novel Indel Mutation

    Science.gov (United States)

    Corbani, S.; Chouery, E.; Fayyad, J.; Fawaz, A.; El Tourjuman, O.; Badens, C.; Lacoste, C.; Delague, V.; Megarbane, A.

    2012-01-01

    Background: Rett syndrome (RTT), an X-linked, dominant, neurodevelopment disorder represents 10% of female subjects with profound intellectual disability. Mutations in the "MECP2" gene are responsible for up to 95% of the classical RTT cases, and nearly 500 different mutations distributed throughout the gene have been reported. Methods:…

  17. Working Group report 3: sensitivity to organic dusts--atopy and gene polymorphisms

    DEFF Research Database (Denmark)

    Kline, JN; Doekes, G; Bønløkke, Jakob

    2004-01-01

    Working Group 3 (Sensitivity to Organic Dusts-Atopy and Gene Polymorphisms) was convened to review the current understanding of how effects of inhaled organic dust may be modified by genetic factors-both those that increase as well as those that may reduce susceptibility. Furthermore, the group...... was asked to suggest areas that require more investigation in this field. The discussion focused on individual sensitivity to inhaled agents as the most important determinant of inter-individual heterogeneiety in responses to exposures. Genetic modifiers are known for a number of pathologic conditions...

  18. Molecular characterization of a maize regulatory gene. Annual progress report, March 1990--November 1991

    Energy Technology Data Exchange (ETDEWEB)

    Wessler, S.R.

    1991-12-01

    Based on initial bombardment studies we have previously concluded that promoter diversity was responsible for the diversity of naturally occurring R alleles. During this period we have found that R is controlled at the level of translation initiation and intron 1 is alternatively spliced. The experiments described in Sections 1 and 2 sought to quantify these effects and to determine whether they contribute to the tissue specific expression of select R alleles. This study was done because very little is understood about the post-transcriptional regulation of plant genes. Section 3 and 4 describe experiments designed to identify important structural components of the R protein.

  19. Characterization of three mnp genes of Fomitiporia mediterranea and report of additional class II peroxidases in the order hymenochaetales.

    Science.gov (United States)

    Morgenstern, Ingo; Robertson, Deborah L; Hibbett, David S

    2010-10-01

    We report the sequence-based characterization and expression patterns of three manganese peroxidase genes from the white rot fungus and grape vine pathogen Fomitiporia mediterranea (Agaricomycotina, Hymenochaetales), termed Fmmnp1, Fmmnp2, and Fmmnp3. The predicted open reading frames (ORFs) are 1,516-, 1,351-, and 1,345-bp long and are interrupted by seven, four, and four introns, respectively. The deduced amino acid sequences encode manganese peroxidases (EC 1.11.1.13) containing 371, 369, and 371 residues, respectively, and are similar to the manganese peroxidases of the model white rot organism Phanerochaete chrysosporium. The expression of the genes is most likely differentially regulated, as revealed by real-time PCR analysis. Phylogenetic analysis reveals that other members of the order Hymenochaetales harbor mnp genes encoding proteins that are related only distantly to those of F. mediterranea. Furthermore, multiple partial lip- and mnp-like sequences obtained for Pycnoporus cinnabarinus (Agaricomycotina, Polyporales) suggest that lignin degradation by white rot taxa relies heavily on ligninolytic peroxidases and is not efficiently achieved by laccases only.

  20. Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report.

    Science.gov (United States)

    Hokanson, Karen E; Ellstrand, Norman C; Dixon, Alfred G O; Kulembeka, Heneriko P; Olsen, Kenneth M; Raybould, Alan

    2016-02-01

    The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the 'wild' (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms.

  1. Complete response in gallbladder cancer to erlotinib plus gemcitabine does not require mutation of the epidermal growth factor receptor gene: a case report

    Directory of Open Access Journals (Sweden)

    Lincer Robert

    2010-10-01

    Full Text Available Abstract Background Gallbladder cancer typically follows an aggressive course, with chemotherapy the standard of care for advanced disease; complete remissions are rarely encountered. The epidermal growth factor receptor (EGFR is a promising therapeutic target but the activity of single agent oral EGFR tyrosine kinase inhibitors is low. There have been no previous reports of chemotherapy plus an EGFR-tyrosine kinase inhibitor (TKI to treat gallbladder cancer or correlations of response with the mutation status of the tyrosine kinase domain of the EGFR gene. Case presentation A 67 year old man with metastatic gallbladder cancer involving the liver and abdominal lymph nodes was treated with gemcitabine (1000 mg/m2 on day 1 and 8 every 21 days as well as daily erlotinib (100 mg. After four cycles of therapy, the CA 19-9 normalized and a PET/CT showed a complete remission; this response was maintained by the end of 12 cycles of therapy. Gemcitabine was then discontinued and single agent erlotinib was continued as maintenance therapy. The disease remains in good control 18 months after initiation of therapy, including 6 months on maintenance erlotinib. The only grade 3 toxicity was a typical EGFR-related skin rash. Because of the remarkable response to erlotinib plus gemcitabine, we performed tumor genotyping of the EGFR gene for response predicting mutations in exons 18, 19 and 21. This disclosed the wild-type genotype with no mutations found. Conclusion This case report demonstrates a patient with stage IV gallbladder cancer who experienced a rarely encountered complete, prolonged response after treatment with an oral EGFR-TKI plus chemotherapy. This response occurred in the absence of an EGFR gene mutation. These observations should inform the design of clinical trials using EGFR-TKIs to treat gallbladder and other biliary tract cancers; such trials should not select patients based on EGFR mutation status.

  2. Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

    Institute of Scientific and Technical Information of China (English)

    Chunxiao Zhang; Ying Gai; Wenqi Wang; Yanyan Zhu; Xuemei Chen; Xiangning Jiang

    2008-01-01

    The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plantgenetic engineering.In the present study,the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5' end of CaMV 35S promoter in the opposite orientation.To qualitatively and quantitatively esti-mate its bi-directional Wanscriptionai function and activity,two visible reporter genes,gusA β-glucuronidase,GUS) and gfp (green fluo-rescent protein,GFP),were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences,respectively.Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants.The expression of two reporter genes showed that the eonsuructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations.The quantitativeestimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excita-tion and emission.The GUS activity varied from 8 to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/mg protein in various lines of transgenic tobacco plants.Higher GUS activity generally coupled with lower GFP content,and vice versa.

  3. Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis.

    Science.gov (United States)

    Ihara, Masaru; Kitamura, Tomokazu; Kumar, Vimal; Park, Chang-Beom; Ihara, Mariko O; Lee, Sang-Jung; Yamashita, Naoyuki; Miyagawa, Shinichi; Iguchi, Taisen; Okamoto, Seiichiro; Suzuki, Yutaka; Tanaka, Hiroaki

    2015-05-19

    The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.

  4. Birt-Hogg-Dubé (BHD) syndrome: report of two novel germline mutations in the folliculin (FLCN) gene.

    Science.gov (United States)

    Palmirotta, Raffaele; Donati, Pietro; Savonarola, Annalisa; Cota, Carlo; Ferroni, Patrizia; Guadagni, Fiorella

    2008-01-01

    Molecular analysis of the folliculin (FLCN) gene was performed in four consenting patients from two families with Birt-Hogg-Dubé (BHD) syndrome, showing the occurrence of two frameshift mutations located respectively in exons 5 (802insA) and 9 (1345delAAAG) of the FLCN gene. A novel homozygous sequence variant in the intron 9 (IVS9 +5C>T) was also found. 1345delAAAG was associated with a wide variety of tumors, including stomach, colon, breast and parotid cancer. Conversely, the family carrying 802insA only had clinical evidence of dermatological lesions. These findings further suggest the relevance of exon 9 mutations in cancer predisposition for BHD.

  5. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Zacharewski, T. [Univ. of Western Ontario, London, Ontario (Canada). Dept. of Pharmacology and Toxicology

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  6. Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

    Science.gov (United States)

    Sommer, Jeffrey R; Alderson, Jon; Laible, Goetz; Petters, Robert M

    2006-06-01

    We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

  7. The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes.

    Science.gov (United States)

    Hube, Florent; Myal, Yvonne; Leygue, Etienne

    2006-05-01

    Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.

  8. Luciferase Genes as Reporter Reactions: How to Use Them in Molecular Biology?

    Science.gov (United States)

    Cevenini, L; Calabretta, M M; Calabria, D; Roda, A; Michelini, E

    2016-01-01

    : The latest advances in molecular biology have made available several biotechnological tools that take advantage of the high detectability and quantum efficiency of bioluminescence (BL), with an ever-increasing number of novel applications in environmental, pharmaceutical, food, and forensic fields. Indeed, BL proteins are being used to develop ultrasensitive binding assays and cell-based assays, thanks to their high detectability and to the availability of highly sensitive BL instruments. The appealing aspect of molecular biology tools relying on BL reactions is their general applicability in both in vitro assays, such as cell cultures or purified proteins, and in vivo settings, such as in whole-animal BL imaging. The aim of this chapter is to provide the reader with an overview of state-of-the-art bioluminescent tools based on luciferase genes, highlighting molecular biology strategies that have been applied so far, together with some selected examples.

  9. Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene.

    Science.gov (United States)

    Yu, T T; Skinner, D Z; Liang, G H; Trick, H N; Huang, B; Muthukrishnan, S

    2000-01-01

    Creeping bentgrass (Agrostis palustris Huds.) is a cool season grass widely used on putting greens in golf courses. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. The objective of our study is to develop an alternative and more efficient approach in transforming the grass using Agrobacterium (strain EHA 101). This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose. Dozens of transgenic plants have been produced from two independent transformed calli. Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings. Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the GFP gene were clearly shown in transgenic plants. These results indicated that Agrobacterium transformation can successfully be applied to creeping bentgrass.

  10. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    Energy Technology Data Exchange (ETDEWEB)

    Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others

    1996-07-01

    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  11. Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

    Directory of Open Access Journals (Sweden)

    Heffer Alison

    2011-03-01

    Full Text Available Abstract Background Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR and gene-specific amplified fragment length polymorphism (AFLP. It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.

  12. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne; Jørgensen, E.C.B.; Larsen, John Christian

    1999-01-01

    Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog...

  13. Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects of Environmental Chemicals

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Bonefeld-Jørgensen, Eva Cecilie; Larsen, John Christian

    1999-01-01

    Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrog...

  14. The application of reporter gene assays for the detection of endocrine disruptors in sport supplements

    NARCIS (Netherlands)

    Plotan, M.; Elliot, C.T.; Scippo, M.L.; Müller, M.; Antignac, J.P.; Malone, E.; Bovee, T.F.H.; Mitchell, S.; Connolly, L.

    2011-01-01

    The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to en

  15. RNA interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus.

    Science.gov (United States)

    Wang, Xiao; Li, Yongfeng; Li, Lian-Feng; Shen, Liang; Zhang, Lingkai; Yu, Jiahui; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2016-04-01

    Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious and often fatal disease of pigs, which leads to significant economic losses in many countries. Viral infection can induce the production of interferons (IFNs), giving rise to the transcription of hundreds of IFN-stimulated genes (ISGs) to exert antiviral effects. Although numerous ISGs have been identified to possess antiviral activities against different viruses, rare anti-CSFV ISGs have been reported to date. In this study, to screen anti-CSFV ISGs, twenty-one ISGs reported previously were individually knocked down using small interfering RNAs (siRNAs) followed by infection with a reporter CSFV expressing Renilla luciferase (Rluc). As a result, four novel anti-CSFV ISGs were identified, including natural-resistance-associated macrophage protein 1 (NRAMP1), cytosolic 5'-nucleotidase III A (NT5C3A), chemokine C-X-C motif ligand 10 (CXCL10), and 2'-5'-oligoadenylate synthetase 1 (OAS1), which were further verified to exhibit antiviral activities against wild-type CSFV. We conclude that the reporter virus is a useful tool for efficient screening anti-CSFV ISGs.

  16. The Ag-195 (C®G mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

    Directory of Open Access Journals (Sweden)

    Schreiber R.

    2001-01-01

    Full Text Available Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G mutation. Furthermore, this is the first time that the -195 (C->G mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

  17. Extra nuchal-type fibroma associated with elastosis, traumatic neuroma, a rare APC gene missense mutation, and a very rare MUTYH gene polymorphism: a case report and review of the literature*.

    Science.gov (United States)

    Linos, Konstantinos; Sedivcová, Monika; Cerna, Katerina; Sima, Radek; Kazakov, Dmitry V; Nazeer, Tipu; Glazyrin, Alexey; Valerian, Brian T; Carlson, J Andrew

    2011-11-01

    We report a case of an extra nuchal-type fibroma in a 51-year-old male suspected to have attenuated familial adenomatous polyposis (Gardner's syndrome), who presented with a longstanding buttock mass excised due to enlargement and pain. Histopathologically, lobules of haphazard, hypocellular, hyalinized collagen bundles replaced the dermis and subcutis and entrapped nerve bundles, mimicking Morton neuroma. Ramifying nerve twigs found around larger nerve fascicles showed the co-existence of traumatic neuroma. Elastic tissue stain revealed elastosis characterized by large, arborizing fibers lying between and within the hyalinized collagen bundles. Modified Masson's trichrome stain showed light blue staining of collagen bundles producing the hyalinized nodules with irregular, light red staining of collagen bundles at their periphery and within tumor collagen. Compression and/or degeneration of collagen and secondary elastosis with later entrapment by tumor collagen could explain this microscopic phenotype. By immunohistochemistry, tumor spindle cells expressed nuclear β-catenin and cyclin D1, mostly within regions of fibrosis implicating activation of the adenomatous polyposis coli (APC)-Wnt pathway. Genetic analysis showed a missense mutation in APC gene (c.7504G>A, p.G2502S in exon 15) and a functional homozygous polymorphism in the MUTYH gene (c.36+325G>C, (IVS1+5G/C)). Nuchal-type fibroma has been associated with Gardner's syndrome and trauma. In this patient, genetic predisposition coupled with repetitive, localized trauma and collagen degeneration may have provided the stimulus for the development of extra nuchal-type fibroma.

  18. Suicidal behavior and haplotypes of the dopamine receptor gene (DRD2 and ANKK1 gene polymorphisms in patients with alcohol dependence--preliminary report.

    Directory of Open Access Journals (Sweden)

    Andrzej Jasiewicz

    Full Text Available Suicide is a significant public health issue and a major cause of death throughout the world. According to WHO it accounts for almost 2% of deaths worldwide. The etiology of suicidal behavior is complex but the results of many studies suggest that genetic determinants are of significant importance. In our study,--we have analyzed selected SNPs polymorphisms in the DRD2 and ANKK1 genes in patients with alcohol dependence syndrome (169 Caucasian subjects including a subgroup of individuals (n = 61 who have experienced at least one suicide attempt. The aim of the study was to verify if various haplotypes of selected genes, comprising Taq1A, Taq1B, and Taq1D single nucleotide polymorphisms (SNP, play any role in the development of alcohol dependence and suicidal behavior. The control group comprised 157 unrelated individuals matched for ethnicity, gender,- and age and included no individuals with mental disorders. All subjects were recruited in the North West region of Poland. The study showed that alcohol dependent subjects with a history of at least one suicidal attempt were characterized by a significantly higher frequency of the T-G-A2 haplotype when compared to individuals in whom alcohol dependence was not associated with suicidal behavior (p = 0.006. It appears that studies based on identifying correlation between SNPs is the future for research on genetic risk factors that contribute to the development of alcohol addiction and other associated disorders. To sum up, there is a necessity to perform further research to explain dependencies between the dopaminergic system, alcohol use disorders and suicidal behavior.

  19. Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

    Science.gov (United States)

    Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

    2012-03-01

    B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

  20. Hepatitis B virus gene mutations in liver diseases: a report from New Delhi.

    Directory of Open Access Journals (Sweden)

    Abdul Malik

    Full Text Available OBJECTIVES: The study was designed to characterize the surface, core promoter, precore/core region sequences for the presence of mutations in hepatitis B virus (HBV associated with different liver diseases. METHODS: 567 HBV associated patients with different liver diseases were enrolled in this study. All samples were analyzed for HBV surface, core promoter, precore/core region mutations and genotypes using PCR and direct sequencing. RESULTS: HBV genotype D (72.8% was the predominant type followed by genotype A (27.2%. The serum viral load of HBV was highest in HBsAg carriers group and lowest in patients with hepatocellular carcinoma. 17.9% patients with cirrhosis and 24.6% hepatocellular carcinoma cases were ADV-resistant with rtA181T/V mutations in the S-gene. A1896T was found more frequently in fulminant hepatic failure compared to acute viral hepatitis patients (p = 0.038. T1753V mutation was significantly higher in patients with cirrhosis of liver (34.6% than in chronic hepatitis (18.9% and hepatocellular carcinoma patients (21.2%; p = 0.001. T1762/A1764 mutation was observed in all the groups. C1914G core gene mutation was associated with the hepatocellular carcinoma (32.2% compared to other groups. HBV genotype D predominated in comparison to genotype A. An increased frequency of precore mutation and BCP double mutations amongst the population studied was also observed. CONCLUSION: Mutations such as T1762/A1764, T1753V and C1914G were usually associated with advanced forms of liver disease and had an increased risk of HCC. The nucleotide variability in the basal core promoter and precore regions possibly plays a role in the progression of HBV disease. Prospective studies on the sequence variations of the preC/C region of the HBV genome and the molecular mechanisms in relation to progression of liver disease would aid in better understanding of the biological significance of HBV strains in India.

  1. Early host cell reactivation of an oxidatively damaged adenovirus-encoded reporter gene requires the Cockayne syndrome proteins CSA and CSB.

    Science.gov (United States)

    Leach, Derrik M; Rainbow, Andrew J

    2011-03-01

    Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for

  2. A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation.

    Science.gov (United States)

    Orzaez, Diego; Medina, Aurora; Torre, Sara; Fernández-Moreno, Josefina Patricia; Rambla, José Luis; Fernández-Del-Carmen, Asun; Butelli, Eugenio; Martin, Cathie; Granell, Antonio

    2009-07-01

    Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.

  3. EPA Releases Scientific Report Showing U.S. Coastal Waters a Mix of Good and Fair Health/Contaminants Post Threat to Fish, Birds, and Wildlife in Most Coastal Waters

    Science.gov (United States)

    WASHINGTON - The U.S. Environmental Protection Agency (EPA) today released the 2010 National Coastal Condition Assessment showing that more than half of the nation's coastal and Great Lakes nearshore waters are rated good for biological and sediment

  4. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

    NARCIS (Netherlands)

    Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.

    2001-01-01

    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 and

  5. Planning a Successful Tech Show

    Science.gov (United States)

    Nikirk, Martin

    2011-01-01

    Tech shows are a great way to introduce prospective students, parents, and local business and industry to a technology and engineering or career and technical education program. In addition to showcasing instructional programs, a tech show allows students to demonstrate their professionalism and skills, practice public presentations, and interact…

  6. Validation of an interferon stimulatory response element reporter gene assay for quantifying type I interferons.

    Science.gov (United States)

    McCoski, S R; Xie, M; Hall, E B; Mercadante, P M; Spencer, T E; Lonergan, P; Ealy, A D

    2014-04-01

    The goal of this