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Sample records for gene conversion induced

  1. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

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    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  2. Special Issue: Gene Conversion in Duplicated Genes

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    Hideki Innan

    2011-06-01

    Full Text Available Gene conversion is an outcome of recombination, causing non-reciprocal transfer of a DNA fragment. Several decades later than the discovery of crossing over, gene conversion was first recognized in fungi when non-Mendelian allelic distortion was observed. Gene conversion occurs when a double-strand break is repaired by using homologous sequences in the genome. In meiosis, there is a strong preference to use the orthologous region (allelic gene conversion, which causes non-Mendelian allelic distortion, but paralogous or duplicated regions can also be used for the repair (inter-locus gene conversion, also referred to as non-allelic and ectopic gene conversion. The focus of this special issue is the latter, interlocus gene conversion; the rate is lower than allelic gene conversion but it has more impact on phenotype because more drastic changes in DNA sequence are involved.

  3. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

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    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  4. Gene conversion in human rearranged immunoglobulin genes.

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    Darlow, John M; Stott, David I

    2006-07-01

    Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.

  5. Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells.

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    Schildkraut, Ezra; Miller, Cheryl A; Nickoloff, Jac A

    2006-04-01

    Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.

  6. Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair.

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    Mehta, Anuja; Beach, Annette; Haber, James E

    2017-02-02

    Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a non-homologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the recombination enhancer, which tethers HMLα near the DSB. Thus, homology shorter than an apparent minimum efficient processing segment can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases 3-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes gene conversion, whereas deletion of the FANCM-related Mph1 helicase promotes BIR. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Mechanisms of Ectopic Gene Conversion

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    P.J. Hastings

    2010-11-01

    Full Text Available Gene conversion (conversion, the unidirectional transfer of DNA sequence information, occurs as a byproduct of recombinational repair of broken or damaged DNA molecules. Whereas excision repair processes replace damaged DNA by copying the complementary sequence from the undamaged strand of duplex DNA, recombinational mechanisms copy similar sequence, usually in another molecule, to replace the damaged sequence. In mitotic cells the other molecule is usually a sister chromatid, and the repair does not lead to genetic change. Less often a homologous chromosome or homologous sequence in an ectopic position is used. Conversion results from repair in two ways. First, if there was a double-strand gap at the site of a break, homologous sequence will be used as the template for synthesis to fill the gap, thus transferring sequence information in both strands. Second, recombinational repair uses complementary base pairing, and the heteroduplex molecule so formed is a source of conversion, both as heteroduplex and when donor (undamaged template information is retained after correction of mismatched bases in heteroduplex. There are mechanisms that favour the use of sister molecules that must fail before ectopic homology can be used. Meiotic recombination events lead to the formation of crossovers required in meiosis for orderly segregation of pairs of homologous chromosomes. These events result from recombinational repair of programmed double-strand breaks, but in contrast with mitotic recombination, meiotic recombinational events occur predominantly between homologous chromosomes, so that transfer of sequence differences by conversion is very frequent. Transient recombination events that do not form crossovers form both between homologous chromosomes and between regions of ectopic homology, and leave their mark in the occurrence of frequent non-crossover conversion, including ectopic conversion.

  8. Chromatin structure regulates gene conversion.

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    W Jason Cummings

    2007-10-01

    Full Text Available Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vlambda pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205, expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vlambda donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vlambda array, and altered the outcome of Vlambda diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.

  9. Gene conversion in the rice genome

    DEFF Research Database (Denmark)

    Xu, Shuqing; Clark, Terry; Zheng, Hongkun;

    2008-01-01

    BACKGROUND: Gene conversion causes a non-reciprocal transfer of genetic information between similar sequences. Gene conversion can both homogenize genes and recruit point mutations thereby shaping the evolution of multigene families. In the rice genome, the large number of duplicated genes...... is not tightly linked to natural selection in the rice genome. To assess the contribution of segmental duplication on gene conversion statistics, we determined locations of conversion partners with respect to inter-chromosomal segment duplication. The number of conversions associated with segmentation is less...

  10. Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers

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    José Sereno

    2014-01-01

    Full Text Available Protocols of conversion from cyclosporin A (CsA to sirolimus (SRL have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups (n=6 were tested during 9 weeks: control, CsA, SRL, and conversion (CsA for 3 weeks followed by SRL for 6 weeks. Classical and emergent serum, urinary, and kidney tissue (gene and protein expression markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson’s trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF-κβ, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF-β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions, while NGAL (serum versus urine seems to be a feasible biomarker of CsA replacement to SRL.

  11. Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers

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    Sereno, José; Nunes, Sara; Rodrigues-Santos, Paulo; Rocha-Pereira, Petronila; Fernandes, João; Teixeira, Frederico; Reis, Flávio

    2014-01-01

    Protocols of conversion from cyclosporin A (CsA) to sirolimus (SRL) have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups (n = 6) were tested during 9 weeks: control, CsA, SRL, and conversion (CsA for 3 weeks followed by SRL for 6 weeks). Classical and emergent serum, urinary, and kidney tissue (gene and protein expression) markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary) as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF-κ β, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF-β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions), while NGAL (serum versus urine) seems to be a feasible biomarker of CsA replacement to SRL. PMID:24971338

  12. Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL

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    Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy

    2001-09-25

    Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. U sing isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells.

  13. Efficient microwave-induced optical frequency conversion

    CERN Document Server

    Kosachiov, D V

    1999-01-01

    Frequency conversion process is studied in a medium of atoms with a $\\Lambda$ configuration of levels, where transition between two lower states is driven by a microwave field. In this system, conversion efficiency can be very high by virtue of the effect of electromagnetically induced transparency (EIT). Depending on intensity of the microwave field, two regimes of EIT are realized: ''dark-state'' EIT for the weak field, and Autler-Townes-type EIT for the strong one. We study both cases via analytical and numerical solution and find optimum conditions for the conversion.

  14. Conversion of homothallic yeast to heterothallism trough HO gene disruption

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    Van Zyl, WH

    1993-04-01

    Full Text Available A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeaststrains to heterothallism through HO gene disruption. An integrative ho:: neo disrupted allele was constructed by cloning a dominant selectable marker...

  15. Gene conversion homogenizes the CMT1A paralogous repeats

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    Hurles Matthew E

    2001-12-01

    Full Text Available Abstract Background Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination. Results Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 × 10-4 and 5.1 × 10-5 per generation for the alternative models. Conclusions This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

  16. Gene conversion homogenizes the CMT1A paralogous repeats.

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    Hurles, M E

    2001-01-01

    Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination. Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs) exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 x 10(-4) and 5.1 x 10(-5) per generation for the alternative models. This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

  17. Microhomology-mediated deletion and gene conversion in African trypanosomes.

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    Glover, Lucy; Jun, Junho; Horn, David

    2011-03-01

    Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.

  18. Polarized gene conversion at the bz locus of maize.

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    Dooner, Hugo K; He, Limei

    2014-09-23

    Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.

  19. Sequential EMT-MET induces neuronal conversion through Sox2.

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    He, Songwei; Chen, Jinlong; Zhang, Yixin; Zhang, Mengdan; Yang, Xiao; Li, Yuan; Sun, Hao; Lin, Lilong; Fan, Ke; Liang, Lining; Feng, Chengqian; Wang, Fuhui; Zhang, Xiao; Guo, Yiping; Pei, Duanqing; Zheng, Hui

    2017-01-01

    Direct neuronal conversion can be achieved with combinations of small-molecule compounds and growth factors. Here, by studying the first or induction phase of the neuronal conversion induced by defined 5C medium, we show that the Sox2-mediated switch from early epithelial-mesenchymal transition (EMT) to late mesenchymal-epithelial transition (MET) within a high proliferation context is essential and sufficient for the conversion from mouse embryonic fibroblasts (MEFs) to TuJ(+) cells. At the early stage, insulin and basic fibroblast growth factor (bFGF)-induced cell proliferation, early EMT, the up-regulation of Stat3 and Sox2, and the subsequent activation of neuron projection. Up-regulated Sox2 then induced MET and directed cells towards a neuronal fate at the late stage. Inhibiting either stage of this sequential EMT-MET impaired the conversion. In addition, Sox2 could replace sequential EMT-MET to induce a similar conversion within a high proliferation context, and its functions were confirmed with other neuronal conversion protocols and MEFs reprogramming. Therefore, the critical roles of the sequential EMT-MET were implicated in direct cell fate conversion in addition to reprogramming, embryonic development and cancer progression.

  20. Self-induced neutrino flavor conversion without flavor mixing

    CERN Document Server

    Chakraborty, Sovan; Izaguirre, Ignacio; Raffelt, Georg

    2016-01-01

    Neutrino-neutrino refraction in dense media can cause self-induced flavor conversion triggered by collective run-away modes of the interacting flavor oscillators. The growth rates were usually found to be of order a typical vacuum oscillation frequency $\\Delta m^2/2E$. However, even in the simple case of a $\

  1. Evidence for gene conversion among immunoglobulin heavy chain variable region genes.

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    Clarke, S H; Rudikoff, S

    1984-03-01

    We have previously reported that the VH region amino acid sequence of a phosphocholine (PC)-binding hybridoma antibody of CBA/J origin, HP101 6G6 (6G6), differs extensively from the VH regions of other PC-binding antibodies. The sequence of 6G6 VH appears to be derived from a gene homologous to the BALB/c V11 gene, a member of the PC VH (T15 VH) gene family not normally used to encode PC-binding antibodies. The 6G6 VH sequence differs from the translated sequence of V11 by six amino acids, four of which occur at the same position in other members of this gene family. This coincidence led to the proposal that the 6G6 VH gene was derived by gene conversion involving three genes of the PC VH gene family. We report here the nucleic acid sequence of the rearranged VH gene of hybridoma 6G6. This sequence supports our previous suggestion of gene conversion by confirming those differences, relative to the BALB/c V11 gene sequence, that are encoded by other members of this gene family, and extends this correlation to include three silent base pair substitutions as well. In addition, 5' noncoding region sequence and Southern blot analysis using probes derived from the coding and 5' noncoding regions confirm that the 6G6 VH gene is likely to be derived from the V11 homologue in CBA/J mice, and suggest that all three genes believed to be involved in the generation of the 6G6 VH gene are present in the CBA/J genome, a prerequisite for their involvement in gene conversion.

  2. Gene conversion between red and defective green opsin gene in blue cone monochromacy

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    Reyniers, E.; Van Thienen, M.N.; De Boulle, K.; Willems, P.J. [Univ. of Antwerp (Belgium)] [and others

    1995-09-20

    Blue cone monochromacy is an X-linked condition in which the function of both the red pigment gene (RCP) and the green pigment gene (GCP) is impaired. Blue cone monochromacy can be due to a red/green gene array rearrangement existing of a single red/green hybrid gene and an inactivating C203R point mutation in both RCP and GCP. The flanking sequences of the C230R mutation in exon 4 of RCP were characteristic for GCP, indicating that this mutation was transferred from GCP into RCP by gene conversion. 23 refs., 3 figs., 1 tab.

  3. Signals of historical interlocus gene conversion in human segmental duplications.

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    Beth L Dumont

    Full Text Available Standard methods of DNA sequence analysis assume that sequences evolve independently, yet this assumption may not be appropriate for segmental duplications that exchange variants via interlocus gene conversion (IGC. Here, we use high quality multiple sequence alignments from well-annotated segmental duplications to systematically identify IGC signals in the human reference genome. Our analysis combines two complementary methods: (i a paralog quartet method that uses DNA sequence simulations to identify a statistical excess of sites consistent with inter-paralog exchange, and (ii the alignment-based method implemented in the GENECONV program. One-quarter (25.4% of the paralog families in our analysis harbor clear IGC signals by the quartet approach. Using GENECONV, we identify 1477 gene conversion tracks that cumulatively span 1.54 Mb of the genome. Our analyses confirm the previously reported high rates of IGC in subtelomeric regions and Y-chromosome palindromes, and identify multiple novel IGC hotspots, including the pregnancy specific glycoproteins and the neuroblastoma breakpoint gene families. Although the duplication history of a paralog family is described by a single tree, we show that IGC has introduced incredible site-to-site variation in the evolutionary relationships among paralogs in the human genome. Our findings indicate that IGC has left significant footprints in patterns of sequence diversity across segmental duplications in the human genome, out-pacing the contributions of single base mutation by orders of magnitude. Collectively, the IGC signals we report comprise a catalog that will provide a critical reference for interpreting observed patterns of DNA sequence variation across duplicated genomic regions, including targets of recent adaptive evolution in humans.

  4. Active tunable plasmonically induced polarization conversion in the THz regime

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    Ling, Furi; Yao, Gang; Yao, Jianquan

    2016-01-01

    A plasmon-induced polarization conversion (PIPC) structure based on periodically patterned graphene was demonstrated in the THz regime. By varying the Fermi level of two connected T-shape graphene strips through the electrostatic gating, the peak frequency and the group index in the transparency window can be tuned, which is good agreement with the coupled Lorentz oscillator model. Due to interference between two polarization selective graphene plasmonic resonances coexisting in the planar metamaterial, polarization conversion can be achieved. The linearly polarized THz wave can be converted to elliptically and right circularly polarized THz wave through varying the relaxation time of electrons in graphene. This novel chip-scale active terahertz device promises essential application opportunities in terahertz sensing and terahertz communications. PMID:27734912

  5. Neutral and Non-Neutral Evolution of Duplicated Genes with Gene Conversion

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    Jeffrey A. Fawcett

    2011-02-01

    Full Text Available Gene conversion is one of the major mutational mechanisms involved in the DNA sequence evolution of duplicated genes. It contributes to create unique patters of DNA polymorphism within species and divergence between species. A typical pattern is so-called concerted evolution, in which the divergence between duplicates is maintained low for a long time because of frequent exchanges of DNA fragments. In addition, gene conversion affects the DNA evolution of duplicates in various ways especially when selection operates. Here, we review theoretical models to understand the evolution of duplicates in both neutral and non-neutral cases. We also explain how these theories contribute to interpreting real polymorphism and divergence data by using some intriguing examples.

  6. Gene conversions in the growth hormone gene family of primates: stronger homogenizing effects in the Hominidae lineage.

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    Petronella, Nicholas; Drouin, Guy

    2011-09-01

    In humans, the growth hormone/chorionic somatomammotropin gene family is composed of five highly similar genes. We characterized the gene conversions that occurred between the growth hormone genes of 11 primate species. We detected 48 conversions using GENECONV and others were only detected using phylogenetic analyses. Gene conversions were detected in all species analyzed, their average size (±standard deviation) is 197.8±230.4 nucleotides, the size of the conversions is correlated with sequence similarity and converted regions are significantly more GC-rich than non-converted regions. Gene conversions have a stronger homogenizing effect in Hominidae genes than in other primate species. They are also less frequent in conserved gene regions and towards functionally important genes. This suggests that the high degree of sequence similarity observed between the growth hormone genes of primate species is a consequence of frequent gene conversions in gene regions which are under little selective constraints. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Identifying concerted evolution and gene conversion in mammalian gene pairs lasting over 100 million years

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    Scherer Stephen W

    2009-07-01

    Full Text Available Abstract Background Concerted evolution occurs in multigene families and is characterized by stretches of homogeneity and higher sequence similarity between paralogues than between orthologues. Here we identify human gene pairs that have undergone concerted evolution, caused by ongoing gene conversion, since at least the human-mouse divergence. Our strategy involved the identification of duplicated genes with greater similarity within a species than between species. These genes were required to be present in multiple mammalian genomes, suggesting duplication early in mammalian divergence. To eliminate genes that have been conserved due to strong purifying selection, our analysis also required at least one intron to have retained high sequence similarity between paralogues. Results We identified three human gene pairs undergoing concerted evolution (BMP8A/B, DDX19A/B, and TUBG1/2. Phylogenetic investigations reveal that in each case the duplication appears to have occurred prior to eutherian mammalian radiation, with exactly two paralogues present in all examined species. This indicates that all three gene duplication events were established over 100 million years ago. Conclusion The extended duration of concerted evolution in multiple distant lineages suggests that there has been prolonged homogenization of specific segments within these gene pairs. Although we speculate that selection for homogenization could have been utilized in order to maintain crucial homo- or hetero- binding domains, it remains unclear why gene conversion has persisted for such extended periods of time. Through these analyses, our results demonstrate additional examples of a process that plays a definite, although unspecified, role in molecular evolution.

  8. Gene conversion-like events in the diversification of human rearranged IGHV3-23*01 gene sequences

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    Bhargavi eDuvvuri

    2012-06-01

    Full Text Available Gene conversion (GCV as a mechanism of immunoglobulin diversification is well established in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. GCV is mediated by activation-induced cytidine deaminase (AID. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23*01 gene sequences from common variable immunodeficiency (CVID patients and healthy individuals to survey ‘GCV-like’ activities. Our search identified strong evidence of GCV-like patterns. Germline VH sequences were identified as potential donors for clustered mutations in rearranged IGHV3-23*01 gene sequences. We identified minimum and maximum sequence identities between donor and recipient sequences that can serve as targets for GCV and our findings are consistent with those reported in literature. We observed that GCV-like tracts are flanked by activation-induced cytidine deaminase (AID hotspot motifs. Structural modeling of IGHV3-23*01 gene sequence revealed that hypermutable bases flanking GCV-like tracts, are in the single stranded DNA (ssDNA of stable stem-loop structures (SLSs. SsDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV323-*01 sequences from healthy individuals compared to that of CVID patients. GCV, unlike SHM, can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events, as identified in this study would be instrumental in protecting humans against pathogens that diversify their genome by

  9. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

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    Alan M Williams

    Full Text Available The activation induced cytosine deaminase (AID mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM, class switch recombination (CSR, and gene conversion (GCV. SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

  10. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

    Science.gov (United States)

    Williams, Alan M.; Maman, Yaakov; Alinikula, Jukka; Schatz, David G.

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  11. Deep genome-wide measurement of meiotic gene conversion using tetrad analysis in Arabidopsis thaliana.

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    Yujin Sun

    Full Text Available Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3.5×10(-4 conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions.

  12. Horizontal acquisition of multiple mitochondrial genes from a parasitic plant followed by gene conversion with host mitochondrial genes

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    Hao Weilong

    2010-12-01

    Full Text Available Abstract Background Horizontal gene transfer (HGT is relatively common in plant mitochondrial genomes but the mechanisms, extent and consequences of transfer remain largely unknown. Previous results indicate that parasitic plants are often involved as either transfer donors or recipients, suggesting that direct contact between parasite and host facilitates genetic transfer among plants. Results In order to uncover the mechanistic details of plant-to-plant HGT, the extent and evolutionary fate of transfer was investigated between two groups: the parasitic genus Cuscuta and a small clade of Plantago species. A broad polymerase chain reaction (PCR survey of mitochondrial genes revealed that at least three genes (atp1, atp6 and matR were recently transferred from Cuscuta to Plantago. Quantitative PCR assays show that these three genes have a mitochondrial location in the one species line of Plantago examined. Patterns of sequence evolution suggest that these foreign genes degraded into pseudogenes shortly after transfer and reverse transcription (RT-PCR analyses demonstrate that none are detectably transcribed. Three cases of gene conversion were detected between native and foreign copies of the atp1 gene. The identical phylogenetic distribution of the three foreign genes within Plantago and the retention of cytidines at ancestral positions of RNA editing indicate that these genes were probably acquired via a single, DNA-mediated transfer event. However, samplings of multiple individuals from two of the three species in the recipient Plantago clade revealed complex and perplexing phylogenetic discrepancies and patterns of sequence divergence for all three of the foreign genes. Conclusions This study reports the best evidence to date that multiple mitochondrial genes can be transferred via a single HGT event and that transfer occurred via a strictly DNA-level intermediate. The discovery of gene conversion between co-resident foreign and native

  13. Evidence of gene conversion in genes encoding the Gal/GalNac lectin complex of Entamoeba.

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    Gareth D Weedall

    2011-06-01

    Full Text Available The human gut parasite Entamoeba histolytica, uses a lectin complex on its cell surface to bind to mucin and to ligands on the intestinal epithelia. Binding to mucin is necessary for colonisation and binding to intestinal epithelia for invasion, therefore blocking this binding may protect against amoebiasis. Acquired protective immunity raised against the lectin complex should create a selection pressure to change the amino acid sequence of lectin genes in order to avoid future detection. We present evidence that gene conversion has occurred in lineages leading to E. histolytica strain HM1:IMSS and E. dispar strain SAW760. This evolutionary mechanism generates diversity and could contribute to immune evasion by the parasites.

  14. Evidence of gene conversion in genes encoding the Gal/GalNac lectin complex of Entamoeba.

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    Gareth D Weedall

    2011-06-01

    Full Text Available The human gut parasite Entamoeba histolytica, uses a lectin complex on its cell surface to bind to mucin and to ligands on the intestinal epithelia. Binding to mucin is necessary for colonisation and binding to intestinal epithelia for invasion, therefore blocking this binding may protect against amoebiasis. Acquired protective immunity raised against the lectin complex should create a selection pressure to change the amino acid sequence of lectin genes in order to avoid future detection. We present evidence that gene conversion has occurred in lineages leading to E. histolytica strain HM1:IMSS and E. dispar strain SAW760. This evolutionary mechanism generates diversity and could contribute to immune evasion by the parasites.

  15. Ectopic Gene Conversions in the Genome of Ten Hemiascomycete Yeast Species

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    Robert T. Morris

    2011-01-01

    Full Text Available We characterized ectopic gene conversions in the genome of ten hemiascomycete yeast species. Of the ten species, three diverged prior to the whole genome duplication (WGD event present in the yeast lineage and seven diverged after it. We analyzed gene conversions from three separate datasets: paralogs from the three pre-WGD species, paralogs from the seven post-WGD species, and common ohnologs from the seven post-WGD species. Gene conversions have similar lengths and frequency and occur between sequences having similar degrees of divergence, in paralogs from pre- and post-WGD species. However, the sequences of ohnologs are both more divergent and less frequently converted than those of paralogs. This likely reflects the fact that ohnologs are more often found on different chromosomes and are evolving under stronger selective pressures than paralogs. Our results also show that ectopic gene conversions tend to occur more frequently between closely linked genes. They also suggest that the mechanisms responsible for the loss of introns in S. cerevisiae are probably also involved in the gene 3'-end gene conversion bias observed between the paralogs of this species.

  16. Channa striatus capsules induces cytokine conversion in pulmonary tuberculosis patients

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    Novita Paliliewu

    2013-06-01

    Full Text Available Objective: This study aimed to investigate whether Channa striatus capsule induces sputum and cytokine conversion in pulmonary tuberculosis (TB patients. Methods: Randomized, placebo-controlled, double-blind pilot study was conducted to pulmonary TB patients who admitted to Department of Internal Medicine, Faculty of Medicine, University of Sam Ratulangi, Manado, North Sulawesi, Indonesia. A total of 36 pulmonary TB patients were randomly divided into two equal groups (n = 18 including one group received standart antituberculosis drugs plus Channa striatus capsule and another group received standart antituberculosis drugs plus placebo. Channa striatus capsule was given at a dose of 2 g each time, 3 times per day, for 12 weeks. The levels of tumor necrosis factor (TNF-α, interferon (IFN-γ, and interleukin (IL-10 were analyses using enzyme linked immunosorbent assay (ELISA method. Results: The rate of positive sputum smear decline was more pronounced in the Channa striatus group but did not reach statistically different value between groups. The levels of TNF-α, IFN-γ, and IL-10 were not significantly different in Channa striatus group compared to placebo group at baseline (week 0. But at week 12, the supplementation of Channa striatus capsule significantly decreased TNF-α, IFN-γ, and IL-10 levels compared to baseline. In placebo groups, there were no significant differences for IL-10 levels at week 12, but the levels of TNF-α and IFN-γ significantly decreased. Conclusion: Adjunctive supplementation of Channa striatus capsules accelerated the beneficial therapeutic effect of TB chemotherapy by improving cytokine response. [J Exp Integr Med 2013; 3(3.000: 237-242

  17. Population-specific differences in gene conversion patterns between human SUZ12 and SUZ12P are indicative of the dynamic nature of interparalog gene conversion.

    Science.gov (United States)

    Mussotter, Tanja; Bengesser, Kathrin; Högel, Josef; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2014-04-01

    Nonallelic homologous gene conversion (NAHGC) resulting from interparalog recombination without crossover represents an important influence on the evolution of duplicated sequences in the human genome. In 17q11.2, different paralogous sequences mediate large NF1 deletions by nonallelic homologous recombination with crossover (NAHR). Among these paralogs are SUZ12 and its pseudogene SUZ12P which harbour the breakpoints of type-2 (1.2-Mb) NF1 deletions. Such deletions are caused predominantly by mitotic NAHR since somatic mosaicism with normal cells is evident in most patients. Investigating whether SUZ12 and SUZ12P have also been involved in NAHGC, we observed gene conversion tracts between these paralogs in both Africans (AFR) and Europeans (EUR). Since germline type-2 NF1 deletions resulting from meiotic NAHR are very rare, the vast majority of the gene conversion tracts in SUZ12 and SUZ12P are likely to have resulted from mitotic recombination during premeiotic cell divisions of germ cells. A higher number of gene conversion tracts were noted within SUZ12 and SUZ12P in AFR as compared to EUR. Further, the distinctive signature of NAHGC (a high number of SNPs per paralog and a high number of shared SNPs between paralogs), a characteristic of many actively recombining paralogs, was observed in both SUZ12 and SUZ12P but only in AFR and not in EUR. A novel polymorphic 2.3-kb deletion in SUZ12P was identified which exhibited a high allele frequency in EUR. We postulate that this interparalog structural difference, together with low allelic recombination rates, could have caused a reduction in NAHGC between SUZ12 and SUZ12P during human evolution.

  18. DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation.

    Science.gov (United States)

    Garcia, P; Gasc, A M; Kyriakidis, X; Baty, D; Sicard, M

    1988-11-01

    In pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5'-ATTCAT----5'-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.

  19. Frequent gene conversion events between the X and Y homologous chromosomal regions in primates

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    Hirai Hirohisa

    2010-07-01

    Full Text Available Abstract Background Mammalian sex-chromosomes originated from a pair of autosomes. A step-wise cessation of recombination is necessary for the proper maintenance of sex-determination and, consequently, generates a four strata structure on the X chromosome. Each stratum shows a specific per-site nucleotide sequence difference (p-distance between the X and Y chromosomes, depending on the time of recombination arrest. Stratum 4 covers the distal half of the human X chromosome short arm and the p-distance of the stratum is ~10%, on average. However, a 100-kb region, which includes KALX and VCX, in the middle of stratum 4 shows a significantly lower p-distance (1-5%, suggesting frequent sequence exchanges or gene conversions between the X and Y chromosomes in humans. To examine the evolutionary mechanism for this low p-distance region, sequences of a corresponding region including KALX/Y from seven species of non-human primates were analyzed. Results Phylogenetic analysis of this low p-distance region in humans and non-human primate species revealed that gene conversion like events have taken place at least ten times after the divergence of New World monkeys and Catarrhini (i.e., Old World monkeys and hominoids. A KALY-converted KALX allele in white-handed gibbons also suggests a possible recent gene conversion between the X and Y chromosomes. In these primate sequences, the proximal boundary of this low p-distance region is located in a LINE element shared between the X and Y chromosomes, suggesting the involvement of this element in frequent gene conversions. Together with a palindrome on the Y chromosome, a segmental palindrome structure on the X chromosome at the distal boundary near VCX, in humans and chimpanzees, may mediate frequent sequence exchanges between X and Y chromosomes. Conclusion Gene conversion events between the X and Y homologous regions have been suggested, mainly in humans. Here, we found frequent gene conversions in the

  20. Radiation-induced gene responses

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Paunesku, T.; Shearin-Jones, P.; Oryhon, J.

    1996-12-31

    In the process of identifying genes that are differentially regulated in cells exposed to ultraviolet radiation (UV), we identified a transcript that was repressed following the exposure of cells to a combination of UV and salicylate, a known inhibitor of NF-kappaB. Sequencing this band determined that it has identify to lactate dehydrogenase, and Northern blots confirmed the initial expression pattern. Analysis of the sequence of the LDH 5` region established the presence of NF-kappaB, Sp1, and two Ap-2 elements; two partial AP- 1; one partial RE, and two halves of E-UV elements were also found. Electromobility shift assays were then performed for the AP-1, NF- kappaB, and E-UV elements. These experiments revealed that binding to NF-kappaB was induced by UV but repressed with salicylic acid; UV did not affect AP-1 binding, but salicylic acid inhibited it alone or following UV exposure; and E-UV binding was repressed by UV, and salicylic acid had little effect. Since the binding of no single element correlated with the expression pattern of LDH, it is likely that multiple elements govern UV/salicylate-mediated expression.

  1. Evolutionary Stasis in Cycad Plastomes and the First Case of Plastome GC-Biased Gene Conversion.

    Science.gov (United States)

    Wu, Chung-Shien; Chaw, Shu-Miaw

    2015-06-27

    In angiosperms, gene conversion has been known to reduce the mutational load of plastid genomes (the plastomes). Particularly, more frequent gene conversions in inverted repeat (IR) than in single copy (SC) regions result in contrasting substitution rates between these two regions. However, little has been known about the effect of gene conversion in the evolution of gymnosperm plastomes. Cycads (Cycadophyta) are the second largest gymnosperm group. Evolutionary study of their plastomes is limited to the basal cycad genus, Cycas. In this study, we addressed three questions. 1) Do the plastomes of other cycad genera evolve slowly as previously observed in the plastome of Cycas taitungensis? 2) Do substitution rates differ between their SC and IR regions? And 3) Does gene conversion occur in the cycad plastomes? If yes, is it AT-biased or GC-biased? Plastomes of eight species from other eight genera of cycads were sequenced. These plastomes are highly conserved in genome organization. Excluding ginkgo, cycad plastomes have significantly lower synonymous and nonsynonymous substitution rates than other gymnosperms, reflecting their evolutionary stasis in nucleotide mutations. In the IRs of cycad plastomes, the reduced substitution rates and GC-biased mutations are associated with a GC-biased gene conversion (gBGC) mechanism. Further investigations suggest that in cycads, gBGC is able to rectify plastome-wide mutations. Therefore, this study is the first to uncover the plastomic gBGC in seed plants. We also propose a gBGC model to interpret the dissimilar evolutionary patterns as well as the compositionally biased mutations in the SC and IR regions of cycad plastomes.

  2. Radiation-Induced High-Temperature Conversion of Cellulose

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    Alexander V. Ponomarev

    2014-10-01

    Full Text Available Thermal decomposition of cellulose can be upgraded by means of an electron-beam irradiation to produce valuable organic products via chain mechanisms. The samples being irradiated decompose effectively at temperatures below the threshold of pyrolysis inception. Cellulose decomposition resembles local “explosion” of the glucopyranose unit when fast elimination of carbon dioxide and water precede formation of residual carbonyl or carboxyl compounds. The dry distillation being performed during an irradiation gives a liquid condensate where furfural and its derivatives are dominant components. Excessively fast heating is adverse, as it results in a decrease of the yield of key organic products because pyrolysis predominates over the radiolytic-controlled decomposition of feedstock. Most likely, conversion of cellulose starts via radiolytic formation of macroradicals do not conform with each other, resulting in instability of the macroradical. As a consequence, glucosidic bond cleavage, elimination of light fragments (water, carbon oxides, formaldehyde, etc. and formation of furfural take place.

  3. Long- and short-patch gene conversions in Streptococcus pneumoniae transformation.

    Science.gov (United States)

    Sicard, M; Lefèvre, J C; Mostachfi, P; Gasc, A M; Méjean, V; Claverys, J P

    1985-01-01

    In pneumococcal transformation some point mutations are integrated by an excision-repair pathway which switches the heteroduplex DNA into homoduplex. This transfer of information is a gene conversion. We have reviewed some of the properties of this system especially those relating to heteroduplex specificity and given evidence that this extends over several kilobases of DNA. We then describe a new process of conversion in pneumococcal transformation which occurs over a very short distance (5 to 27 base-pairs) and is triggered by a single site mutation resulting from the transversion 5'-ATTCAT...to 5'...ATTAAT... Only one of the two heteroduplexes 5'...A...3'/3'...G...5', is converted.

  4. A new type of resonant neutrino conversions induced by magnetic fields

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    Sahu, S; Valle, José W F

    1995-01-01

    We consider resonant neutrino conversions in magnetised matter, such as a degenerate electron gas. We show how magnetisation effects caused by axial vector interactions of neutrinos with the charged leptons in the medium can induce a new type of resonant neutrino conversion which may occur even in situations where the MSW effect does not occur, such as the case of degenerate or inverted neutrino mass spectra. Our new resonance may simultaneously affect anti-neutrino \\bar{\

  5. Recombination dynamics of a human Y-chromosomal palindrome: rapid GC-biased gene conversion, multi-kilobase conversion tracts, and rare inversions.

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    Pille Hallast

    Full Text Available The male-specific region of the human Y chromosome (MSY includes eight large inverted repeats (palindromes in which arm-to-arm similarity exceeds 99.9%, due to gene conversion activity. Here, we studied one of these palindromes, P6, in order to illuminate the dynamics of the gene conversion process. We genotyped ten paralogous sequence variants (PSVs within the arms of P6 in 378 Y chromosomes whose evolutionary relationships within the SNP-defined Y phylogeny are known. This allowed the identification of 146 historical gene conversion events involving individual PSVs, occurring at a rate of 2.9-8.4×10(-4 events per generation. A consideration of the nature of nucleotide change and the ancestral state of each PSV showed that the conversion process was significantly biased towards the fixation of G or C nucleotides (GC-biased, and also towards the ancestral state. Determination of haplotypes by long-PCR allowed likely co-conversion of PSVs to be identified, and suggested that conversion tract lengths are large, with a mean of 2068 bp, and a maximum in excess of 9 kb. Despite the frequent formation of recombination intermediates implied by the rapid observed gene conversion activity, resolution via crossover is rare: only three inversions within P6 were detected in the sample. An analysis of chimpanzee and gorilla P6 orthologs showed that the ancestral state bias has existed in all three species, and comparison of human and chimpanzee sequences with the gorilla outgroup confirmed that GC bias of the conversion process has apparently been active in both the human and chimpanzee lineages.

  6. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    Science.gov (United States)

    Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  7. Gene conversion limits divergence of mammalian TLR1 and TLR6

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    Dunoyer-Geindre Sylvie

    2007-08-01

    Full Text Available Abstract Background Toll-like receptors (TLR recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris. Results The N-terminal sequences of the orthologous proteins showed greater similarity than corresponding paralog sequences. However, we identified a region of 300 amino acids towards the C-terminus of TLR1 and TLR6, where paralogs had a greater degree of sequence identity than orthologs. Preservation of DNA sequence identity of paralogs in this region was observed in all nine mammalian species investigated, and is due to independent gene conversion events. The regions having undergone gene conversion in each species are almost identical and encode the leucine-rich repeat motifs 16 to 19, the C-terminal cap motif, the transmembrane domain and most of the intracellular Toll/interleukin-1 receptor (TIR domain. Conclusion Our results show that, for a specific conserved region, divergence of TLR1 and TLR6 is limited by gene conversion, most likely because of the need for co-evolution with multiple intracellular and extracellular binding partners. Thus, gene conversion provides a mechanism for limiting the divergence of functional regions of protein paralogs, while allowing other domains to evolve diversified functions.

  8. Gene conversion occurs within the mating-type locus of Cryptococcus neoformans during sexual reproduction.

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    Sheng Sun

    2012-07-01

    Full Text Available Meiotic recombination of sex chromosomes is thought to be repressed in organisms with heterogametic sex determination (e.g. mammalian X/Y chromosomes, due to extensive divergence and chromosomal rearrangements between the two chromosomes. However, proper segregation of sex chromosomes during meiosis requires crossing-over occurring within the pseudoautosomal regions (PAR. Recent studies reveal that recombination, in the form of gene conversion, is widely distributed within and may have played important roles in the evolution of some chromosomal regions within which recombination was thought to be repressed, such as the centromere cores of maize. Cryptococcus neoformans, a major human pathogenic fungus, has an unusually large mating-type locus (MAT, >100 kb, and the MAT alleles from the two opposite mating-types show extensive nucleotide sequence divergence and chromosomal rearrangements, mirroring characteristics of sex chromosomes. Meiotic recombination was assumed to be repressed within the C. neoformans MAT locus. A previous study identified recombination hot spots flanking the C. neoformans MAT, and these hot spots are associated with high GC content. Here, we investigated a GC-rich intergenic region located within the MAT locus of C. neoformans to establish if this region also exhibits unique recombination behavior during meiosis. Population genetics analysis of natural C. neoformans isolates revealed signals of homogenization spanning this GC-rich intergenic region within different C. neoformans lineages, consistent with a model in which gene conversion of this region during meiosis prevents it from diversifying within each lineage. By analyzing meiotic progeny from laboratory crosses, we found that meiotic recombination (gene conversion occurs around the GC-rich intergenic region at a frequency equal to or greater than the meiotic recombination frequency observed in other genomic regions. We discuss the implications of these findings with

  9. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

    OpenAIRE

    Johnson, Roger D.; Jasin, Maria

    2000-01-01

    In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contras...

  10. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

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    Suvi Jain

    2016-04-01

    Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.

  11. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Suvi Jain

    2016-04-01

    Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.

  12. Porphyrin Induced Laser Deactivation of Trypsinogen-Trypsin Conversion

    Science.gov (United States)

    Perido, Joanna; Brancaleon, Lorenzo

    2015-03-01

    Pancreatitis is caused by the inflammation of the pancreas, where the digestive enzyme trypsin is activated from the precursor enzyme trypsinogen while still in the pancreas. The presence of trypsin in the pancreas causes auto-activation of trypsinogen, resulting in greater inflammation and auto-digestion of the pancreas. In severe cases, this cascade effect can lead to organ failure, diabetes, and pancreatic cancer. Our hypothesis is that if trypsinogen is prevented from auto-activating into trypsin, then this cascade can be stopped. We propose to do this by inducing conformational changes in trypsinogen when bound to a photoactive porphyrin dye. Porphyrins are comprised of four linked heterocyclic groups forming a flat ring, and bind well with proteins such as trypsinogen. In this study we used spectroscopic techniques to probe the binding of meso-tetrakis (4-sulfonatephenyl) porphyrin (TSPP) to trypsinogen in vitro, as a preliminary step to then prompt and characterize conformational changes of trypsinogen through irradiation. If conformational changes are detected the trypsinogen will be tested for trypsin inactivation. This investigation may provide promising initial results to the possible use of porphyrins as an inhibitor of the self-activation of trypsinogen into trypsin, and a potential inhibitor of pancreatitis. MARC*U-STAR.

  13. Closely linked H2B genes in the marine copepod, Tigriopus californicus indicate a recent gene duplication or gene conversion event.

    Science.gov (United States)

    Brown, D; Cook, A; Wagner, M; Wells, D

    1992-01-01

    Two nonallelic histone gene clusters were characterized in the marine copepod, Tigriopus californicus. The DNA sequence of one of the clusters reveals six genes in the contiguous arrangement of H2B, H1, H3, H4, H2B and H2A. The order of genes within the second cluster is H3, H4, H2B and H2A. There is no evidence for the presence of an H1 gene in this cluster. Comparison of the three copepod H2B genes reveals a high degree of similarity between the 5' upstream regions and between the amino terminal halves of the two H2B genes found within the same cluster. From these data we infer that gene duplication and/or gene conversion events occurred within this cluster in the recent past.

  14. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  15. Conversion of syngas to higher alcohols over Cu-Fe-Zr catalysts induced by ethanol

    Institute of Scientific and Technical Information of China (English)

    Hongtao Zhang; Xiaomei Yang; Lipeng Zhou; Yunlai Su; Zhongmin Liu

    2009-01-01

    Ethanol induced method was applied to prepare Cu-Fe-Zr catalysts for conversion of syngas to higher alcohols. The catalytic performance of the catalysts induced by ethanol was superior to that of the catalyst prepared by the conventional precipitation method. Among various procedures for ethanol induced method,it was found that incorporation of ethanol in the precipitation process was the better. After incorporation of ethanol,the crystal size of CuO decreased and the reduction of copper species became easier. The better activity of Cu-Fe-Zr catalysts prepared by ethanol induced procedures was probably caused by the higher dispersion of Cu species.

  16. Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates.

    Science.gov (United States)

    Palamara, Pier Francesco; Francioli, Laurent C; Wilton, Peter R; Genovese, Giulio; Gusev, Alexander; Finucane, Hilary K; Sankararaman, Sriram; Sunyaev, Shamil R; de Bakker, Paul I W; Wakeley, John; Pe'er, Itsik; Price, Alkes L

    2015-12-01

    The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10(-8) per base per generation and a rate of 1.26 × 10(-9) for conversion as 5.99 × 10(-6). We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction.

  17. Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

    NARCIS (Netherlands)

    Bolder, S.G.; Vasbinder, A.; Sagis, L.M.C.; Linden, van der E.

    2007-01-01

    Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion

  18. Optically induced mode conversion in graded-index fibers using ultra-short laser pulses

    CERN Document Server

    Hellwig, Tim; Fallnich, Carsten

    2013-01-01

    We propose the use of graded-index few-mode fibers for mode-conversion by long-period gratings (LPG) transiently written by ultrashort laser pulses using the optical Kerr effect. The mode inter- action is studied by numerically solving the multi-mode coupled nonlinear Schroedinger equations. We present highly efficient conversion of the LP 01 - into the LP 11 -mode preserving the pulse shape in contrast to previous results in step-index fibers. Furthermore, mode conversion using different wavelengths for inducing and probing the LPG is shown. Due to the flat phase-matching curve of the examined modes in the graded-index fiber, mode-conversion can be observed for probe center wavelengths of 1100nm up to 1800nm with a write beam centered around 1030nm. Therefore, a complete separation of the probe from the write beam should be possible as well as the application of optically induced guided mode conversion for all optical modulation across a broad wavelength range.

  19. Mammalian BEX, WEX and GASP genes: Coding and non-coding chimaerism sustained by gene conversion events

    Directory of Open Access Journals (Sweden)

    Ponting Chris P

    2005-10-01

    Full Text Available Abstract Background The identification of sequence innovations in the genomes of mammals facilitates understanding of human gene function, as well as sheds light on the molecular mechanisms which underlie these changes. Although gene duplication plays a major role in genome evolution, studies regarding concerted evolution events among gene family members have been limited in scope and restricted to protein-coding regions, where high sequence similarity is easily detectable. Results We describe a mammalian-specific expansion of more than 20 rapidly-evolving genes on human chromosome Xq22.1. Many of these are highly divergent in their protein-coding regions yet contain a conserved sequence motif in their 5' UTRs which appears to have been maintained by multiple events of concerted evolution. These events have led to the generation of chimaeric genes, each with a 5' UTR and a protein-coding region that possess independent evolutionary histories. We suggest that concerted evolution has occurred via gene conversion independently in different mammalian lineages, and these events have resulted in elevated G+C levels in the encompassing genomic regions. These concerted evolution events occurred within and between genes from three separate protein families ('brain-expressed X-linked' [BEX], WWbp5-like X-linked [WEX] and G-protein-coupled receptor-associated sorting protein [GASP], which often are expressed in mammalian brains and associated with receptor mediated signalling and apoptosis. Conclusion Despite high protein-coding divergence among mammalian-specific genes, we identified a DNA motif common to these genes' 5' UTR exons. The motif has undergone concerted evolution events independently of its neighbouring protein-coding regions, leading to formation of evolutionary chimaeric genes. These findings have implications for the identification of non protein-coding regulatory elements and their lineage-specific evolution in mammals.

  20. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    Science.gov (United States)

    Melis, Anastasios; Mitra, Mautusi

    2010-06-29

    The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

  1. Complex signatures of locus-specific selective pressures and gene conversion on Human Growth Hormone/Chorionic Somatomammotropin genes.

    Science.gov (United States)

    Sedman, Laura; Padhukasahasram, Badri; Kelgo, Piret; Laan, Maris

    2008-10-01

    Reduced birth weight and slow neonatal growth are risks correlated with the development of common diseases in adulthood. The Human Growth Hormone/Chorionic Somatomammotropin (hGH/CSH) gene cluster (48 kb) at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placental genes (GH2, CSH1, CSH2, and CSHL1) may contribute to common variation in intrauterine and infant growth, and also to the regulation of feto-maternal and adult glucose metabolism. In contrast to GH1, there are limited genetic data on the hGH/CSH genes expressed in utero. We report the first survey of sequence variation encompassing all five hGH/CSH genes. Resequencing identified 113 SNPs/indels (ss86217675-ss86217787 in dbSNP) including 66 novel variants, and revealed remarkable differences in diversity patterns among the homologous duplicated genes as well as between the study populations of European (Estonians), Asian (Han Chinese), and African (Mandenkalu) ancestries. A dominant feature of the hGH/CSH region is hyperactive gene conversion, with the rate exceeding tens to hundreds of times the rate of reciprocal crossing-over and resulting in near absence of linkage disequilibrium. The initiation of gene conversion seems to be uniformly distributed because the data do not predict any recombination hotspots. Signatures of different selective constraints acting on each gene indicate functional specification of the hGH/CSH genes. Most strikingly, the GH2 coding for placental growth hormone shows strong intercontinental diversification (F(ST)=0.41-0.91; p<10(-6)) indicative of balancing selection, whereas the flanking CSH1 exhibits low population differentiation (F(ST)=0.03-0.09), low diversity (non-Africans, pi=8-9 x 10(-5); Africans, pi=8.2 x 10(-4)), and one dominant haplotype worldwide, consistent with purifying selection. The results imply that the success of an association study targeted to duplicated genes may be enhanced by prior resequencing of the study population in order

  2. Contrasting evolutionary histories of MHC class I and class II loci in grouse—Effects of selection and gene conversion

    Science.gov (United States)

    Minias, Piotr; Bateson, Zachary W; Whittingham, Linda A; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O

    2016-01-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

  3. TMEM231 Gene Conversion Associated with Joubert and Meckel-Gruber Syndromes in the Same Family.

    Science.gov (United States)

    Maglic, Dino; Stephen, Joshi; Malicdan, May Christine V; Guo, Jennifer; Fischer, Roxanne; Konzman, Daniel; Mullikin, James C; Gahl, William A; Vilboux, Thierry; Gunay-Aygun, Meral

    2016-11-01

    Joubert and Meckel-Gruber syndromes (JS and MGS) are ciliopathies with overlapping features. JS patients manifest the "molar tooth sign" on brain imaging and variable eye, kidney, and liver disease. MGS presents with polycystic kidneys, occipital encephalocele, and polydactyly; it is typically perinatally fatal. Both syndromes are genetically heterogeneous; some genes cause either syndrome. Here, we report two brothers married to unrelated women. The first brother had three daughters with JS and a son with polycystic kidneys who died at birth. The second brother's wife had a fetal demise due to MGS. Whole exome sequencing identified TMEM231 NM_001077416.2: c.784G>A; p.(Asp262Asn) in all children and the wife of the first brother; the second brother's wife had a c.406T>G;p.(Trp136Gly) change. In-depth analysis uncovered a rare gene conversion event in TMEM231, leading to loss of exon 4, in all the affected children of first brother. We believe that the combination of this gene conversion with different missense mutations led to a spectrum of phenotypes that span JS and MGS. © 2016 WILEY PERIODICALS, INC.

  4. Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity.

    Science.gov (United States)

    Lo, Yi-Chen; Paffett, Kimberly S; Amit, Or; Clikeman, Jennifer A; Sterk, Rosa; Brenneman, Mark A; Nickoloff, Jac A

    2006-06-01

    RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.

  5. The surprising negative correlation of gene length and optimal codon use--disentangling translational selection from GC-biased gene conversion in yeast.

    Science.gov (United States)

    Stoletzki, Nina

    2011-04-11

    Surprisingly, in several multi-cellular eukaryotes optimal codon use correlates negatively with gene length. This contrasts with the expectation under selection for translational accuracy. While suggested explanations focus on variation in strength and efficiency of translational selection, it has rarely been noticed that the negative correlation is reported only in organisms whose optimal codons are biased towards codons that end with G or C (-GC). This raises the question whether forces that affect base composition--such as GC-biased gene conversion--contribute to the negative correlation between optimal codon use and gene length. Yeast is a good organism to study this as equal numbers of optimal codons end in -GC and -AT and one may hence compare frequencies of optimal GC- with optimal AT-ending codons to disentangle the forces. Results of this study demonstrate in yeast frequencies of GC-ending (optimal AND non-optimal) codons decrease with gene length and increase with recombination. A decrease of GC-ending codons along genes contributes to the negative correlation with gene length. Correlations with recombination and gene expression differentiate between GC-ending and optimal codons, and also substitution patterns support effects of GC-biased gene conversion. While the general effect of GC-biased gene conversion is well known, the negative correlation of optimal codon use with gene length has not been considered in this context before. Initiation of gene conversion events in promoter regions and the presence of a gene conversion gradient most likely explain the observed decrease of GC-ending codons with gene length and gene position.

  6. GC-biased gene conversion impacts ribosomal DNA evolution in vertebrates, angiosperms, and other eukaryotes.

    Science.gov (United States)

    Escobar, Juan S; Glémin, Sylvain; Galtier, Nicolas

    2011-09-01

    Ribosomal DNA (rDNA) is one of the most conserved genes in eukaryotes. The multiples copies of rDNA in the genome evolve in a concerted manner, through unequal crossing over and/or gene conversion, two mechanisms related to homologous recombination. Recombination increases local GC content in several organisms through a process known as GC-biased gene conversion (gBGC). gBGC has been well characterized in mammals, birds, and grasses, but its phylogenetic distribution across the tree of life is poorly understood. Here, we test the hypothesis that recombination affects the evolution of base composition in 18S rDNA and examine the reliability of this thoroughly studied molecule as a marker of gBGC in eukaryotes. Phylogenetic analyses of 18S rDNA in vertebrates and angiosperms reveal significant heterogeneity in the evolution of base composition across both groups. Mammals, birds, and grasses experience increases in the GC content of the 18S rDNA, consistent with previous genome-wide analyses. In addition, we observe increased GC contents in Ostariophysi ray-finned fishes and commelinid monocots (i.e., the clade including grasses), suggesting that the genomes of these two groups have been affected by gBGC. Polymorphism analyses in rDNA confirm that gBGC, not mutation bias, is the most plausible explanation for these patterns. We also find that helix and loop sites of the secondary structure of ribosomal RNA do not evolve at the same pace: loops evolve faster than helices, whereas helices are GC richer than loops. We extend analyses to major lineages of eukaryotes and suggest that gBGC might have also affected base composition in Giardia (Diplomonadina), nudibranch gastropods (Mollusca), and Asterozoa (Echinodermata).

  7. The roles of gene duplication, gene conversion and positive selection in rodent Esp and Mup pheromone gene families with comparison to the Abp family.

    Science.gov (United States)

    Karn, Robert C; Laukaitis, Christina M

    2012-01-01

    Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining K(a)/K(s) for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with K(a)/K(s) >1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication.

  8. A Metagenomic Perspective on Changes to Nutrient-cycling Genes Following Forest-to-agriculture Conversion in the Amazon Basin

    Science.gov (United States)

    Meyer, K. M.; Womack, A. M.; Rodrigues, J.; Nüsslein, K.; Bohannan, B. J. M.

    2014-12-01

    Forest-to-agriculture conversion has been shown to alter nutrient cycling and the community composition of soil microorganisms. However, few studies have looked simultaneously at how the abundance, composition, and diversity of microbial genes involved in nutrient cycling change with conversion. We used shotgun metagenomic sequencing to analyze soil from primary rainforest and converted cattle pasture sampled at the Fazenda Nova Vida in Rondônia, Brazil. The diversity, richness, and evenness of nutrient cycling genes were significantly higher in the pasture, and the composition of nutrient cycling communities differed significantly between land use types. These results largely mirror taxonomic shifts following Amazon rainforest conversion, which tends to increase diversity, richness, and evenness of soil microbial communities. The abundance of genes related to N cycling and methane flux differed between land use types. Methanotrophy genes decreased in abundance in the pasture, whereas methanogenesis genes were not significantly different between land use types. These changes could underlie the commonly observed shift from methane sink to source following forest-to-agriculture conversion. Multiple genes in the nitrogen cycle also differed with land use, including genes related to N-fixation and ammonification. Metagenomics provides a unique perspective on the consequences of land use change on microbial community structure and function.

  9. Codelivery of antitumor drug and gene by a pH-sensitive charge-conversion system.

    Science.gov (United States)

    Guan, Xiuwen; Li, Yanhui; Jiao, Zixue; Lin, Lin; Chen, Jie; Guo, Zhaopei; Tian, Huayu; Chen, Xuesi

    2015-02-11

    In the present study, a gene and drug codelivery system was developed by electrostatic binding of polyethylenimine-poly(l-lysine)-poly(l-glutamic acid) (PELG), polyethylenimine (PEI), cis-aconityl-doxorubicin (CAD), and DNA. Zeta potential and drug release analysis confirmed the pH-responsive charge conversion and acid-sensitive drug release functional properties of the PELG/PEI/(DNA+CAD) system. Gel retardation assay and transfection experiment showed the codelivery system had effective DNA binding ability and good transfection efficiency on HepG2 cells. The therapeutic gene p53 was further employed to study its combinational effects with CAD. Cytotoxicity assay showed the half inhibitory concentration (IC50) of the PELG/PEI/(p53+CAD) codelivery system was lower than that of the gene or the drug delivery system. Confocal laser scanning microscopy (CLSM) showed that the drug and gene could be delivered into the cells simultaneously. A significant increase of p53 gene expression was achieved after HepG2 cells treated by PELG/PEI/(p53+CAD) codelivery system. The apoptosis experiment indicated clearly that the codelivery system could lead an effective apoptosis on tumor cells, which was beneficial for the treatment of cancer. The biodistribution and tumor accumulation of the codelivery system was explored via in vivo imaging in subcutaneous xenograft and in situ tumor models. The tumor and some major organs were excised and imaged, and the results showed that the codelivery system can accumulate efficiently in tumor for both tumor models. It can be suggested from the above results that the PELG/PEI/(DNA+CAD) codelivery system will have great potential applications in cancer therapy.

  10. Tetracycline inducible gene manipulation in serotonergic neurons.

    Directory of Open Access Journals (Sweden)

    Tillmann Weber

    Full Text Available The serotonergic (5-HT neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA mouse line (TPH2-tTA that allows temporal and spatial control of tetracycline (Ptet controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ. In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox. Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20 were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We

  11. The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana

    Science.gov (United States)

    Wijnker, Erik; Velikkakam James, Geo; Ding, Jia; Becker, Frank; Klasen, Jonas R; Rawat, Vimal; Rowan, Beth A; de Jong, Daniël F; de Snoo, C Bastiaan; Zapata, Luis; Huettel, Bruno; de Jong, Hans; Ossowski, Stephan; Weigel, Detlef; Koornneef, Maarten; Keurentjes, Joost JB; Schneeberger, Korbinian

    2013-01-01

    Knowledge of the exact distribution of meiotic crossovers (COs) and gene conversions (GCs) is essential for understanding many aspects of population genetics and evolution, from haplotype structure and long-distance genetic linkage to the generation of new allelic variants of genes. To this end, we resequenced the four products of 13 meiotic tetrads along with 10 doubled haploids derived from Arabidopsis thaliana hybrids. GC detection through short reads has previously been confounded by genomic rearrangements. Rigid filtering for misaligned reads allowed GC identification at high accuracy and revealed an ∼80-kb transposition, which undergoes copy-number changes mediated by meiotic recombination. Non-crossover associated GCs were extremely rare most likely due to their short average length of ∼25–50 bp, which is significantly shorter than the length of CO-associated GCs. Overall, recombination preferentially targeted non-methylated nucleosome-free regions at gene promoters, which showed significant enrichment of two sequence motifs. DOI: http://dx.doi.org/10.7554/eLife.01426.001 PMID:24347547

  12. IDconverter and IDClight: Conversion and annotation of gene and protein IDs

    Directory of Open Access Journals (Sweden)

    Díaz-Uriarte Ramón

    2007-01-01

    Full Text Available Abstract Background Researchers involved in the annotation of large numbers of gene, clone or protein identifiers are usually required to perform a one-by-one conversion for each identifier. When the field of research is one such as microarray experiments, this number may be around 30,000. Results To help researchers map accession numbers and identifiers among clones, genes, proteins and chromosomal positions, we have designed and developed IDconverter and IDClight. They are two user-friendly, freely available web server applications that also provide additional functional information by mapping the identifiers on to pathways, Gene Ontology terms, and literature references. Both tools are high-throughput oriented and include identifiers for the most common genomic databases. These tools have been compared to other similar tools, showing that they are among the fastest and the most up-to-date. Conclusion These tools provide a fast and intuitive way of enriching the information coming out of high-throughput experiments like microarrays. They can be valuable both to wet-lab researchers and to bioinformaticians.

  13. Suppression of self-induced flavor conversion in the supernova accretion phase.

    Science.gov (United States)

    Sarikas, Srdjan; Raffelt, Georg G; Hüdepohl, Lorenz; Janka, Hans-Thomas

    2012-02-10

    Self-induced flavor conversions of supernova (SN) neutrinos can strongly modify the flavor-dependent fluxes. We perform a linearized flavor stability analysis with accretion-phase matter profiles of a 15M[symbol: see text] spherically symmetric model and corresponding neutrino fluxes. We use realistic energy and angle distributions, the latter deviating strongly from quasi-isotropic emission, thus accounting for both multiangle and multienergy effects. For our matter and neutrino density profile we always find stable conditions: flavor conversions are limited to the usual Mikheyev-Smirnov-Wolfenstein effect. In this case one may distinguish the neutrino mass hierarchy in a SN neutrino signal if the mixing angle θ13 is as large as suggested by recent experiments.

  14. Converse Piezoelectric Effect Induced Transverse Deflection of a Free-Standing ZnO Microbelt

    KAUST Repository

    Hu, Youfan

    2009-07-08

    We demonstrate the first electric field induced transverse deflection of a single-crystal, free-standing ZnO microbelt as a result of converse piezoelectric effect. For a microbelt growing along the c-axis, a shear stress in the a-c plane can be induced when an electric field E is applied along the a-axis of the wurtzite structure. As amplified by the large aspect ratio of the microbelt that grows along the c-axis, the strain localized near the root can be detected via the transverse deflection perpendicular to the ZnO microbelt. After an experimental approach was carefully designed and possible artifacts were ruled out, the experimentally observed degree of deflection of the microbelt agrees well with the theoretically expected result. The device demonstrated has potential applications as transverse actuators/sensors/switches and electric field induced mechanical deflectors. © 2009 American Chemical Society.

  15. Gravity-Induced Gene Expression in Plants.

    Science.gov (United States)

    Sederoff, Heike; Heber, Steffen; Howard, Brian; Myburg-Nichols, Henrietta; Hammond, Rebecca; Salinas-Mondragon, Raul; Brown, Christopher S.

    Plants sense changes in their orientation towards the vector of gravity and respond with directional growth. Several metabolites in the signal transduction cascade have been identified. However, very little is known about the interaction between these sensing and signal transduction events and even less is known about their role in the differential growth response. Gravity induced changes in transcript abundance have been identified in Arabidopsis whole seedlings and root apices (Moseyko et al. 2002; Kimbrough et al. 2004). Gravity induced transcript abundance changes can be observed within less than 1 min after stimulation (Salinas-Mondragon et al. 2005). Gene expression however requires not only transcription but also translation of the mRNA. Translation can only occur when mRNA is associated with ribosomes, even though not all mRNA associated with ribosomes is actively translated. To approximate translational capacity we quantified whole genome transcript abundances in corn stem pulvini during the first hour after gravity stimulation in total and poly-ribosomal fractions. As in Arabidopsis root apices, transcript abundances of several clusters of genes responded to gravity stimulation. The vast majority of these transcripts were also found to associate with polyribosomes in the same temporal and quantitative pattern. These genes are transcriptionally regulated by gravity stimulation, but do not exhibit translational regulation. However, a small group of genes showed increased transcriptional regulation after gravity stimulation, but no association with polysomes. These transcripts likely are translationally repressed. The mechanism of translational repression for these transcripts is unknown. Based on the hypothesis that the genes essential for gravitropic responses should be expressed in most or all species, we compared the temporal gravity induced expression pattern of all orthologs identified between maize and Arabidopsis. A small group of genes showed high

  16. Photorhabdus luminescens genes induced upon insect infection

    Directory of Open Access Journals (Sweden)

    Jung Kirsten

    2008-05-01

    Full Text Available Abstract Background Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. Results A differential fluorescence induction (DFI approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18 were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known

  17. Supernova neutrino halo and the suppression of self-induced flavor conversion

    CERN Document Server

    Sarikas, Srdjan; Raffelt, Georg; Hüdepohl, Lorenz; Janka, Hans-Thomas

    2012-01-01

    Neutrinos streaming from a supernova (SN) core occasionally scatter in the envelope, producing a small "neutrino halo" with a much broader angle distribution than the primary flux originating directly from the core. Cherry et al. (2012) have recently pointed out that, during the accretion phase, the halo actually dominates neutrino-neutrino refraction at distances exceeding some 100 km. However, the multi-angle matter effect (which increases if the angle distribution is broader) still appears to suppress self-induced flavor conversion during the accretion phase.

  18. OrgConv: detection of gene conversion using consensus sequences and its application in plant mitochondrial and chloroplast homologs

    Directory of Open Access Journals (Sweden)

    Hao Weilong

    2010-03-01

    Full Text Available Abstract Background The ancestry of mitochondria and chloroplasts traces back to separate endosymbioses of once free-living bacteria. The highly reduced genomes of these two organelles therefore contain very distant homologs that only recently have been shown to recombine inside the mitochondrial genome. Detection of gene conversion between mitochondrial and chloroplast homologs was previously impossible due to the lack of suitable computer programs. Recently, I developed a novel method and have, for the first time, discovered recurrent gene conversion between chloroplast mitochondrial genes. The method will further our understanding of plant organellar genome evolution and help identify and remove gene regions with incongruent phylogenetic signals for several genes widely used in plant systematics. Here, I implement such a method that is available in a user friendly web interface. Results OrgConv (Organellar Conversion is a computer package developed for detection of gene conversion between mitochondrial and chloroplast homologous genes. OrgConv is available in two forms; source code can be installed and run on a Linux platform and a web interface is available on multiple operating systems. The input files of the feature program are two multiple sequence alignments from different organellar compartments in FASTA format. The program compares every examined sequence against the consensus sequence of each sequence alignment rather than exhaustively examining every possible combination. Making use of consensus sequences significantly reduces the number of comparisons and therefore reduces overall computational time, which allows for analysis of very large datasets. Most importantly, with the significantly reduced number of comparisons, the statistical power remains high in the face of correction for multiple tests. Conclusions Both the source code and the web interface of OrgConv are available for free from the OrgConv website http

  19. Electromagnetically induced transparency and wide-band wavelength conversion in silicon nitride microdisk optomechanical resonators

    CERN Document Server

    Liu, Yuxiang; Aksyuk, Vladimir; Srinivasan, Kartik

    2013-01-01

    We demonstrate optomechanically-mediated electromagnetically-induced transparency and wavelength conversion in silicon nitride (Si3N4) microdisk resonators. Fabricated devices support whispering gallery optical modes with a quality factor (Q) of 10^6, and radial breathing mechanical modes with a Q=10^4 and a resonance frequency of 625 MHz, so that the system is in the resolved sideband regime. Placing a strong optical control field on the red (blue) detuned sideband of the optical mode produces coherent interference with a resonant probe beam, inducing a transparency (absorption) window for the probe. This is observed for multiple optical modes of the device, all of which couple to the same mechanical mode, and which can be widely separated in wavelength due to the large bandgap of Si3N4. These properties are exploited to demonstrate frequency upconversion and downconversion of optical signals between the 1300 nm and 980 nm bands.

  20. Identification of Candidate Genes and Biosynthesis Pathways Related to Fertility Conversion by Wheat KTM3315A Transcriptome Profiling

    Directory of Open Access Journals (Sweden)

    Lingli Zhang

    2017-04-01

    Full Text Available The Aegilops kotschyi thermo-sensitive cytoplasmic male sterility (K-TCMS system may facilitate hybrid wheat (Triticum aestivum L. seed multiplication and production. The K-TCMS line is completely male sterile during the normal wheat-growing season, whereas its fertility can be restored in a high-temperature environment. To elucidate the molecular mechanisms responsible for male sterility/fertility conversion and candidate genes involved with pollen development in K-TCMS, we employed RNA-seq to sequence the transcriptomes of anthers from K-TCMS line KTM3315A during development under sterile and fertile conditions. We identified 16840 differentially expressed genes (DEGs in different stages including15157 known genes (15135 nuclear genes and 22 plasmagenes and 1683 novel genes. Bioinformatics analysis identified possible metabolic pathways involved with fertility based on KEGG pathway enrichment of the DEGs expressed in fertile and sterile plants. We found that most of the genes encoding key enzyme in the phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were significant upregulated in uninucleate, binuclate or trinucleate stage, which both interact with MYB transcription factors, and that link between all play essential roles in fertility conversion. The relevant DEGs were verified by quantitative RT-PCR. Thus, we suggested that phenylpropanoid biosynthesis and jasmonate biosynthesis pathways were involved in fertility conversion of K-TCMS wheat. This will provide a new perspective and an effective foundation for the research of molecular mechanisms of fertility conversion of CMS wheat. Fertility conversion mechanism in thermo-sensitive cytoplasmic male sterile/fertile wheat involves the phenylpropanoid biosynthesis pathway, jasmonate biosynthesis pathway, and MYB transcription factors.

  1. Self-induced flavor conversion of supernova neutrinos on small scales

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, S. [Max-Planck-Institut für Physik (Werner-Heisenberg-Institut),Föhringer Ring 6, 80805 München (Germany); Hansen, R. S. [Department of Physics and Astronomy, University of Aarhus,8000 Aarhus C (Denmark); Izaguirre, I.; Raffelt, G.G. [Max-Planck-Institut für Physik (Werner-Heisenberg-Institut),Föhringer Ring 6, 80805 München (Germany)

    2016-01-15

    Self-induced flavor conversion of supernova (SN) neutrinos is a generic feature of neutrino-neutrino dispersion. The corresponding run-away modes in flavor space can spontaneously break the original symmetries of the neutrino flux and in particular can spontaneously produce small-scale features as shown in recent schematic studies. However, the unavoidable “multi-angle matter effect” shifts these small-scale instabilities into regions of matter and neutrino density which are not encountered on the way out from a SN. The traditional modes which are uniform on the largest scales are most prone for instabilities and thus provide the most sensitive test for the appearance of self-induced flavor conversion. As a by-product we clarify the relation between the time evolution of an expanding neutrino gas and the radial evolution of a stationary SN neutrino flux. Our results depend on several simplifying assumptions, notably stationarity of the solution, the absence of a “backward” neutrino flux caused by residual scattering, and global spherical symmetry of emission.

  2. Self-induced flavor conversion of supernova neutrinos on small scales

    CERN Document Server

    Chakraborty, Sovan; Izaguirre, Ignacio; Raffelt, Georg

    2016-01-01

    Self-induced flavor conversion of supernova (SN) neutrinos is a generic feature of neutrino-neutrino dispersion. The corresponding run-away modes in flavor space can spontaneously break the original symmetries of the neutrino flux and in particular can spontaneously produce small-scale features as shown in recent schematic studies. However, the unavoidable "multi-angle matter effect" shifts these small-scale instabilities into regions of matter and neutrino density which are not encountered on the way out from a SN. The traditional modes which are uniform on the largest scales are most prone for instabilities and thus provide the most sensitive test for the appearance of self-induced flavor conversion. As a by-product we clarify the relation between the time evolution of an expanding neutrino gas and the radial evolution of a stationary SN neutrino flux. Our results depend on several simplifying assumptions, notably stationarity of the solution, the absence of a "backward" neutrino flux caused by residual sca...

  3. Gene genealogies indicates abundant gene conversions and independent evolutionary histories of the mating-type chromosomes in the evolutionary history of Neurospora tetrasperma

    Directory of Open Access Journals (Sweden)

    Whittle Carrie A

    2010-07-01

    Full Text Available Abstract Background The self-fertile filamentous ascomycete Neurospora tetrasperma contains a large (~7 Mbp and young (mat chromosomes. The objective of the present study is to reveal the evolutionary history, including key genomic events, associated with the various regions of the mat chromosomes among ten strains representing all the nine known species (lineages contained within the N. tetrasperma species complex. Results Comparative analysis of sequence divergence among alleles of 24 mat-linked genes (mat A and mat a indicates that a large region of suppressed recombination exists within the mat chromosome for each of nine lineages of N. tetrasperma sensu latu. The recombinationally suppressed region varies in size and gene composition among lineages, and is flanked on both ends by normally recombining regions. Genealogical analyses among lineages reveals that eight gene conversion events have occurred between homologous mat A and mat a-linked alleles of genes located within the region of restricted recombination during the evolutionary history of N. tetrasperma. Conclusions We conclude that the region of suppressed recombination in the mat chromosomes has likely been subjected to independent contraction and/or expansion during the evolutionary history of the N. tetrasperma species complex. Furthermore, we infer that gene conversion events are likely a common phenomenon within this recombinationally suppressed genomic region. We argue that gene conversions might provide an efficient mechanism of adaptive editing of functional genes, including the removal of deleterious mutations, within the young recombinationally suppressed region of the mat chromosomes.

  4. Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin.

    Science.gov (United States)

    Zhang, Huijuan; Xiao, Xiao; Wang, Peng; Pang, Siping; Qu, Feng; Ai, Xicheng; Zhang, Jianping

    2009-09-15

    The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)(2) (S12) have been investigated by means of circular dichroism (CD), UV-visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K(+), and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K(+). Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)(3)T) in the absence of K(+) show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4.

  5. Shaking alone induces de novo conversion of recombinant prion proteins to β-sheet rich oligomers and fibrils.

    Directory of Open Access Journals (Sweden)

    Carol L Ladner-Keay

    Full Text Available The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl, high temperature, phospholipids, or mildly acidic conditions (pH 4. Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2 and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE, electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA and quaking induced conversion (QuIC.

  6. Conversion of the pathogenic fungus Colletotrichum magna to a nonpathogenic, endophytic mutualist by gene disruption

    Science.gov (United States)

    Redman, R.S.; Ranson, J.C.; Rodriguez, R.J.

    1999-01-01

    Hygromycin-resistant transformants of the cucurbit pathogen Colletotrichum magna (teleomorph: Glomerella magna) were generated by restriction enzyme-mediated integration (REMI) transformation. A rapid pathogenicity assay involving watermelon (Citrullus lanatus) seedlings was developed and 14,400 REMI transformants were screened and assessed for their ability to cause disease, colonize plant tissues, and confer disease resistance against wild-type C. magna. A total of 176 nonpathogenic REMI mutants capable of colonizing cucurbit plants were isolated and assigned to three groups based on their ability to confer disease resistance: phenotype A, 80 to 100% disease protection; phenotype B, 10 to 65% disease protection; and phenotype C, 0 to 4% disease protection. Molecular and genetic analyses of one REMI mutant (R1) indicated that the nonpathogenic phenotype A resulted from a single-site integration. R1 showed a 1:1 segregation of hygromycin resistance and nonpathogenicity and all hygromycin-resistant progeny were nonpathogenic. The integrated vector and 5.5 kb of flanking fungal genomic DNA were isolated from R1 and designated pGMR1. To verify that pGMR1 contained pathogenicity gene sequences, a wild-type isolate of C. magna was transformed with pGMR1 to induce gene disruptions by homologous integration. Approximately 47% of the pGMR1 transformants expressed phenotype A, indicating homologous integration and gene disruption.

  7. 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

    Science.gov (United States)

    Cayer, Marie-Pierre; Drouin, Mathieu; Proulx, Maryse; Jung, Daniel

    2010-04-15

    2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

  8. Streptococcus mitis induces conversion of Helicobacter pylori to coccoid cells during co-culture in vitro.

    Directory of Open Access Journals (Sweden)

    Yalda Khosravi

    Full Text Available Helicobacter pylori (H. pylori is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptococcus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.

  9. Heat-inducible RNAi for gene functional analysis in plants.

    Science.gov (United States)

    Masclaux, Frédéric; Galaud, Jean-Philippe

    2011-01-01

    Controlling gene expression during plant development is an efficient method to explore gene function and RNA interference (RNAi) is now considered as a powerful technology for gene functional analysis. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability or plant growth and alternative silencing strategies avoiding these limitations should be preferred. Tissue-specific and inducible promoters, able to control gene expression at spatial and/or temporal level, can be used to circumvent viability problems. In this chapter, after a rapid overview of the inducible promoters currently used for transgenic approaches in plants, we describe a method we have developed to study gene function by heat-inducible RNAi. This system is easy to use and complementary to those based on chemical gene inducer treatments and might be useful for both research and biotechnological applications.

  10. Photon Self-Induced Spin to Orbital Conversion in TGG crystal at high laser power

    CERN Document Server

    Mosca, S; Karimi, E; Piccirillo, B; Marrucci, L; De Rosa, R; Genin, E; Milano, L; Santamato, E

    2010-01-01

    In this paper, we present experimental evidence of a newly discovered third-order nonlinear optical process Self-Induced Spin-to-Orbital Conversion (SISTOC) of the photon angular momentum. This effect is the physical mechanism at the origin of the depolarization of very intense laser beams propagating in isotropic materials. The SISTOC process, like self-focusing, is triggered by laser heating leading to a radial temperature gradient in the medium. In this work we tested the occurrence of SISTOC in a terbium gallium garnet (TGG) rod for an impinging laser power of about 100~W. To study the SISTOC process we used different techniques: polarization analysis, interferometry and tomography of the photon orbital angular momentum. Our results confirm, in particular, that the apparent depolarization of the beam is due to the occurrence of maximal entanglement between the spin and orbital angular momentum of the photons undergoing the SISTOC process. This explanation of the true nature of the depolarization mechanism...

  11. Laser-induced down-conversion parameters of singly and doubly doped ZnS phosphors

    Indian Academy of Sciences (India)

    H S Bhatti; Rajesh Sharma; N K Verma

    2005-09-01

    Singly and doubly doped ZnS phosphors have been synthesized using flux method. Laser-induced photoluminescence has been observed in ZnS-doped phosphors when these were excited by the pulsed UV N2 laser radiation. Due to down-conversion phenomenon, fast phosphorescence emission in the visible region is recorded in milliseconds time domain for ZnS:Mn while in the case of ZnS:Mn:killer (Fe, Co and Ni) the lifetime reduces to microseconds time domain. Experimentally observed luminescent emission parameters of excited states such as, lifetimes, trap-depth values and decay constants have been reported here at room temperature. The high efficiency and fast recombination times observed in doped ZnS phosphors make these materials very attractive for optoelectronic applications.

  12. Repeat-induced gene silencing in mammals.

    Science.gov (United States)

    Garrick, D; Fiering, S; Martin, D I; Whitelaw, E

    1998-01-01

    In both plants and Drosophila melanogaster, expression from a transgenic locus may be silenced when repeated transgene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the lox/Cre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

  13. Biased gene conversion and GC-content evolution in the coding sequences of reptiles and vertebrates.

    Science.gov (United States)

    Figuet, Emeric; Ballenghien, Marion; Romiguier, Jonathan; Galtier, Nicolas

    2014-12-19

    Mammalian and avian genomes are characterized by a substantial spatial heterogeneity of GC-content, which is often interpreted as reflecting the effect of local GC-biased gene conversion (gBGC), a meiotic repair bias that favors G and C over A and T alleles in high-recombining genomic regions. Surprisingly, the first fully sequenced nonavian sauropsid (i.e., reptile), the green anole Anolis carolinensis, revealed a highly homogeneous genomic GC-content landscape, suggesting the possibility that gBGC might not be at work in this lineage. Here, we analyze GC-content evolution at third-codon positions (GC3) in 44 vertebrates species, including eight newly sequenced transcriptomes, with a specific focus on nonavian sauropsids. We report that reptiles, including the green anole, have a genome-wide distribution of GC3 similar to that of mammals and birds, and we infer a strong GC3-heterogeneity to be already present in the tetrapod ancestor. We further show that the dynamic of coding sequence GC-content is largely governed by karyotypic features in vertebrates, notably in the green anole, in agreement with the gBGC hypothesis. The discrepancy between third-codon positions and noncoding DNA regarding GC-content dynamics in the green anole could not be explained by the activity of transposable elements or selection on codon usage. This analysis highlights the unique value of third-codon positions as an insertion/deletion-free marker of nucleotide substitution biases that ultimately affect the evolution of proteins.

  14. Propane decomposition and conversion into other hydrocarbons using metal target assisted laser induced plasma

    Science.gov (United States)

    Moosakhani, A.; Parvin, P.; Reyhani, A.; Mortazavi, S. Z.

    2017-01-01

    It is shown that the propane molecules are strongly decomposed in the metal assisted laser induced plasma based on the nano-catalytic adsorption. A Q-Switched Nd:YAG laser is employed to irradiate the propane gas filled in the control chamber in the presence of the reactive metals such as Ni, Fe, Pd, and Cu in order to study the effect of catalysts during the decomposition. The catalytic targets simultaneously facilitate the plasma formation and the decomposition events leading to generate a wide distribution of the light and heavy hydrocarbon molecules, mainly due to the recombination processes. Fourier transform infrared spectroscopy and gas chromatography instruments support the findings by detecting the synthetic components. Furthermore, the optical emission spectroscopy of the laser induced plasma emissions realizes the real time monitoring of the reactions taking place during each laser shot. The subsequent recombination events give rise to the generation of a variety of the hydrocarbon molecules. The dissociation rate, conversion ratio, selectivity, and yield as well as the performance factor arise mainly from the catalytic effects of the metal species. Moreover, the ablation rate of the targets of interest is taken into account as a measure of the catalytic reactivity due to the abundance of the metal species ablated from the target. This leads to assess the better performance factor for Pd among four metal catalysts of interest during propane decomposition. Finally, the molecules such as ethane and ethylene are identified as the stable abundant species created during the successive molecular recombination processes.

  15. Analysis of crossover breakpoints yields new insights into the nature of the gene conversion events associated with large NF1 deletions mediated by nonallelic homologous recombination.

    Science.gov (United States)

    Bengesser, Kathrin; Vogt, Julia; Mussotter, Tanja; Mautner, Victor-Felix; Messiaen, Ludwine; Cooper, David N; Kehrer-Sawatzki, Hildegard

    2014-02-01

    Large NF1 deletions are mediated by nonallelic homologous recombination (NAHR). An in-depth analysis of gene conversion operating in the breakpoint-flanking regions of large NF1 deletions was performed to investigate whether the rate of discontinuous gene conversion during NAHR with crossover is increased, as has been previously noted in NAHR-mediated rearrangements. All 20 germline type-1 NF1 deletions analyzed were mediated by NAHR associated with continuous gene conversion within the breakpoint-flanking regions. Continuous gene conversion was also observed in 31/32 type-2 NF1 deletions investigated. In contrast to the meiotic type-1 NF1 deletions, type-2 NF1 deletions are predominantly of post-zygotic origin. Our findings therefore imply that the mitotic as well as the meiotic NAHR intermediates of large NF1 deletions are processed by long-patch mismatch repair (MMR), thereby ensuring gene conversion tract continuity instead of the discontinuous gene conversion that is characteristic of short-patch repair. However, the single type-2 NF1 deletion not exhibiting continuous gene conversion was processed without MMR, yielding two different deletion-bearing chromosomes, which were distinguishable in terms of their breakpoint positions. Our findings indicate that MMR failure during NAHR, followed by post-meiotic/mitotic segregation, has the potential to give rise to somatic mosaicism in human genomic rearrangements by generating breakpoint heterogeneity.

  16. The role of induced mutation in conversion of photoperiod dependence in cotton.

    Science.gov (United States)

    Abdurakhmonov, Ibrokhim Y; Kushanov, Fakhriddin N; Djaniqulov, Fayzulla; Buriev, Zabardast T; Pepper, Alan E; Fayzieva, Nilufar; Mavlonov, Gafurjon T; Saha, Sukumar; Jenkins, Jonnie N; Abdukarimov, Abdusattor

    2007-01-01

    Wild cotton germplasm resources are largely underutilized because of photoperiod-dependent flowering of "exotic" cottons. The objectives of this work were to explore the genome-wide effect of induced mutation in photoperiod-converted induced cotton mutants, estimating the genetic change between mutant and wild-type cottons using simple sequence repeats (SSRs) as well as understand the pattern of SSR mutation in induced mutagenesis. Three groups of photoperiod-converted radiomutants ((32)P) including their wild-type parental lines, A- and D-genome diploids, and typically grown cotton cultivars were screened with 250 cotton SSR primer pairs. Forty SSRs revealed the same SSR mutation profile in, at least, 2 independent mutant lines that were different from the original wild types. Induced mutagenesis both increased and decreased the allele sizes of SSRs in mutants with the higher mutation rate in SSRs containing dinucleotide motifs. Genetic distance obtained based on 141 informative SSR alleles ranged from 0.09 to 0.60 in all studied cotton genotypes. Genetic distance within all photoperiod-converted induced mutants was in a 0.09-0.25 range. The genetic distance among photoperiod-converted mutants and their originals ranged from 0.28 to 0.50, revealing significant modification of mutants from their original wild types. Typical Gossypium hirsutum cultivar, Namangan-77, revealed mutational pattern similar to induced radiomutants in 40 mutated SSR loci, implying possible pressure to these SSR loci not only in radiomutagenesis but also during common breeding process. Outcomes of the research should be useful in understanding the photoperiod-related mutations, and markers might help in mapping photoperiodic flowering genes in cotton.

  17. Electrochemical and conversion electron Moessbauer study of corrosion induced by acid rain

    Energy Technology Data Exchange (ETDEWEB)

    Vertes, C.; Lakatos-Varsanyi, M.; Vertes, A. (Dept. of Physical Chemistry and of Nuclear Chemistry, Eoetvoes Univ., Budapest (Hungary)); Meisel, W.; Guetlich, P. (Inst. of Inorganic Chemistry and Analytical Chemistry, Univ. Mainz (Germany))

    1993-04-01

    The passivation of low carbon steel was studied in aqueous solution of 0.5M Na[sub 2]SO[sub 4]+0.001M NaHSO[sub 3] (pH 3.5, 6.5 and 8.5) which can be considered as a model of acid rain. The used conversion electron Moessbauer spectroscopy (CEMS) with the complementary electrochemical investigations proved that the sulfite ions induce pitting corrosion at pH 3.5 and 6.5, while the measurements showed much weaker pitting at pH 8.5. The compositions and thicknesses of the passive films formed during the electrochemical treatments are determined from the CEM spectra. Only [gamma]-FeOOH was found on the surface of the samples at pH 6.5 and 8.5. Nevertheless, at pH 3.5 the sextet belonging to Fe[sub 3]C appears in the spectra, and also FeSO[sub 4].H[sub 2]O could be detected in low concentration. (orig.).

  18. Virus-induced gene silencing in soybean and common bean.

    Science.gov (United States)

    Zhang, Chunquan; Whitham, Steven A; Hill, John H

    2013-01-01

    Plant viral vectors are useful for transient gene expression as well as for downregulation of gene expression via virus-induced gene silencing (VIGS). When used in reverse genetics approaches, VIGS offers a convenient way of transforming genomic information into knowledge of gene function. Efforts to develop and improve plant viral vectors have expanded their applications and have led to substantial advances needed to facilitate gene function studies in major row crops. Here, we describe a DNA-based Bean pod mottle virus (BPMV) vector system for both gene expression and VIGS in soybean and common bean.

  19. No evidence for 'break-induced replication' in a higher plant – but break-induced conversion may occur

    Directory of Open Access Journals (Sweden)

    Ingo eSchubert

    2011-04-01

    Full Text Available ‘Break-induced replication’ (BIR is considered as one way to repair DNA double-strand breaks (DSBs. BIR is defined as replication of the proximal break-ends up to the end of the broken chromosome using an undamaged homologous double-stranded template and mimicking a non-reciprocal translocation. This phenomenon was detected by genetic experiments in yeast. BIR is assumed to occur also in mammals, but experimental evidence is not yet at hand. We have studied chromosomes of the field bean, Vicia faba L., as to the occurrence of BIR after DSB induction during S and G2 phase. Simultaneous incorporation of the base analogue ethynyldeoxyuridine (EdU revealed no chromosomal replication pattern indicative of BIR. Thus, if occurring at all, BIR does not play a major role for DSB repair in higher plants with large chromosome arms. However, the frequency of interstitial asymmetric EdU incorporation within heterochromatic regions, visible on metaphase chromosomes, increased after chromosome breakage during S and G2 phase. Such asymmetric labelling could be interpreted as conservative replication up to the next replicon, circumventing a DSB and yielding an interstitial conversion-like event.

  20. The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

    Directory of Open Access Journals (Sweden)

    Bradley L. Bearson

    2014-02-01

    Full Text Available Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the U.S. during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness

  1. Low-temperature affected LC-PUFA conversion and associated gene transcript level in Nannochloropsis oculata CS-179

    Science.gov (United States)

    Ma, Xiaolei; Zhang, Lin; Zhu, Baohua; Pan, Kehou; Li, Si; Yang, Guanpin

    2011-09-01

    Nannochloropsis oculata CS-179, a marine eukaryotic unicellular microalga, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs). Culture temperature affected cell growth and the composition of LC-PUFAs. At an initial cell density of 1.5 × 106 cell mL-1, the highest growth was observed at 25°C and the cell density reached 3 × 107 cell mL-1 at the beginning of logarithmic phase. The content of LC-PUFAs varied with culture temperature. The highest content of LC-PUFAs (43.96%) and EPA (36.6%) was gained at 20°C. Real-time PCR showed that the abundance of Δ6-desaturase gene transcripts was significantly different among 5 culture temperatures and the highest transcript level (15°C) of Nanoc-D6D took off at cycle 21.45. The gene transcript of C20-elongase gene was higher at lower temperatures (10, 15, and 20°C), and the highest transcript level (20°C) of Nanoc-E took off at cycle 21.18. The highest conversion rate (39.3%) of Δ6-desaturase was also gained at 20°C. But the conversion rate of Nanoc-E was not detected. The higher content of LC-PUFAs was a result of higher gene transcript level and higher enzyme activity. Compared with C20-elongase gene, Δ6-desaturase gene transcript and enzyme activity varied significantly with temperature. It will be useful to study the mechanism of how the content of LC-PUFAs is affected by temperature.

  2. Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

    NARCIS (Netherlands)

    Romero, I.; Tikunov, Y.M.; Bovy, A.G.

    2011-01-01

    Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltrat

  3. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Science.gov (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  4. Evidence for somatic gene conversion and deletion in bipolar disorder, Crohn's disease, coronary artery disease, hypertension, rheumatoid arthritis, type-1 diabetes, and type-2 diabetes.

    Science.gov (United States)

    Ross, Kenneth Andrew

    2011-02-03

    During gene conversion, genetic information is transferred unidirectionally between highly homologous but non-allelic regions of DNA. While germ-line gene conversion has been implicated in the pathogenesis of some diseases, somatic gene conversion has remained technically difficult to investigate on a large scale. A novel analysis technique is proposed for detecting the signature of somatic gene conversion from SNP microarray data. The Wellcome Trust Case Control Consortium has gathered SNP microarray data for two control populations and cohorts for bipolar disorder (BD), cardiovascular disease (CAD), Crohn's disease (CD), hypertension (HT), rheumatoid arthritis (RA), type-1 diabetes (T1D) and type-2 diabetes (T2D). Using the new analysis technique, the seven disease cohorts are analyzed to identify cohort-specific SNPs at which conversion is predicted. The quality of the predictions is assessed by identifying known disease associations for genes in the homologous duplicons, and comparing the frequency of such associations with background rates. Of 28 disease/locus pairs meeting stringent conditions, 22 show various degrees of disease association, compared with only 8 of 70 in a mock study designed to measure the background association rate (P conversion could be a significant causative factor in each of the seven diseases. The specific genes provide potential insights about disease mechanisms, and are strong candidates for further study.

  5. Genetic Polymorphisms of Mc4R and IGF2 Gene Association with Feed Conversion Efficiency Traits in Beef Cattle

    Directory of Open Access Journals (Sweden)

    Xin-hua Du§, Cui Chen§, Zheng-rong Yuan, Li-min Zhang, Xiao-jie Chen, Yan-hui Wang, Xue Gao, Lu-pei Zhang, Hui-jiang Gao, Jun-ya Li and Shang-zhong Xu*

    2013-11-01

    Full Text Available Melanocortin-4 receptor (MC4R gene is part of the central melanocortin pathway located in the hypothalamus, an area of the brain in which appetite is regulated. Insulin-like growth factor 2 (IGF2 gene plays a role in muscle growth, myoblast proliferation and differentiation. Thus, they are candidate genes for feed conversion efficiency (FCE. The study was to investigate the effects of variants in cattle MC4R and IGF2 gene on FCE traits including residual feed intake (RFI, feed conversion ratio (FCR and average daily gain (ADG. We screened single nucleotide polymorphisms (SNPs of the two genes in 118 Simmental bulls by DNA-pool sequencing and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS analysis. C1069G locus of MC4R and four SNPs (C2209T, G18587C, A22950T and G26920T of IGF2 were identified in the population. The χ2 test showed that only MC4R-C1069G, IGF2-C2209T and IGF2-G18587C loci fitted with Hardy-Weinberg equilibrium (P>0.05. General linear model (GLM was used to analyze differences between genotypes. The results showed that only IGF2-G18587C locus has a significant effect on ADG (P0.05. CC and GG genotypes were the dominant genotypes; individual with CC or GG genotype had a larger ADG than GC (P<0.05.

  6. The genes coding for the conversion of carbazole to catechol are flanked by IS6100 elements in Sphingomonas sp. strain XLDN2-5.

    Directory of Open Access Journals (Sweden)

    Zhonghui Gai

    Full Text Available BACKGROUND: Carbazole is a recalcitrant compound with a dioxin-like structure and possesses mutagenic and toxic activities. Bacteria respond to a xenobiotic by recruiting exogenous genes to establish a pathway to degrade the xenobiotic, which is necessary for their adaptation and survival. Usually, this process is mediated by mobile genetic elements such as plasmids, transposons, and insertion sequences. FINDINGS: The genes encoding the enzymes responsible for the degradation of carbazole to catechol via anthranilate were cloned, sequenced, and characterized from a carbazole-degrading Sphingomonas sp. strain XLDN2-5. The car gene cluster (carRAaBaBbCAc and fdr gene were accompanied on both sides by two copies of IS6100 elements, and organized as IS6100::ISSsp1-ORF1-carRAaBaBbCAc-ORF8-IS6100-fdr-IS6100. Carbazole was converted by carbazole 1,9a-dioxygenase (CARDO, CarAaAcFdr, meta-cleavage enzyme (CarBaBb, and hydrolase (CarC to anthranilate and 2-hydroxypenta-2,4-dienoate. The fdr gene encoded a novel ferredoxin reductase whose absence resulted in lower transformation activity of carbazole by CarAa and CarAc. The ant gene cluster (antRAcAdAbAa which was involved in the conversion of anthranilate to catechol was also sandwiched between two IS6100 elements as IS6100-antRAcAdAbAa-IS6100. Anthranilate 1,2-dioxygenase (ANTDO was composed of a reductase (AntAa, a ferredoxin (AntAb, and a two-subunit terminal oxygenase (AntAcAd. Reverse transcription-PCR results suggested that carAaBaBbCAc gene cluster, fdr, and antRAcAdAbAa gene cluster were induced when strain XLDN2-5 was exposed to carbazole. Expression of both CARDO and ANTDO in Escherichia coli required the presence of the natural reductases for full enzymatic activity. CONCLUSIONS/SIGNIFICANCE: We predict that IS6100 might play an important role in the establishment of carbazole-degrading pathway, which endows the host to adapt to novel compounds in the environment. The organization of the car

  7. Role of Ectopic Gene Conversion in the Evolution of a Candida krusei Pleiotropic Drug Resistance Transporter Family

    Science.gov (United States)

    Lamping, Erwin; Zhu, Jing-yi; Niimi, Masakazu; Cannon, Richard David

    2017-01-01

    Gene duplications enable the evolution of novel gene function, but strong positive selection is required to preserve advantageous mutations in a population. This is because frequent ectopic gene conversions (EGCs) between highly similar, tandem-duplicated, sequences, can rapidly remove fate-determining mutations by replacing them with the neighboring parent gene sequences. Unfortunately, the high sequence similarities between tandem-duplicated genes severely hamper empirical studies of this important evolutionary process, because deciphering their correct sequences is challenging. In this study, we employed the eukaryotic model organism Saccharomyces cerevisiae to clone and functionally characterize all 30 alleles of an important pair of tandem-duplicated multidrug efflux pump genes, ABC1 and ABC11, from seven strains of the diploid pathogenic yeast Candida krusei. Discovery and functional characterization of their closest ancestor, C. krusei ABC12, helped elucidate the evolutionary history of the entire gene family. Our data support the proposal that the pleiotropic drug resistance (PDR) transporters Abc1p and Abc11p have evolved by concerted evolution for ∼134 MY. While >90% of their sequences remained identical, very strong purifying selection protected six short DNA patches encoding just 18 core amino acid (aa) differences in particular trans membrane span (TMS) regions causing two distinct efflux pump functions. A proline-kink change at the bottom of Abc11p TMS3 was possibly fate determining. Our data also enabled the first empirical estimates for key parameters of eukaryotic gene evolution, they provided rare examples of intron loss, and PDR transporter phylogeny confirmed that C. krusei belongs to a novel, yet unnamed, third major Saccharomycotina lineage. PMID:28159755

  8. A riboswitch-based inducible gene expression system for mycobacteria.

    Directory of Open Access Journals (Sweden)

    Jessica C Seeliger

    Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

  9. Sex Conversion Induced by Hydrostatic Pressure in the Marine Copepod Tigriopus californicus.

    Science.gov (United States)

    Vacquier, V D; Belser, W L

    1965-12-17

    High hydrostatic pressure applied to the naupliar larval stages of the marine copepod Tigriopus californicus converts some individuals that would have become males into females. The copepodid stages are not sensitive to pressureinduced conversion.

  10. Laser-induced radial birefringence and spin-to-orbital optical angular momentum conversion in silver-doped glasses

    CERN Document Server

    Amjad, Jafar Mostafavi; Slussarenko, Sergei; Karimi, Ebrahim; Marrucci, Lorenzo; Santamato, Enrico; 10.1063/1.3610474

    2012-01-01

    Samples of Ag$^+$/Na$^{+}$ ion-exchanged glass that have been subject to intense laser irradiation may develop novel optical properties, as a consequence of the formation of patterns of silver nanoparticles and other structures. Here, we report the observation of a laser-induced permanent transverse birefringence, with the optical axis forming a radial pattern, as revealed by the spin-to-orbital angular momentum conversion occurring in a probe light beam. The birefringence pattern can be modeled well as resulting from thermally-induced stresses arising in the silver-doped glass during laser exposure, although the actual mechanism leading to the permanent anisotropy is probably more complex.

  11. Laser-induced radial birefringence and spin-to-orbital optical angular momentum conversion in silver-doped glasses

    Science.gov (United States)

    Amjad, Jafar Mostafavi; Khalesifard, Hamid Reza; Slussarenko, Sergei; Karimi, Ebrahim; Marrucci, Lorenzo; Santamato, Enrico

    2011-07-01

    Samples of Ag+/Na+ ion-exchanged glass that have been subject to intense laser irradiation may develop novel optical properties, as a consequence of the formation of patterns of silver nanoparticles and other structures. Here, we report the observation of a laser-induced permanent transverse birefringence, with the optical axis forming a radial pattern, as revealed by the spin-to-orbital angular momentum conversion occurring in a probe light beam. The birefringence pattern can be modeled well as resulting from thermally-induced stresses arising in the silver-doped glass during laser exposure, although the actual mechanism leading to the permanent anisotropy is probably more complex.

  12. The Wnt-target gene Dlk-1 is regulated by the Prmt5-associated factor Copr5 during adipogenic conversion

    Directory of Open Access Journals (Sweden)

    Conception Paul

    2015-02-01

    Full Text Available Protein arginine methyl transferase 5 (Prmt5 regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB and of primary embryo fibroblasts (Mefs was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and β-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.

  13. A heuristic Bayesian method for segmenting DNA sequence alignments and detecting evidence for recombination and gene conversion.

    Science.gov (United States)

    Kedzierska, Anna; Husmeier, Dirk

    2006-01-01

    We propose a heuristic approach to the detection of evidence for recombination and gene conversion in multiple DNA sequence alignments. The proposed method consists of two stages. In the first stage, a sliding window is moved along the DNA sequence alignment, and phylogenetic trees are sampled from the conditional posterior distribution with MCMC. To reduce the noise intrinsic to inference from the limited amount of data available in the typically short sliding window, a clustering algorithm based on the Robinson-Foulds distance is applied to the trees thus sampled, and the posterior distribution over tree clusters is obtained for each window position. While changes in this posterior distribution are indicative of recombination or gene conversion events, it is difficult to decide when such a change is statistically significant. This problem is addressed in the second stage of the proposed algorithm, where the distributions obtained in the first stage are post-processed with a Bayesian hidden Markov model (HMM). The emission states of the HMM are associated with posterior distributions over phylogenetic tree topology clusters. The hidden states of the HMM indicate putative recombinant segments. Inference is done in a Bayesian sense, sampling parameters from the posterior distribution with MCMC. Of particular interest is the determination of the number of hidden states as an indication of the number of putative recombinant regions. To this end, we apply reversible jump MCMC, and sample the number of hidden states from the respective posterior distribution.

  14. Coalescent Times and Patterns of Genetic Diversity in Species with Facultative Sex: Effects of Gene Conversion, Population Structure, and Heterogeneity.

    Science.gov (United States)

    Hartfield, Matthew; Wright, Stephen I; Agrawal, Aneil F

    2016-01-01

    Many diploid organisms undergo facultative sexual reproduction. However, little is currently known concerning the distribution of neutral genetic variation among facultative sexual organisms except in very simple cases. Understanding this distribution is important when making inferences about rates of sexual reproduction, effective population size, and demographic history. Here we extend coalescent theory in diploids with facultative sex to consider gene conversion, selfing, population subdivision, and temporal and spatial heterogeneity in rates of sex. In addition to analytical results for two-sample coalescent times, we outline a coalescent algorithm that accommodates the complexities arising from partial sex; this algorithm can be used to generate multisample coalescent distributions. A key result is that when sex is rare, gene conversion becomes a significant force in reducing diversity within individuals. This can reduce genomic signatures of infrequent sex (i.e., elevated within-individual allelic sequence divergence) or entirely reverse the predicted patterns. These models offer improved methods for assessing null patterns of molecular variation in facultative sexual organisms.

  15. Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

    Science.gov (United States)

    Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

    2011-07-18

    Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed.

  16. Effects of immunological challenge induced by lipopolysaccharide on skeletal muscle fiber type conversion of piglets.

    Science.gov (United States)

    Jia, A F; Feng, J H; Zhang, M H; Chang, Y; Li, Z Y; Hu, C H; Zhen, L; Zhang, S S; Peng, Q Q

    2015-11-01

    The objective of this study was to investigate the effects of immunological challenge on the skeletal muscle fiber type conversion of piglets. Sixteen Large White weaned barrows (28 ± 3 d, 8.22 ± 0.89 kg BW) were allotted by weight and litter to 2 groups: the control group and the lipopolysaccharide (LPS) group. Saline (control) or LPS was injected intravenously via a jugular catheter on d 1, 3, 5, 7, 9, 11, 13, and 15 at an initial dosage of 80 μg/kg BW, which was increased by 30% at each subsequent injection. Blood samples were collected via the jugular catheter 3 h after the LPS challenge on d -1, 1, 5, 9, and 13. Muscle tissue samples were collected from the LM after exsanguination on d 15. The LPS challenge increased the plasma IL-6, tumor necrosis factor-α (TNF-α), cortisol, IL-1β, and haptoglobin concentrations on d 1 and 5 ( 19.47% ( < 0.05) during d 1 to 4, d 5 to 8, and d 9 to 15, respectively. In the LM of LPS-challenged piglets, myosin heavy chain 1 (MyHC1) mRNA and protein expression tended to be reduced ( = 0.08, 0.09), whereas mRNA, mRNA, and MyHC2 protein expression increased ( < 0.05). The LPS challenge reduced succinic dehydrogenase (SDH) activity ( < 0.05) and increased lactate dehydrogenase (LDH) activity ( < 0.05) in the LM of piglets. Compared with those in the control group, transcriptional peroxisome proliferator-activated receptor coactivator-α () mRNA ( < 0.05), calcineurin (CaN) mRNA, and protein expression were reduced ( < 0.05), and PGC-α protein expression tended to be reduced ( = 0.08) in the LM of LPS-challenged piglets. These results show that immunological challenge induced by LPS resulted in a shift from type I to type II fibers in the LM of piglets, which may be mediated by the downregulation of the CaN/PGC-α signaling pathway.

  17. Screening of hypoxia-inducible genes in sporadic ALS.

    LENUS (Irish Health Repository)

    Cronin, Simon

    2008-10-01

    Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

  18. Energy Conversion Application of Chemicurrents Induced in Metal-Semiconductor Nanostructured Devices

    Science.gov (United States)

    Dasari, Suhas Kiran

    Hydrogen is one the most attractive and suitable energy systems for generation of power in the future with high efficiencies and renewable properties. Nanoscale materials, because of their surface and physical properties are the promising candidates for the development of high performance energy conversion devices, essential components to ensure the efficient operation of the infrastructure and to facilitate the wide spread implementation of hydrogen technologies. This work realizes the use of solid state energy conversion concept to develop metal-semiconductor, metal-oxide architecture devices for electrolyte free conversion of chemical energy to electrical energy by hydrogen oxidation process. This investigation addresses the synthesis of these nanostructure devices by selection of suitable system material combinations, electrical and surface morphological characterization leading to the successful implementation in generation of chemicurrents. Also, the hydrogen oxidation process on each nanostructure device is elucidated with the help of corresponding mechanisms and the performance of each system developed was evaluated based on the resulting output efficiency. The two systems (metal-semiconductor and metal-oxide) realized, showed excellent chemical to electrical energy conversion abilities. Compared to metal-semiconductor nanostructure devices, metal-oxide systems exhibited better energy conversion abilities for indefinitely long duration of time at room temperature. The electron yield observed in considered metal-oxide systems can be sufficient for their use in practical applications. A continued realization of these metal-oxide systems with different material combinations would lead to more ecologically friendly and sustainable energy economics.

  19. Targeted delivery of Bcl-2 conversion gene by MPEG-PCL-PEI-FA cationic copolymer to combat therapeutic resistant cancer.

    Science.gov (United States)

    Li, Zibiao; Liu, Xuan; Chen, Xiaohong; Chua, Ming Xuan; Wu, Yun-Long

    2017-07-01

    Deregulation of anti-apoptosis Bcl-2 protein expression was a key feature in human cancers with therapeutic resistance. Nuclear receptor Nur77 could induce the conformation change of Bcl-2 protein and converted it into an apoptosis inducer by "enemy to friend" strategy. However, the safe and effective delivery of this gene to combat therapeutic resistant cancer remained largely unexplored. In this report, we designed an amphiphilic cationic MPEG-PCL-PEI-FA copolymer, comprising biocompatible and hydrophilic methoxy-poly(ethylene glycol) (MPEG), biodegradable and hydrophobic poly(ε-caprolactone) (PCL), cationic poly(ethylene imine) (PEI) segments, and folic acid (FA) as targeting group, as a high efficient Nur77 gene carrier to folate receptor (FR) highly expressed and therapeutic resistant HeLa/Bcl-2 cancer cells. Interestingly, due to the incorporation of PCL and PEG segments, this MPEG-PCL-PEI-FA copolymer showed less toxicity but better gene transfection efficiency than non-viral gene carrier gold standard PEI (25kDa). This might be due to the formation of micelles to stabilize polyplex for enhanced gene transfection ability. More importantly, MPEG-PCL-PEI-FA copolymer exhibited excellent growth inhibition ability on therapeutic resistant HeLa/Bcl-2 cancer cells, which was FR overexpressed HeLa cervical cancer cells with high expression of Bcl-2 protein, thanks to its FA induced targeting ability, high gene transfection efficiency, and low cytotoxicity. This work signifies the first time that cationic amphiphilic MPEG-PCL-PEI-FA copolymers could be utilized for the gene delivery to therapeutic resistant cancer cells with high expression of anti-apoptosis Bcl-2 protein and the positive results are encouraging for the further design of polymeric platforms for combating drug resistant tumors. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Mechanisms of radiation-induced gene responses

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Paunesku, T.

    1996-10-01

    In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts; however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.

  1. Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Harada, F.; Kimura, A.; Iwanaga, T.; Shimozawa, K.; Yata, J.; Sasazuki,T.

    1987-11-01

    Genomic DNAs from twelve Japanese patients with steroid 21-hydroxylase deficiency were analyzed by Southern blot hybridization. A 3.7-kilobase (kb) Taq I and a 1.7-kb Pvu II restriction endonuclease fragment that correspond to a 21-OHase B gene were absent from the DNA of two unrelated patients with the salt-wasting form of the disease. However, a 10.5-kb Bgl II fragment corresponding to the region encompassing the 21-OHase B gene was still present in these two patients. The genes encoding 21-OHase were cloned from one of these two patients, who was homozygous by descent for HLA-A26;B39;C4A3;C4B1;DR4. Restriction endonuclease mapping as well as partial nucleotide sequencing analysis revealed that the 21-OHase B gene of the patient has been converted to the pseudogene, 21-OHase A, as far as the critical 0.5-kb sequence was concerned. Thus, the defect was due to both chromosomes each carrying two copies of 21-OHase A pseudogene and lacking functional 21-OHase B gene.

  2. The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion.

    Science.gov (United States)

    Hernando, Henar; Shannon-Lowe, Claire; Islam, Abul B; Al-Shahrour, Fatima; Rodríguez-Ubreva, Javier; Rodríguez-Cortez, Virginia C; Javierre, Biola M; Mangas, Cristina; Fernández, Agustín F; Parra, Maribel; Delecluse, Henri-Jacques; Esteller, Manel; López-Granados, Eduardo; Fraga, Mario F; López-Bigas, Nuria; Ballestar, Esteban

    2013-01-15

    Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth. We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation. Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.

  3. Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene

    NARCIS (Netherlands)

    Kaay, Jeroen van der; Draijer, Richard; Haastert, Peter J.M. van

    1990-01-01

    Dictyostelium discoideum cells that overexpress a ras gene with a Gly12 → Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher th

  4. Effect of hydrostatic pressure and magnetic field on electromagnetically induced transparency based nonlinear frequency conversion in quantum ring

    Science.gov (United States)

    Gumber, Sukirti; Gambhir, Monica; Jha, Pradip Kumar; Mohan, Man

    2016-10-01

    We study the combined effect of hydrostatic pressure and magnetic field on electromagnetically induced transparency in quantum ring. The high flexibility in size and shape of ring makes it possible to fabricate a nearly perfect two-dimensional quantum structure. We also explore the dependence of frequency conversion, measured in terms of third order nonlinear susceptibility χ(3) , on coupling field, hydrostatic pressure and magnetic field. Although, a dip in χ(3) is observed with the introduction of strong coupling field, it renders the ring structure transparent to generated wave thus effectively enhancing the output of nonlinear frequency conversion process. At a fixed coupling strength, the output can be further enhanced by increasing the magnetic field while it shows an inverse relationship with pressure. These parameters, being externally controlled, provide an easy handle to control the output of quantum ring which can be used as frequency converter in communication networks.

  5. Salmonella induces prominent gene expression in the rat colon

    Directory of Open Access Journals (Sweden)

    Roosing Susanne

    2007-09-01

    Full Text Available Abstract Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point. As fructo-oligosaccharides (FOS affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase, antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2, inflammation (e.g. calprotectin, oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2 and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9. Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap, showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in

  6. Inducible gene expression and environmentally regulated genes in lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1996-01-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transc

  7. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  8. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.

    Science.gov (United States)

    Halliday, Gary M

    2005-04-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.

  9. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Halliday, Gary M. [Dermatology Research Laboratories, Division of Medicine, Melanoma and Skin Cancer Research Institute, Royal Prince Alfred Hospital at the University of Sydney, Sydney, NSW (Australia)]. E-mail: garyh@med.usyd.edu.au

    2005-04-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.

  10. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  11. Transcription dynamics of inducible genes modulated by negative regulations.

    Science.gov (United States)

    Li, Yanyan; Tang, Moxun; Yu, Jianshe

    2015-06-01

    Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.

  12. Roles of factorial noise in inducing bimodal gene expression

    Science.gov (United States)

    Liu, Peijiang; Yuan, Zhanjiang; Huang, Lifang; Zhou, Tianshou

    2015-06-01

    Some gene regulatory systems can exhibit bimodal distributions of mRNA or protein although the deterministic counterparts are monostable. This noise-induced bimodality is an interesting phenomenon and has important biological implications, but it is unclear how different sources of expression noise (each source creates so-called factorial noise that is defined as a component of the total noise) contribute separately to this stochastic bimodality. Here we consider a minimal model of gene regulation, which is monostable in the deterministic case. Although simple, this system contains factorial noise of two main kinds: promoter noise due to switching between gene states and transcriptional (or translational) noise due to synthesis and degradation of mRNA (or protein). To better trace the roles of factorial noise in inducing bimodality, we also analyze two limit models, continuous and adiabatic approximations, apart from the exact model. We show that in the case of slow gene switching, the continuous model where only promoter noise is considered can exhibit bimodality; in the case of fast switching, the adiabatic model where only transcriptional or translational noise is considered can also exhibit bimodality but the exact model cannot; and in other cases, both promoter noise and transcriptional or translational noise can cooperatively induce bimodality. Since slow gene switching and large protein copy numbers are characteristics of eukaryotic cells, whereas fast gene switching and small protein copy numbers are characteristics of prokaryotic cells, we infer that eukaryotic stochastic bimodality is induced mainly by promoter noise, whereas prokaryotic stochastic bimodality is induced primarily by transcriptional or translational noise.

  13. Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes.

    Science.gov (United States)

    Iacovides, Demetris; Rizki, Gizem; Lapathitis, Georgios; Strati, Katerina

    2016-07-29

    The insufficient ability of specialized cells such as neurons, cardiac myocytes, and epidermal cells to regenerate after tissue damage poses a great challenge to treat devastating injuries and ailments. Recent studies demonstrated that a diverse array of cell types can be directly derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or somatic cells by combinations of specific factors. The use of iPSCs and direct somatic cell fate conversion, or transdifferentiation, holds great promise for regenerative medicine as these techniques may circumvent obstacles related to immunological rejection and ethical considerations. However, producing iPSC-derived keratinocytes requires a lengthy two-step process of initially generating iPSCs and subsequently differentiating into skin cells, thereby elevating the risk of cellular damage accumulation and tumor formation. In this study, we describe the reprogramming of mouse embryonic fibroblasts into functional keratinocytes via the transient expression of pluripotency factors coupled with directed differentiation. The isolation of an iPSC intermediate is dispensable when using this method. Cells derived with this approach, termed induced keratinocytes (iKCs), morphologically resemble primary keratinocytes. Furthermore they express keratinocyte-specific markers, downregulate mesenchymal markers as well as the pluripotency factors Oct4, Sox2, and Klf4, and they show important functional characteristics of primary keratinocytes. iKCs can be further differentiated by high calcium administration in vitro and are capable of regenerating a fully stratified epidermis in vivo. Efficient conversion of somatic cells into keratinocytes could have important implications for studying genetic skin diseases and designing regenerative therapies to ameliorate devastating skin conditions.

  14. Interlocus gene conversion explains at least 2.7% of single nucleotide variants in human segmental duplications.

    Science.gov (United States)

    Dumont, Beth L

    2015-06-16

    Interlocus gene conversion (IGC) is a recombination-based mechanism that results in the unidirectional transfer of short stretches of sequence between paralogous loci. Although IGC is a well-established mechanism of human disease, the extent to which this mutagenic process has shaped overall patterns of segregating variation in multi-copy regions of the human genome remains unknown. One expected manifestation of IGC in population genomic data is the presence of one-to-one paralogous SNPs that segregate identical alleles. Here, I use SNP genotype calls from the low-coverage phase 3 release of the 1000 Genomes Project to identify 15,790 parallel, shared SNPs in duplicated regions of the human genome. My approach for identifying these sites accounts for the potential redundancy of short read mapping in multi-copy genomic regions, thereby effectively eliminating false positive SNP calls arising from paralogous sequence variation. I demonstrate that independent mutation events to identical nucleotides at paralogous sites are not a significant source of shared polymorphisms in the human genome, consistent with the interpretation that these sites are the outcome of historical IGC events. These putative signals of IGC are enriched in genomic contexts previously associated with non-allelic homologous recombination, including clear signals in gene families that form tandem intra-chromosomal clusters. Taken together, my analyses implicate IGC, not point mutation, as the mechanism generating at least 2.7% of single nucleotide variants in duplicated regions of the human genome.

  15. 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.

  16. A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

    Indian Academy of Sciences (India)

    Ramkumar Sambasivan; Grace K Pavlath; Jyotsna Dhawan

    2008-03-01

    Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent genetrap lines revealed arrest-dependent induction of gal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines, insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY – a BRCA2-interacting protein, p8/com1– a p300HAT-binding protein and MLL5 – a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

  17. Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

    Science.gov (United States)

    Yoshioka, R; Soga, K; Wakabayashi, K; Takeba, G; Hoson, T

    2003-01-01

    Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

  18. Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster.

    Science.gov (United States)

    Lin, Chun-Chieh; Potter, Christopher J

    2016-08-01

    Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the H: omology A: ssisted C: RISPR K: nock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available.

  19. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Science.gov (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  20. Dynamics of Homology Searching During Gene Conversion in Saccharomyces cerevisiae Revealed by Donor Competition

    Science.gov (United States)

    Coïc, Eric; Martin, Joshua; Ryu, Taehyun; Tay, Sue Yen; Kondev, Jané; Haber, James E.

    2011-01-01

    One of the least understood aspects of homologous recombination is the process by which the ends of a double-strand break (DSB) search the entire genome for homologous templates that can be used to repair the break. We took advantage of the natural competition between the alternative donors HML and HMR employed during HO endonuclease-induced switching of the budding yeast MAT locus. The strong mating-type-dependent bias in the choice of the donors is enforced by the recombination enhancer (RE), which lies 17 kb proximal to HML. We investigated factors that improve the use of the disfavored donor. We show that the normal heterochromatic state of the donors does not impair donor usage, as donor choice is not affected by removing this epigenetic silencing. In contrast, increasing the length of homology shared by the disfavored donor increases its use. This result shows that donor choice is not irrevocable and implies that there are several encounters between the DSB ends and even the favored donor before recombination is accomplished. The increase by adding more homology is not linear; these results can be explained by a thermodynamic model that determines the energy cost of using one donor over the other. An important inference from this analysis is that when HML is favored as the donor, RE causes a reduction in its effective genomic distance from MAT from 200 kb to ∼20 kb, which we hypothesize occurs after the DSB is created, by epigenetic chromatin modifications around MAT. PMID:21954161

  1. Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse

    Directory of Open Access Journals (Sweden)

    Kimura Shioko

    2012-12-01

    Full Text Available Abstract Background The CCAAT/enhancer binding proteins (C/EBPs play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known. Methods A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined. Results A-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse. Conclusions The DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.

  2. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  3. Function of DNA methyltransferase 3a in lead (Pb(2+) )-Induced Cyclooxygenase-2 gene.

    Science.gov (United States)

    Tsai, Yao-Ting; Chang, Che-Mai; Wang, Jaw-Yuan; Hou, Ming-Feng; Wang, Ju-Ming; Shiurba, Robert; Chang, Wen-Chang; Chang, Wei-Chiao

    2015-09-01

    Lead ions (Pb(2+) ) are toxic industrial pollutants associated with chronic inflammatory diseases in humans and animals. Previously, we found that Pb(2+) ions induce COX-2 gene expression via the EGF receptor/nuclear factor-κB signal transduction pathway in epidermoid carcinoma cell line A431. In this study, to see whether Pb(2+) ions affect COX-2 expression by epigenetic mechanisms, we looked at the mRNAs of DNA methyltransferases (DNMTs) using real-time PCR of total RNA from these cells. Cells exposed to Pb(2+) had low levels of DNMT3a mRNA, whereas the levels of DNMT1 and DNMT3b mRNAs remained unchanged. Pretreatment of cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5 μM) followed by Pb(2+) (1 μM) significantly increased levels of COX-2 mRNA compared with cells treated with Pb(2+) alone. Overexpression of tumor suppressor gene Rb correlated with an increase in COX-2 mRNA and a decrease in DNMT3a mRNA. Conversely, overexpression of transcription factor E2F1 correlated with a decrease in COX-2 mRNA and an increase in DMNT3a mRNA. Pretreatment with EGFR inhibitors AG1478 and PD153035 significantly limited Pb(2+) -induced reduction in DNMT3a mRNA. In addition, gene knockdown of DNMT3a with short hairpin RNA correlated with increased COX-2 mRNA induced by Pb(2+) . Our findings suggest Pb(2+) ions induce COX-2 expression indirectly by reducing DNMT3a methylation of the COX-2 promoter via transcription factors Rb and E2F1. © 2014 Wiley Periodicals, Inc.

  4. Non-reciprocal nonlinear optic induced transparency and frequency conversion on a chip

    CERN Document Server

    Guo, Xiang; Jung, Hojoong; Tang, Hong X

    2015-01-01

    Developments in photonic chips have spurred photon based classical and quantum information processing, attributing to the high stability and scalability of integrated photonic devices [1, 2]. Optical nonlinearity [3] is indispensable in these complex photonic circuits, because it allows for classical and quantum light sources, all-optical switch, modulation, and non-reciprocity in ambient environments. It is commonly known that nonlinear interactions are often greatly enhanced in the microcavities [4]. However, the manifestations of coherent photon-photon interaction in a cavity, analogous to the electromagnetically induced transparency [5], have never been reported on an integrated platform. Here, we present an experimental demonstration of the coherent photon-photon interaction induced by second order optical nonlinearity (\\chi^{(2)} ) on an aluminum nitride photonic chip. The non-reciprocal nonlinear optic induced transparency is demonstrated as a result of the coherent interference between photons with di...

  5. Induced focusing and conversion of a Gaussian beam into an elliptic Gaussian beam

    Indian Academy of Sciences (India)

    Manoj Mishra; Swapan Konar

    2005-09-01

    We have presented an investigation of the induced focusing in Kerr media of two laser beams, the pump beam and the probe beam, which could be either Gaussian or elliptic Gaussian or a combination of the two. We have used variational formalism to derive relevant beam-width equations. Among several important findings, the finding that a very week probe beam can be guided and focused when power of both beams are well below their individual threshold for self-focusing, is a noteworthy one. It has been found that induced focusing is not possible for laser beams of any wavelength and beam radius. In case both beams are elliptic Gaussian, we have shown that when power of both beams is above a certain threshold value then the effective radius of both beams collapses and collapse distance depends on power. Moreover, it has been found that induced focusing can be employed to convert a circular Gaussian beam into an elliptic Gaussian beam.

  6. Differential Gene Expression in Chemically Induced Mouse Lung Adenomas

    Directory of Open Access Journals (Sweden)

    Ruisheng Yao

    2003-01-01

    Full Text Available Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60% of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK, α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240 and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of

  7. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

    Science.gov (United States)

    Nagamatsu, Atsushi; Masuta, Chikara; Senda, Mineo; Matsuura, Hideyuki; Kasai, Atsushi; Hong, Jin-Sung; Kitamura, Keisuke; Abe, Jun; Kanazawa, Akira

    2007-11-01

    Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

  8. Induced Pluripotent Stem Cell Technology and Direct Conversion : New Possibilities to Study and Treat Parkinson's Disease

    NARCIS (Netherlands)

    Roessler, Reinhard; Boddeke, Erik; Copray, Sjef

    Recent developments in in vitro disease modeling and regenerative medicine have placed induced pluripotent stem cells (iPSCs) in the center of attention as a unique source to study Parkinson's disease. After only 5 years of intensive research, human iPSCs can be generated without viral integration

  9. The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

    Directory of Open Access Journals (Sweden)

    Andrew W. Taylor

    2011-01-01

    Full Text Available The neuropeptide alpha-melanocyte stimulating hormone (α-MSH has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+  CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

  10. Induced Pluripotent Stem Cell Technology and Direct Conversion : New Possibilities to Study and Treat Parkinson's Disease

    NARCIS (Netherlands)

    Roessler, Reinhard; Boddeke, Erik; Copray, Sjef

    2013-01-01

    Recent developments in in vitro disease modeling and regenerative medicine have placed induced pluripotent stem cells (iPSCs) in the center of attention as a unique source to study Parkinson's disease. After only 5 years of intensive research, human iPSCs can be generated without viral integration a

  11. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    Directory of Open Access Journals (Sweden)

    Tsutomu Motohashi

    2016-03-01

    Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

  12. Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase,respectively, both essential for the conversion of rifamycin SV into B

    Institute of Scientific and Technical Information of China (English)

    Hua Yuan; Wei Zhao; Yi Zhong; Jin Wang; Zhongiun Qin; Xiaoming Ding; Guo-Ping Zhao

    2011-01-01

    Amycolatopsis mediterranei produces an important antibiotic rifamycin,the biosynthesis of which involves many unusual modifications.Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B.In this study,we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion.Expression of merely rif15 and rif16 in a rif cluster null mutant ofA.mediterranei U32 was able to convert rifamycin SV into B.However,this Rifl5- and Rifl6-mediated conversion was only detected in intact cells of A.meidterranei,but not in Streptomyce coelicolor or Mycobacterium smegmatis,suggesting that yet-characterized gene(s) in A.mediterranei other than those encoded by the rif cluster should be involved in this process.

  13. CW laser-induced photothermal conversion and shape transformation of gold nanodogbones in hydrated chitosan films

    Energy Technology Data Exchange (ETDEWEB)

    Ratto, Fulvio, E-mail: f.ratto@ifac.cnr.it; Matteini, Paolo [National Research Council of Italy, Institute of Applied Physics (Italy); Cini, Alberto [University of Florence, Department of Physics and Astronomy (Italy); Centi, Sonia [University of Florence, Department of Clinical Physiopathology (Italy); Rossi, Francesca [National Research Council of Italy, Institute of Applied Physics (Italy); Fusi, Franco [University of Florence, Department of Clinical Physiopathology (Italy); Pini, Roberto [National Research Council of Italy, Institute of Applied Physics (Italy)

    2011-09-15

    We investigate the photothermal conversion and transformation of gold nanoparticles with an initial dogbone shape after dispersion in hydrated chitosan films, which is a representative model of biological tissue, and excitation by a CW diode laser for 1 min. Gold nanodogbones are observed to undergo a distinct modification above a sharp threshold of {approx}11 W cm{sup -2} and 110 Degree-Sign C. Surprisingly, the very same modification is achieved up to at least 36 W cm{sup -2} and 250 Degree-Sign C. We use an analytical model derived from Gans theory to associate the change in color of the films with the change in shape statistics of these gold nanoparticles. This model proves both convenient and dependable. We interpret the photothermal transformation as a rearrangement of particles with a dogbone shape and an aspect ratio of 4.1 into rods with an aspect ratio of 2.5, where material from the end lobes of the dogbones may relocate to the waists of the rods. In turn, additional transitions to stable gold nanospheres may exhibit fairly slower kinetics.

  14. Mobile drama in an instrumented museum: inducing group conversation via coordinated narratives

    Science.gov (United States)

    Callaway, Charles; Stock, Oliviero; Dekoven, Elyon; Noy, Kinneret; Citron, Yael; Dobrin, Yael

    2012-03-01

    Museum visits can be more enjoyable to small groups if they can be both social and educational experiences. One very rewarding aspect of a visit, especially those involving small groups such as families, is the unmediated group discussion that can ensue during a shared cultural experience. We present a situated, mobile museum system that delivers an hour-long drama to museum visitors. It perceives and analyzes group behavior, uses the result to dynamically deliver coordinated dramatic narrative presentations about the nearby museum exhibit, with the expected result of stimulating group discussion. To accomplish this, our drama-based presentations contain small, complementary differences in the content delivered to each participant, leveraging the narrative tension/release cycle of drama to naturally lead visitors to fill in missing pieces by interacting with friends, thus initiating a conversation. We present two evaluations for these story variations, one in a closed, non-mobile environment, and the other a formative evaluation to gauge how well the methodology used in the non-mobile evaluation performs in evaluating the fully implemented system in a real museum environment.

  15. Bisphenol A 3,4-quinone induces the conversion of xanthine dehydrogenase into oxidase in vitro.

    Science.gov (United States)

    Sakuma, Satoru; Nakanishi, Masahiko; Morinaga, Kazuhiro; Fujitake, Mihoyo; Wada, Shun-ichi; Fujimoto, Yohko

    2010-01-01

    In the present study, we assessed the influence of bisphenol A (BPA) and bisphenol A 3,4-quinone (BPAQ) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in the rat liver in vitro. BPA up to 100 micromol/L did not affect the XO and XD activities in the partially purified cytosolic fraction from rat liver, whereas BPAQ (2-10 micromol/L) dose-dependently enhanced the XO activity concomitant with a decrease in the XD activity, implying that BPAQ, but not BPA, can convert XD into the reactive oxygen species (ROS) producing the form XO. Furthermore, it was found that BPAQ could increase the generation of ROS and oxidize the guanine moiety of deoxyguanosine in the DNA of primary rat hepatocyte cultures. These results suggest that BPAQ has the potential to convert XD into XO in the liver, which in turn may lead to ROS generation and oxidative DNA damage in this region. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  16. Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley Stripe Mosaic Virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  17. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  18. The dilp2/5 genes control diapause inducibility

    OpenAIRE

    Schiesari, Luca

    2015-01-01

    Many holometabolous insects hibernate by triggering diapause, an “actively-induced” dormancy that blocks developmental functions. Yet, the nature of signals enhancing the plasticity of developmental system and underlying diapause inducibility is still elusive. We show that the “Insulin/IGF” dilp2/5 genes, encoding for developmental hormones, antagonize diapause switch in D. melanogaster and their modulation is pivotal in sensitizing the developmental system to environmental perturbations. Fun...

  19. Screening Helicobacter pylori genes induced during infection of mouse stomachs

    Institute of Scientific and Technical Information of China (English)

    Aparna Singh; Nathaniel Hodgson; Ming Yan; Jungsoo Joo; Lei Gu; Hong Sang; Emmalena Gregory-Bryson

    2012-01-01

    AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology (IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced (ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only

  20. Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

    Directory of Open Access Journals (Sweden)

    Sally K. Mak

    2012-01-01

    Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC lines from patients with Parkinson’s disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

  1. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).

    Science.gov (United States)

    Zhang, Can-kui; Lang, Ping; Dane, Fenny; Ebel, Robert C; Singh, Narendra K; Locy, Robert D; Dozier, William A

    2005-03-01

    Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

  2. Development and modelisation of a hydro-power conversion system based on vortex induced vibration

    Science.gov (United States)

    Lefebure, David; Dellinger, Nicolas; François, Pierre; Mosé, Robert

    2016-11-01

    The Vortex Induced Vibration (VIV) phenomenon leads to mechanical issues concerning bluff bodies immerged in fluid flows and have therefore been studied by numerous authors. Moreover, an increasing demand for energy implies the development of alternative, complementary and renewable energy solutions. The main idea of EauVIV project consists in the use of VIV rather than its deletion. When rounded objects are immerged in a fluid flow, vortices are formed and shed on their downstream side, creating a pressure imbalance resulting in an oscillatory lift. A convertor modulus consists of an elastically mounted, rigid cylinder on end-springs, undergoing flow- induced motion when exposed to transverse fluid-flow. These vortices induce cyclic lift forces in opposite directions on the circular bar and cause the cylinder to vibrate up and down. An experimental prototype was developed and tested in a free-surface water channel and is already able to recover energy from free-stream velocity between 0.5 and 1 m.s -1. However, the large number of parameters (stiffness, damping coefficient, velocity of fluid flow, etc.) associated with its performances requires optimization and we choose to develop a complete tridimensionnal numerical model solution. A 3D numerical model has been developed in order to represent the real system behavior and improve it through, for example, the addition of parallel cylinders. The numerical model build up was carried out in three phases. The first phase consists in establishing a 2D model to choose the turbulence model and quantify the dependence of the oscillations amplitudes on the mesh size. The second corresponds to a 3D simulation with cylinder at rest in first time and with vertical oscillation in a second time. The third and final phase consists in a comparison between the experimental system dynamic behavior and its numerical model.

  3. Virus-induced Gene Silencing in Eggplant (Solanum melongena)

    Institute of Scientific and Technical Information of China (English)

    HaipingLiu; Daqi Fu; Benzhong Zhu; Huaxue Yan; Xiaoying Shen; Jinhua Zuo; Yi Zhu; Yunbo Luo

    2012-01-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions.The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays.Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses.In this paper,TRV-mediated VIGS was successfully elicited in eggplant.We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene.Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation,indicating that VIGS can be used to silence genes in eggplant.To further illustrate the reliability of VIGS in eggplant,we selected Chl H,Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method.Suppression of Chl H and Su led to yellow leaves,while the depletion of CLA1 resulted in albino.In conclusion,four genes,PDS,Chl H,Su (Sulfur),CLA1,were down-regulated significantly by VIGS,indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

  4. Octylphenol induced gene expression in testes of Frog, Rana chensinensis.

    Science.gov (United States)

    Li, Xinyi; Liu, Jia; Zhang, Yuhui

    2016-06-01

    Octylphenol (OP) is an endocrine-disrupting chemical (EDC), which can disrupt the reproductive system. To understand the effect of OP, a subtractive cDNA library was constructed using suppression subtractive hybridization (SSH) to identify alterations of gene transcription in the testes of the frog Rana chensinensis after OP exposure. Two hundred positive clones were selected and 134 sequences of gene fragments were produced from the subtractive library randomly. These genes were identified to be involved in metabolic process, cellular process, biological regulation, stimulus, immune system and female pregnancy process. In order to verify the efficiency of the subtractive cDNA library, PSG9 and PAPP-A were analyzed further as two representatives of differentially expressed transcription genes using semi-quantitative RT-PCR. Our result was the first successful construction of the subtractive cDNA library in frog testes after OP treatment. Based on this cDNA library, OP was shown to affect multiple physiological processes including inducing immune response, disrupting the steroid hormone synthesis and influencing spermatogenesis in the testis by up-regulation of specific genes.

  5. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    Directory of Open Access Journals (Sweden)

    Lan-Huan Meng

    Full Text Available Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS and Chlorophyll H subunit (ChlH of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  6. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    Science.gov (United States)

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  7. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    Science.gov (United States)

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  8. Bitumen fume-induced gene expression profile in rat lung.

    Science.gov (United States)

    Gate, Laurent; Langlais, Cristina; Micillino, Jean-Claude; Nunge, Hervé; Bottin, Marie-Claire; Wrobel, Richard; Binet, Stéphane

    2006-08-15

    Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 degrees C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure.

  9. Recombination Rate Variation Modulates Gene Sequence Evolution Mainly via GC-Biased Gene Conversion, Not Hill-Robertson Interference, in an Avian System.

    Science.gov (United States)

    Bolívar, Paulina; Mugal, Carina F; Nater, Alexander; Ellegren, Hans

    2016-01-01

    The ratio of nonsynonymous to synonymous substitution rates (ω) is often used to measure the strength of natural selection. However, ω may be influenced by linkage among different targets of selection, that is, Hill-Robertson interference (HRI), which reduces the efficacy of selection. Recombination modulates the extent of HRI but may also affect ω by means of GC-biased gene conversion (gBGC), a process leading to a preferential fixation of G:C ("strong," S) over A:T ("weak," W) alleles. As HRI and gBGC can have opposing effects on ω, it is essential to understand their relative impact to make proper inferences of ω. We used a model that separately estimated S-to-S, S-to-W, W-to-S, and W-to-W substitution rates in 8,423 avian genes in the Ficedula flycatcher lineage. We found that the W-to-S substitution rate was positively, and the S-to-W rate negatively, correlated with recombination rate, in accordance with gBGC but not predicted by HRI. The W-to-S rate further showed the strongest impact on both dN and dS. However, since the effects were stronger at 4-fold than at 0-fold degenerated sites, likely because the GC content of these sites is farther away from its equilibrium, ω slightly decreases with increasing recombination rate, which could falsely be interpreted as a consequence of HRI. We corroborated this hypothesis analytically and demonstrate that under particular conditions, ω can decrease with increasing recombination rate. Analyses of the site-frequency spectrum showed that W-to-S mutations were skewed toward high, and S-to-W mutations toward low, frequencies, consistent with a prevalent gBGC-driven fixation bias.

  10. The relationship of host-mediated induced resistance to polymorphism in gene-for-gene relationships.

    Science.gov (United States)

    Tellier, Aurélien; Brown, James K M

    2008-01-01

    Gene-for-gene relationships are a common feature of plant-parasite interactions. Polymorphism at host resistance and parasite avirulence loci is maintained if there is negative, direct frequency-dependent selection on alleles of either gene. More specifically, selection of this kind is generated when the disease is polycyclic with frequent auto-infection. When an incompatible interaction occurs between a resistant host and an avirulent parasite, systemic defenses are triggered, rendering the plant more resistant to a later attack by another parasite. However, induced resistance (IR) incurs a fitness cost to the plant. Here, the effect of IR on polymorphism in gene-for-gene interactions is investigated. First, in an infinite population model in which parasites have two generations per host generation, increasing the fitness cost of IR increases selection for susceptible plants at low disease severity, while increasing the effectiveness of IR against further parasite attacks enhances selection for resistant plants at high disease severity. This reduces the possibility of polymorphism being maintained in host and parasite populations. In finite population models, the number of plants varies over time as a function of the disease burden of the population. Polymorphism in gene-for-gene relationships is then more stable at high disease prevalence and severity if IR reactions are more costly when there is competition for resources between plants.

  11. Inducible gene manipulations in brain serotonergic neurons of transgenic rats.

    Directory of Open Access Journals (Sweden)

    Tillmann Weber

    Full Text Available The serotonergic (5-HT system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP, in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.

  12. Putting the diet back into diet-induced obesity: diet-induced hypothalamic gene expression.

    Science.gov (United States)

    Mercer, Julian G; Archer, Zoë A

    2008-05-06

    A wealth of detailed mechanistic information relating to obesity and body weight regulation has emerged from study of single gene mutation models, and continues to be generated by engineered rodent models targeting specific genes. However, as an early step in translational research, many researchers are turning to models of diet-induced obesity. Interpretation of data generated from such models is not aided by the variety of diets and rodent strains employed in these studies and a strong case could be made for rationalisation. Differences in experimental protocol, which may deploy a single obligatory solid diet, a choice of solid diets, or liquid/solid combinations, and which may or may not allow a preferred macronutrient composition to be selected, mean that different models of diet-induced obesity achieve that obesity by different routes. The priority should be to mimic the palatability- and choice-driven over-consumption that probably underlies the majority of human obesity. Some of the hypothalamic energy balance genes apparently 'recognise' developing diet-induced obesity as indicated by counter-regulatory changes in expression levels. However, substantial changes in gene expression on long-term exposure to obesogenic diets are not able to prevent weight gain. Forebrain reward systems are widely assumed to be overriding hypothalamic homeostatic energy balance systems under these circumstances. More mechanism-based research at the homeostatic/reward/diet interface may enable diets to be manipulated with therapeutic benefit, or define the contribution of these interactions to susceptibility to diet-induced obesity.

  13. Hydrothermal conversion of graphite to carbon nanotubes (CNTs) induced by bubble collapse

    Science.gov (United States)

    Zhang, Yong; Liu, Fang

    2016-11-01

    Cu-Fe-CNTs and Ni-Fe-CNTs coatings were deposited on gray cast iron by a hydrothermal approach. It was demonstrated that, the flaky graphite of gray cast iron was exfoliated to graphene nanosheets under hydrothermal reactions, and graphene nanosheets were scrolled to CNTs. After high temperature treatments, the volume losses of Cu-Fe-CNTs and Ni-Fe-CNTs coatings were 52.6 % and 40.0 % of gray cast iron substrate at 60 min wear tests, respectively, obviously increasing the wear properties of gray cast iron. During hydrothermal reactions, water jets and shock waves were produced by bubble collapse. Induced by the water jets and shock waves, exfoliation of flaky graphite was performed, producing exfoliated graphene nanosheets. Attacked by the radially distributed water jets and shock waves, graphene nanosheets were curved, shaped to semicircle morphology and eventually scrolled to tubular CNTs.

  14. A model-based analysis of GC-biased gene conversion in the human and chimpanzee genomes.

    Directory of Open Access Journals (Sweden)

    John A Capra

    Full Text Available GC-biased gene conversion (gBGC is a recombination-associated process that favors the fixation of G/C alleles over A/T alleles. In mammals, gBGC is hypothesized to contribute to variation in GC content, rapidly evolving sequences, and the fixation of deleterious mutations, but its prevalence and general functional consequences remain poorly understood. gBGC is difficult to incorporate into models of molecular evolution and so far has primarily been studied using summary statistics from genomic comparisons. Here, we introduce a new probabilistic model that captures the joint effects of natural selection and gBGC on nucleotide substitution patterns, while allowing for correlations along the genome in these effects. We implemented our model in a computer program, called phastBias, that can accurately detect gBGC tracts about 1 kilobase or longer in simulated sequence alignments. When applied to real primate genome sequences, phastBias predicts gBGC tracts that cover roughly 0.3% of the human and chimpanzee genomes and account for 1.2% of human-chimpanzee nucleotide differences. These tracts fall in clusters, particularly in subtelomeric regions; they are enriched for recombination hotspots and fast-evolving sequences; and they display an ongoing fixation preference for G and C alleles. They are also significantly enriched for disease-associated polymorphisms, suggesting that they contribute to the fixation of deleterious alleles. The gBGC tracts provide a unique window into historical recombination processes along the human and chimpanzee lineages. They supply additional evidence of long-term conservation of megabase-scale recombination rates accompanied by rapid turnover of hotspots. Together, these findings shed new light on the evolutionary, functional, and disease implications of gBGC. The phastBias program and our predicted tracts are freely available.

  15. New CYP2A6 gene deletion and conversion variants in a population of Black African descent.

    Science.gov (United States)

    Mwenifumbo, Jill C; Zhou, Qian; Benowitz, Neal L; Sellers, Edward M; Tyndale, Rachel F

    2010-02-01

    Cytochrome P450 2A6 (CYP2A6) is a human enzyme best known for metabolizing nicotine and nitrosamine precarcinogens. Our aim was to discover and characterize new CYP2A6 alleles in a population of Black African descent. We used cloning, sequencing and genotyping of genomic DNA to discover new variants, and in vivo nicotine pharmacokinetic phenotyping to characterize the functional effect of the new alleles. Four new CYP2A6 alleles, CYP2A6*4G, *4H, *1B4 and *1L, were discovered and characterized in a population of Black African descent. The two new deletion alleles, CYP2A6*4G and *4H, are distinguished by different crossover junctions at 7.9 and 7.8 kb downstream of the CYP2A6 +1ATG start site, respectively; their combined allele frequency is 1.6%. The new gene conversion alleles, CYP2A6*1B4 and CYP2A6*1L, contain 27 and 10 bp of CYP2A7 sequence in the CYP2A6 3 -flanking region, respectively; their combined allele frequency is 7.3%. CYP2A6*4 appears to associate with lower CYP2A6 activity in vivo, while CYP2A6*1L does not; however, CYP2A6*1L confounds genotyping assays that use the 2A6R3 and 2A6R4 primers. As new variants are discovered, the relationships between CYP2A6 genotype, nicotine metabolism, smoking behaviors and tobacco-related cancer risk will be further clarified.

  16. Correlation of photothermal conversion on the photo-induced deformation of amorphous carbon nitride films prepared by reactive sputtering

    Energy Technology Data Exchange (ETDEWEB)

    Harata, T.; Aono, M., E-mail: aono@nda.ac.jp; Kitazawa, N.; Watanabe, Y. [Department of Materials Science and Engineering, National Defense Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa 239-8686 (Japan)

    2014-08-04

    The photo-induced deformation of hydrogen-free amorphous carbon nitride (a-CN{sub x}) films was investigated under visible-light illumination. The films gave rise to photothermal conversion by irradiation. In this study, we investigated the effects of thermal energy generated by irradiation on the deformation of a-CN{sub x}/ultrathin substrate bimorph specimens. The films were prepared on both ultrathin Si and SiO{sub 2} substrates by reactive radio-frequency magnetron sputtering from a graphite target in the presence of pure nitrogen gas. The temperature of the film on the SiO{sub 2} substrate increased as the optical band-gap of the a-CN{sub x} was decreased. For the film on Si, the temperature remained constant. The deformation degree of the films on Si and SiO{sub 2} substrates were approximately the same. Thus, the deformation of a-CN{sub x} films primarily induced by photon energy directly.

  17. Role of 5-HT2C receptor gene variants in antipsychotic-induced weight gain

    Directory of Open Access Journals (Sweden)

    Brandl EJ

    2011-08-01

    Full Text Available Tessa JM Wallace, Clement C Zai, Eva J Brandl, Daniel J MüllerNeurogenetics Section, Center for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Toronto, ON, CanadaAbstract: Antipsychotic-induced weight gain is a serious side effect of antipsychotic medication that can lead to increased morbidity, mortality, and non-compliance in patients. Numerous single nucleotide polymorphisms have been studied for association with antipsychotic-induced weight gain in an attempt to find genetic predictors of this side effect. An ability to predict this side effect could lead to personalized treatment plans for predisposed individuals, which could significantly decrease the prevalence and severity of weight gain. Variations in the serotonin receptor 2c gene (HTR2C have emerged as promising candidates for prediction of antipsychotic-induced weight gain. Specifically, the well-studied -759C/T promoter polymorphism has been associated with weight gain in diverse populations, although some studies have reported no association. This discrepancy is likely due to heterogeneity in study design with respect to ethnicity, treatment duration, and other variables. Notably, the association between HTR2C and antipsychotic-induced weight gain appears strongest in short-term studies on patients with limited or no previous antipsychotic treatment. Other, less extensively studied promoter polymorphisms (-697C/G, -997G/A, and -1165A/G have also emerged as potential predictors of antipsychotic-induced weight gain. Conversely, the well-studied intronic polymorphism Cys23Ser does not appear to be associated. With further research on both HTR2C and other genetic and environmental predictors of antipsychotic-induced weight gain, a predictive test could one day be created to screen patients and provide preventative or alternative treatment for those who are predisposed to this serious side effect.Keywords: HTR2C, pharmacogenomics, promoter polymorphism

  18. Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

    Directory of Open Access Journals (Sweden)

    Verónica S Di Stilio

    Full Text Available Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS of a PHYTOENE DESATURASE ortholog (TdPDS can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05, as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.

  19. Light-Induced Conversion of Chemical Permeability to Enhance Electron and Molecular Transfer in Nanoscale Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Balgley, Renata; de Ruiter, Graham; Evmenenko, Guennadi; Bendikov, Tatyana; Lahav, Michal; van der Boom, Milko E.

    2016-12-21

    In this paper, we demonstrate how photochemically enhancing the permeability of metal–organic assemblies results in a significant enhancement of the electrochemical activity of metal complexes located within the assembly. The molecular assemblies consist of different layers of redox-active metal complexes ([M(mbpy-py)3][PF6]2; M = Ru or Os) that are separated by redox-inactive spacers consisting of 1,4-bis[2-(4-pyridyl)ethenyl]benzene (BPEB) and PdCl2 of variable thicknesses (0–13.4 nm). UV-irradiation (λ = 254 nm) of our assemblies induces a photochemical reaction in the redox-inactive spacer increasing the permeability of the assembly. The observed increase was evident by trapping organic (nBu4NBF4) and inorganic (NiCl2) salts inside the assemblies, and by evaluating the electrochemical response of quinones absorbed inside the molecular assemblies before and after UV irradiation. The increase in permeability is reflected by higher currents and a change in the directionality of electron transfer, i.e., from mono- to bidirectional, between the redox-active metal complexes and the electrode surface. The supramolecular structure of the assemblies dominates the overall electron transfer properties and overrules possible electron transfer mediated by the extensive π-conjugation of its individual organic components.

  20. MicroRNA-138 enhances TRAIL-induced apoptosis through interferon-stimulated gene 15 downregulation in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zuo, Chaohui; Sheng, Xinyi; Liu, Zhuo; Ma, Min; Xiong, Shuhan; Deng, Hongyu; Li, Sha; Yang, Darong; Wang, Xiaohong; Xiao, Hua; Quan, Hu; Xia, Man

    2017-06-01

    Hepatocellular carcinoma is a leading cause of cancer-related mortality worldwide. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a potential target for cancer therapy. However, many cancer cells are resistant to TRAIL-induced apoptosis and its mechanism is not well understood. In this study, to identify potential therapeutic targets for TRAIL-resistant cancer cells, we compared the expression levels of interferon-stimulated gene 15 in TRAIL-sensitive and TRAIL-resistant hepatocellular carcinoma cell lines. Western blot analysis showed that interferon-stimulated gene 15 expression levels were significantly higher in resistant HLCZ01and Huh7 cells than in sensitive LH86 and SMMC-7721 cells. Interferon-stimulated gene 15 knockdown in resistance cells led to TRAIL sensitivity. Conversely, interferon-stimulated gene 15 overexpression in sensitive cells resulted in TRAIL resistance. Our bioinformatics search detected a putative target sequence for microRNA miR-138 in the 3' untranslated region of the interferon-stimulated gene 15. Real-time quantitative polymerase chain reaction analysis demonstrated that miR-138 was significantly downregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. Forced expression of miR-138 in resistant cells decreased both messenger RNA and protein levels of interferon-stimulated gene 15, and when exposed to TRAIL, activated poly(adenosine diphosphate-ribose) polymerase, indicating sensitization to TRAIL. The results suggested that miR-138 regulates the interferon-stimulated gene 15 expression by directly targeting the 3' untranslated region of interferon-stimulated gene 15 and modulates the sensitivity to TRAIL-induced apoptosis. MiR-138 may be a target for therapeutic intervention in TRAIL-based drug treatments of resistant hepatocellular carcinoma or could be a biomarker to select patients who may benefit from the treatment.

  1. Reactive ion etching (RIE) induced p- to n-type conversion in extrinsically doped p-type HgCdTe

    Energy Technology Data Exchange (ETDEWEB)

    Musca, C.A.; Smith, E.P.G.; Siliquini, J.F.; Dell, J.M.; Antoszewski, J.; Faraone, L. [Univ. of Western Australia, Nedlands, Western Australia (Australia). Dept. of Electrical and Electronic Engineering; Piotrowski, J. [Vigo System Ltd., Warsaw (Poland)

    1998-12-31

    Mercury annealing of reactive ion etching (RIE) induced p- to n-type conversion in extrinsically doped p-type epitaxial layers of HgCdTe (x = 0.31) has been used to reconvert n-type conversion sustained during RIE processing. For the RIE processing conditions used (400 mT, CH{sub 4}/H{sub 2}, 90 W) p- to n-type conversion was observed using laser beam induced current (LBIC) measurements. After a sealed tube mercury anneal at 200 C for 17 hours, LBIC measurements clearly indicated no n-type converted region remained. Subsequent Hall measurements confirmed that the material consisted of a p-type layer, with electrical properties equivalent to that of the initial as-grown wafer (N{sub A}-N{sub D} = 2 {times} 10{sup 16} cm{sup {minus}3}, {mu} = 350 cm{sup 2}.V{sup {minus}1}.s{sup {minus}1}).

  2. Tomato leaf spatial expression of stress-induced Asr genes.

    Science.gov (United States)

    Maskin, Laura; Maldonado, Sara; Iusem, Norberto D

    2008-12-01

    Asr1 and Asr2 are water stress-inducible genes belonging to the Asr gene family, which transcriptionally regulate a sugar transporter gene, at least in grape. Using an in situ RNA hybridization methodology, we determined that, in basal conditions, expression of Asr2 in tomato leaves is detected in the phloem tissue, particularly in companion phloem cells. When plants are exposed to water stress, Asr2 expression is contained in companion cells but expands occasionally to mesophyll cells. In contrast, Asr1 transcript localization seems to be sparse in leaf vascular tissue under both non-stress and stress conditions. The occurrence of Asr transcripts precisely in companion cells is in accordance with the cell type specificity reported for hexose-transporter protein molecules in grape encoded by the only Asr-target gene known to date. The results are discussed in light of the reported scarcity of plasmodesmata between companion cells and the rest of leaf tissue in the family Solanaceae.

  3. Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

    Science.gov (United States)

    The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

  4. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  5. Conversational Dominance.

    Science.gov (United States)

    Esau, Helmut; Poth, Annette

    Details of conversational behavior can often not be interpreted until the social interaction, including the rights and obligations of the participants, their intent, the topic, etc., has been defined. This paper presents a model of conversation in which the conversational image a person presents in a given conversational situation is a function of…

  6. Inducible gene expression and environmentally regulated genes in lactic acid bacteria.

    Science.gov (United States)

    Kok, J

    1996-10-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed.

  7. Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion.

    Science.gov (United States)

    Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP(Sc)). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP(Sc) results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP(Sc). However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP(Sc) can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.

  8. Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen.

    Science.gov (United States)

    Hamerski, D; Matern, U

    1988-01-15

    Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.

  9. Pristane-induced granulocyte recruitment promotes phenotypic conversion of macrophages and protects against diffuse pulmonary hemorrhage in Mac-1 deficiency.

    Science.gov (United States)

    Shi, Yiqin; Tsuboi, Naotake; Furuhashi, Kazuhiro; Du, Qiuna; Horinouchi, Asuka; Maeda, Kayaho; Kosugi, Tomoki; Matsuo, Seiichi; Maruyama, Shoichi

    2014-11-15

    Diffuse pulmonary hemorrhage (DPH) is an uncommon but critical complication of systemic lupus erythematosus. Peritoneal administration of 2,6,10,14-tetramethylpentadecane (pristane) can recapitulate a lupus-like syndrome in mice, which can develop into DPH within a few weeks, especially in C57BL/6 mice. Mac-1 (CD11b/CD18), a leukocyte adhesion molecule, is known to play a role in inflammation by regulating migration of leukocytes into injured tissue. In this study, we aimed to clarify the role of Mac-1 in pristane-induced DPH, using Mac-1(-/-) and wild-type (WT) mice on a C57BL/6 background. After pristane injection, Mac-1(-/-) mice showed reduced prevalence of DPH and attenuated peritonitis compared with WT mice. Analysis of the peritoneal lavage on days 5 and 10 after pristane treatment revealed increased numbers of eosinophils and alternatively activated macrophages, but decreased numbers of neutrophils and classically activated macrophages in Mac-1(-/-) mice compared with WT. Enhanced production of IL-4 and IL-13, both key mediators of macrophage polarization toward the mannose receptor(+) (MMR(+)) phenotype, was observed in the peritoneal cavity of Mac-1(-/-) mice. Depletion of neutrophils and eosinophils or adoptive transfer of classically activated macrophages resulted in the exacerbation of pristane-mediated DPH in both WT and Mac-1(-/-) mice. Moreover, peritoneal transfer of F4/80(high)MMR(+) alternatively activated macrophages successfully reduced the prevalence of DPH in WT mice. Collectively, Mac-1 promoted acute inflammatory responses in the peritoneal cavity and the lungs by downregulating granulocyte migration and subsequent phenotypic conversion of macrophages in a pristane-induced systemic lupus erythematosus model. Copyright © 2014 by The American Association of Immunologists, Inc.

  10. Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing.

    Science.gov (United States)

    Romero, Irene; Tikunov, Yury; Bovy, Arnaud

    2011-07-01

    Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs.

  11. Anti-vascular agent Combretastatin A-4-P modulates Hypoxia Inducible Factor-1 and gene expression

    Directory of Open Access Journals (Sweden)

    Currie Margaret J

    2006-12-01

    Full Text Available Abstract Background A functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia. Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1, controlling angiogenic and metastatic pathways. Methods We investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro. Results CA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor κB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A, was increased. Conclusion Our findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.

  12. Virus-induced silencing of a tobacco deoxyhypusine synthase gene

    Institute of Scientific and Technical Information of China (English)

    WANG Hongzhi; MA Rongcai; LI Ruifen; WANG Guoying; WEI Jianhua

    2005-01-01

    A cDNA fragment corresponding to deoxyhypusine synthase gene NbDHS was isolated and cloned into potato virus X (PVX) vector for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing (VIGS). Plants agroinfected with recombinant virus vector PVX-NbDHS exhibited an increase in leaf biomass, delay in natural leaf senescence and flowering time, and decrease in leaf chlorophyll content. Semi-quantitative RT-PCR and Northern analysis showed that the transcript level of DHS was significantly lower in PVX-NbDHS infected plants. At the same time, the expression for eIF-5A, the target proteins of DHS in N. benthamiana, was concomitantly suppressed by semi-quantitative RT-PCR and Western analysis. From the phenotypic feature of the infected plants and the reduced expression abundance of DHS and eIF-5A, we concluded that NbDHS plays important roles in plant growth, development and senescence. The possible application of DHS gene in genetic modification of crops and horticultural plants was discussed.

  13. Lymphocyte Activation Gene-3 (LAG-3 negatively regulates environmentally-induced autoimmunity.

    Directory of Open Access Journals (Sweden)

    Vibha Jha

    Full Text Available Environmental factors including drugs, mineral oils and heavy metals such as lead, gold and mercury are triggers of autoimmune diseases in animal models or even in occupationally exposed humans. After exposure to subtoxic levels of mercury (Hg, genetically susceptible strains of mice develop an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hyperglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of Hg. Lymphocyte Activation Gene-3 (LAG-3 is a CD4-related, MHC-class II binding molecule expressed on activated T cells and NK cells which maintains lymphocyte homeostatic balance via various inhibitory mechanisms. In our model, administration of anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in serum IgE level. In addition, LAG-3-deficient B6.SJL mice not only had increased susceptibility to Hg-induced autoimmunity but were also unresponsive to tolerance induction. Conversely, adoptive transfer of wild-type CD4(+ T cells was able to partially rescue LAG-3-deficient mice from the autoimmune disease. Further, in LAG-3-deficient mice, mercury elicited higher amounts of IL-6, IL-4 and IFN-γ, cytokines known to play a critical role in mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant.

  14. The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis*

    Science.gov (United States)

    DiStefano, Marina T.; Danai, Laura V.; Roth Flach, Rachel J.; Chawla, Anil; Pedersen, David J.; Guilherme, Adilson; Czech, Michael P.

    2015-01-01

    The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation. PMID:25922078

  15. Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ziqing Liu

    2016-03-01

    Full Text Available Direct conversion of fibroblasts into induced cardiomyocytes (iCMs offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome

  16. L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes

    Science.gov (United States)

    Lobel, Lior; Burg-Golani, Tamar; Sigal, Nadejda; Rose, Jessica; Livnat-Levanon, Nurit; Lewinson, Oded; Herskovits, Anat A.

    2017-01-01

    The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism. PMID:28114430

  17. Electrocatalytic Production of C3-C4 Compounds by Conversion of CO2 on a Chloride-Induced Bi-Phasic Cu2O-Cu Catalyst.

    Science.gov (United States)

    Lee, Seunghwa; Kim, Dahee; Lee, Jaeyoung

    2015-12-01

    Electrocatalytic conversion of carbon dioxide (CO2) has recently received considerable attention as one of the most feasible CO2 utilization techniques. In particular, copper and copper-derived catalysts have exhibited the ability to produce a number of organic molecules from CO2. Herein, we report a chloride (Cl)-induced bi-phasic cuprous oxide (Cu2O) and metallic copper (Cu) electrode (Cu2OCl) as an efficient catalyst for the formation of high-carbon organic molecules by CO2 conversion, and identify the origin of electroselectivity toward the formation of high-carbon organic compounds. The Cu2OCl electrocatalyst results in the preferential formation of multi-carbon fuels, including n-propanol and n-butane C3-C4 compounds. We propose that the remarkable electrocatalytic conversion behavior is due to the favorable affinity between the reaction intermediates and the catalytic surface.

  18. High-performance alternating current field-induced chromatic-stable white polymer electroluminescent devices employing a down-conversion layer

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Yingdong; Chen, Yonghua; Smith, Gregory M. [Center for Nanotechnology and Molecular Materials, Department of Physics, Wake Forest University, Winston-Salem, NC 27105 (United States); Sun, Hengda; Yang, Dezhi [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Nie, Wanyi; Li, Yuan; Huang, Wenxiao [Center for Nanotechnology and Molecular Materials, Department of Physics, Wake Forest University, Winston-Salem, NC 27105 (United States); Ma, Dongge [State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Carroll, David L., E-mail: carroldl@wfu.edu [Center for Nanotechnology and Molecular Materials, Department of Physics, Wake Forest University, Winston-Salem, NC 27105 (United States)

    2015-05-15

    In this work, a high-performance alternating current (AC) filed-induced chromatic-stable white polymer electroluminescence (WFIPEL) device was fabricated by combining a fluorophor Poly(9,9-dioctylfluorene) (PFO)-based blue device with a yellow down-conversion layer (YAG:Ce). A maximum luminance of this down-conversion FIPEL device achieves 3230 cd m{sup −2}, which is 1.41 times higher than the device without the down-conversion layer. A maximum current efficiency and power efficiency of the down-conversion WFIPEL device reach 19.7 cd A{sup −1} at 3050 cd m{sup −2} and 5.37 lm W{sup −1} at 2310 cd m{sup −2} respectively. To the best of our knowledge, the power efficiency is one of the highest reports for the WFIPEL up to now. Moreover, Commison Internationale de L’Eclairage (CIE) coordinates of (0.28, 0.30) is obtained by adjusting the thickness of the down-conversion layer to 30 μm and it is kept stable over the entire AC-driven voltage range. We believe that this AC-driven, down-conversion, WFIPEL device may offer an easy way towards future flat and flexible lighting sources. - Highlights: • A high-performance AC filed-induced chromatic-stable white polymer electroluminescence (WFIPEL) device was fabricated. • A maximum luminance, current efficiency, and power efficiency achieves 3230 cd m{sup −2}, 19.7 cd A{sup −1}, and 5.37 lm W{sup −1}, respectively. • The power efficiency is one of the highest reports for the WFIPEL up to now. • The EL spectrum kept very stable over the entire AC-driven voltage range.

  19. Conversion disorder

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000954.htm Conversion disorder To use the sharing features on this page, please enable JavaScript. Conversion disorder is a mental condition in which a person ...

  20. Detailed computational analysis revealed mutation V210I on PrP induced conformational conversion on β2-α2 loop and α2-α3.

    Science.gov (United States)

    Chandrasekaran, P; Rajasekaran, R

    2016-10-20

    The development of fatal transmissible spongiform encephalopathies (TSE) is associated with the conformational conversion of the normal cellular prion protein, PrP(C), into its pathogenic isoform, PrP(Sc). The present study revealed the structural consequences that induce the conversion of PrP(C)→ PrP(Sc) upon mutation V210I linked with genetic Creutzfeldt-Jakob disease (CJD) using the classical molecular dynamics (MD) approach. Similar to the experimental results, the mutant showed biased disruption in the local folding of α2 and the complete distortion of α3. In addition, substitution of bulkier Ile at position 210 induced reorientations of several residues that were the constituent of hydrophobic cores, thereby influencing α2-α3 inter-helical interactions. In addition, the β2-α2 loop was greatly altered due to the loss of π-π interactions of the residue Tyr(169) with Phe(175), Tye(163), Tyr(162), and Tyr(218), facilitating more conformational flexibility, which may be involved in the conversion of PrP(C)→ PrP(Sc). This study afforded a detailed structure and dynamic properties of the mutant, which were consistent with the experimental results, providing an insight into the molecular basis for the conversion of PrP(C)→ PrP(Sc), which could be used for the development of antiprion drugs.

  1. Chemical memory reactions induced bursting dynamics in gene expression.

    Science.gov (United States)

    Tian, Tianhai

    2013-01-01

    Memory is a ubiquitous phenomenon in biological systems in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many memorial phenomena are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict to the extant stochastic approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. To tackle the challenge, I proposed a novel theory consisting of the memory chemical master equations and memory stochastic simulation algorithm. A stochastic model for single-gene expression was proposed to illustrate the key function of memory reactions in inducing bursting dynamics of gene expression that has been observed in experiments recently. The importance of memory reactions has been further validated by the stochastic model of the p53-MDM2 core module. Simulations showed that memory reactions is a major mechanism for realizing both sustained oscillations of p53 protein numbers in single cells and damped oscillations over a population of cells. These successful applications of the memory modeling framework suggested that this innovative theory is an effective and powerful tool to study memory process and conditional chemical reactions in a wide range of complex biological systems.

  2. Fetal Globin Gene Inducers: Novel Agents & New Potential

    Science.gov (United States)

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  3. Deoxynivalenol-Induced Proinflammatory Gene Expression: Mechanisms and Pathological Sequelae

    Directory of Open Access Journals (Sweden)

    James J. Pestka

    2010-06-01

    Full Text Available The trichothecene mycotoxin deoxynivalenol (DON is commonly encountered in human cereal foods throughout the world as a result of infestation of grains in the field and in storage by the fungus Fusarium. Significant questions remain regarding the risks posed to humans from acute and chronic DON ingestion, and how to manage these risks without imperiling access to nutritionally important food commodities. Modulation of the innate immune system appears particularly critical to DON’s toxic effects. Specifically, DON induces activation of mitogen-activated protein kinases (MAPKs in macrophages and monocytes, which mediate robust induction of proinflammatory gene expression—effects that can be recapitulated in intact animals. The initiating mechanisms for DON-induced ribotoxic stress response appear to involve the (1 activation of constitutive protein kinases on the damaged ribosome and (2 autophagy of the chaperone GRP78 with consequent activation of the ER stress response. Pathological sequelae resulting from chronic low dose exposure include anorexia, impaired weight gain, growth hormone dysregulation and aberrant IgA production whereas acute high dose exposure evokes gastroenteritis, emesis and a shock-like syndrome. Taken together, the capacity of DON to evoke ribotoxic stress in mononuclear phagocytes contributes significantly to its acute and chronic toxic effects in vivo. It is anticipated that these investigations will enable the identification of robust biomarkers of effect that will be applicable to epidemiological studies of the human health effects of this common mycotoxin.

  4. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

    Science.gov (United States)

    Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

    2017-09-26

    Magnaportheoryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M.oryzae-derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M.oryzae was examined by targeting three predicted pathogenicity genes, MoABC1,MoMAC1 and MoPMK1. Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M.oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

  5. Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

    Science.gov (United States)

    Yahara, Koji; Horie, Ryota; Kobayashi, Ichizo; Sasaki, Akira

    2007-01-01

    The necessity to repair genome damage has been considered to be an immediate factor responsible for the origin of sex. Indeed, attack by a cellular restriction enzyme of invading DNA from several bacteriophages initiates recombinational repair by gene conversion if there is homologous DNA. In this work, we modeled the interaction between a bacteriophage and a bacterium carrying a restriction enzyme as antagonistic coevolution. We assume a locus on the bacteriophage genome has either a restriction-sensitive or a restriction-resistant allele, and another locus determines whether it is recombination/repair proficient or defective. A restriction break can be repaired by a co-infecting phage genome if one of them is recombination/repair proficient. We define the fitness of phage (resistant/sensitive and repair-positive/-negative) genotypes and bacterial (restriction-positive/-negative) genotypes by assuming random encounter of the genotypes, with given probabilities of single and double infections, and the costs of resistance, repair, and restriction. Our results show the evolution of the repair allele depends on \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}b_{1}/b_{0},\\end{equation*}\\end{document} the ratio of the burst size \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}b_{1}\\end{equation*}\\end{document} under damage to host cell physiology induced by an unrepaired double-strand break to the default burst size \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage

  6. Human DNA methyltransferase gene-transformed yeasts display an inducible flocculation inhibited by 5-aza-2'-deoxycytidine.

    Science.gov (United States)

    Sugiyama, Kei-Ichi; Takamune, Makiko; Furusawa, Hiroko; Honma, Masamitsu

    2015-01-09

    Mammalian DNA methyltransferases (DNMTs) play an important role in establishing and maintaining the proper regulation of epigenetic information. However, it remains unclear whether mammalian DNMTs can be functionally expressed in yeasts, which probably lack endogenous DNMTs. We cotransformed the budding yeast Saccharomyces cerevisiae with the human DNMT1 gene, which encodes a methylation maintenance enzyme, and the DNMT3A/3B genes, which encode de novo methylation enzymes, in an expression vector also containing the GAL1 promoter, which is induced by galactose, and examined the effects of the DNMT inhibitor 5-aza-2'-deoxycytidine (5AZ) on cell growth. Transformed yeast strains grown in galactose- and glucose-containing media showed growth inhibition, and their growth rate was unaffected by 5AZ. Conversely, 5AZ, but not 2'-deoxycytidine, dose-dependently interfered with the flocculation exhibited by DNMT-gene transformants grown in glucose-containing medium. Further investigation of the properties of this flocculation indicated that it may be dependent on the expression of a Flocculin-encoding gene, FLO1. Taken together, these findings suggest that DNMT-gene transformed yeast strains functionally express these enzymes and represent a useful tool for in vivo screening for DNMT inhibitors.

  7. Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2

    Energy Technology Data Exchange (ETDEWEB)

    Porter, G.; Westmoreland, J.; Priebe, S. [National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States)] [and others

    1996-06-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad{sup +} vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. 67 refs., 5 figs., 4 tabs.

  8. BRD4 regulates fructose-inducible lipid accumulation-related genes in the mouse liver.

    Science.gov (United States)

    Yamada, Aki; Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2016-10-01

    Fructose intake induces hepatic steatosis by activating fat synthesis. In this study, we searched for genes that showed acute induction in the livers of mice force-fed with fructose, and examined how this induction is regulated. We identified genes induced at 6h after the fructose force-feeding using a microarray and quantitative real-time RT-PCR. Histone acetylation and an acetylated histone binding protein bromodomain containing (BRD)4 binding around the fructose-inducible genes were examined using a chromatin immunoprecipitation assay. We examined whether (+)-JQ1, an inhibitor of the binding between the BRD4 and acetylated histones, inhibited the expressions of fructose-inducible genes, histone acetylation and BRD4 binding around the genes. We identified upregulated genes related to lipid accumulation, such as Cyp8b1, Dak and Plin5, in mice force-fed with fructose compared with those force-fed with glucose. Acetylation of histones H3 and H4, and BRD4 binding around the transcribed region of those fructose-inducible genes, were enhanced by fructose force-feeding. Meanwhile, (+)-JQ1 treatment reduced expressions of fructose-inducible genes, histone acetylation and BRD4 binding around these genes. Acute induction of genes related to lipid accumulation in the livers of mice force-fed with fructose is associated with the induction of histone acetylation and BRD4 binding around these genes. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.

    1999-01-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were tr

  10. Independent stratum formation on the avian sex chromosomes reveals inter-chromosomal gene conversion and predominance of purifying selection on the W chromosome.

    Science.gov (United States)

    Wright, Alison E; Harrison, Peter W; Montgomery, Stephen H; Pointer, Marie A; Mank, Judith E

    2014-11-01

    We used a comparative approach spanning three species and 90 million years to study the evolutionary history of the avian sex chromosomes. Using whole transcriptomes, we assembled the largest cross-species dataset of W-linked coding content to date. Our results show that recombination suppression in large portions of the avian sex chromosomes has evolved independently, and that long-term sex chromosome divergence is consistent with repeated and independent inversions spreading progressively to restrict recombination. In contrast, over short-term periods we observe heterogeneous and locus-specific divergence. We also uncover four instances of gene conversion between both highly diverged and recently evolved gametologs, suggesting a complex mosaic of recombination suppression across the sex chromosomes. Lastly, evidence from 16 gametologs reveal that the W chromosome is evolving with a significant contribution of purifying selection, consistent with previous findings that W-linked genes play an important role in encoding sex-specific fitness.

  11. Impaired Bcl3 up-regulation leads to enhanced lipopolysaccharide-induced interleukin (IL)-23P19 gene expression in IL-10(-/-) mice.

    Science.gov (United States)

    Mühlbauer, Marcus; Chilton, Paula M; Mitchell, Thomas C; Jobin, Christian

    2008-05-23

    Genetic and biochemical analyses show that IL-23p19 plays a central role in mediating bacteria-induced colitis in interleukin-10-deficient (IL-10(-/-)) mice. The molecular mechanisms responsible for the dysregulated innate host response leading to enhanced IL-23 gene expression in IL-10(-/-) mice are poorly understood. In this study, we investigated the role of Bcl3 in controlling LPS-induced IL-23p19 gene expression in bone marrow-derived dendritic cells (BMDC) isolated from IL-10(-/-) mice. We report higher IL-23p19 mRNA accumulation and protein secretion in LPS-stimulated BMDC isolated from IL-10(-/-) compared with WT mice. Lipopolysaccharide (LPS)-induced B cell leukemia 3 (Bcl3) expression was strongly impaired (90% decrease) in IL-10(-/-) BMDC compared with WT BMDC. Chromatin immunoprecipitation demonstrated enhanced RelA binding to the IL-23p19 promoter in IL-10(-/-) compared with WT BMDC. Bcl3 overexpression decreased LPS-induced IL-23p19 gene expression in IL-10(-/-) BMDC, which correlated with enhanced NF-kappaB p50 binding and decreased RelA binding to the gene promoter. Conversely, Bcl3 knockdown enhanced LPS-induced IL-23p19 gene expression in WT BMDC. Moreover, LPS-induced IL-23p19 gene expression was significantly enhanced in Bcl3(-/-) BMDC compared with WT BMDC. In conclusion, enhanced LPS-induced IL-23p19 gene expression in IL-10(-/-) mice is due to impaired Bcl3 expression leading to diminished p50 and enhanced RelA recruitment to the IL-23p19 promoter.

  12. Gene conversion in the CYP11B2 gene encoding P450c11AS is associated with, but does not cause, the syndrome of corticosterone methyloxidase II deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Fardella, C.E.; Hum, D.W.; Rodriguez, H. [Univ. of California, San Francisco, CA (United States)]|[Univ. of Colorado, Denver, CO (United States)] [and others

    1996-01-01

    Cytochrome P450c11AS (aldosterone synthase) has 11{beta}hydroxylase, 18-hydroxylase, and 18-oxidase activities and is expressed solely in the adrenal zona glomerulosa. Corticosterone methyloxidase II (CMOII) deficiency denotes a rare disorder of adrenal steroidogenesis in which only the 18-oxidase activity of P450c11AS is disrupted, while the 11{beta}-hydroxylase and 18-hydroxylase activities persist. Such patients have elevated serum concentrations of corticosterone and 18-hydroxycorticosterone and very low or unmeasurable concentrations of aldosterone, often resulting in a clinical salt-losing crisis in infancy. We have sought mutations causing CMOII deficiency in outbred populations. In three of four unrelated P450c11AS alleles from two unrelated patients with CMOII deficiency, we found a gene conversion event in which exons 3 and 4 of the CYP11B2 gene encoding P450c11AS were changed to the sequence of the nearby CYP11B1 gene, which encodes the related enzyme P450c11{beta}. This conversion resulted in a mutant P450c11AS protein carrying three changes. We built seven vectors expressing P450c11AS carrying each mutation singly, each of the three possible pairs of mutations, and the triple mutation as found in the proband. The activities in steroidogenic MA-10 and JEG-3 cells were 10- to 20-fold higher. In these systems all of the mutants retained normal 18-oxidase activity, indicating that the detected gene conversion event is associated with but does not cause CMOII deficiency. None of the four CPY11B2 alleles in these two patients bore other identifiable mutations. These patients might have mutations in the promoters or other noncoding regions, or mutations in genes other than CYP11B2 may cause the syndrome of CMOII deficiency. 37 refs., 2 figs., 2 tabs.

  13. Gene cloning of an efficiency oleate hydratase from Stenotrophomonas nitritireducens for polyunsaturated fatty acids and its application in the conversion of plant oils to 10-hydroxy fatty acids.

    Science.gov (United States)

    Kang, Woo-Ri; Seo, Min-Ju; Shin, Kyung-Chul; Park, Jin-Byung; Oh, Deok-Kun

    2017-01-01

    Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis-9 polyunsaturated fatty acids (PUFAs) to 10-hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double-bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double-bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full-length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10-hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α-linolenic acid and/or γ-linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10-hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA-containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74-82. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  15. XPS investigation of ion beam induced conversion of GaAs(0 0 1) surface into GaN overlayer

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Praveen [Surface Physics and Nanostructure Group, National Physical Laboratory, New Delhi 110012 (India); Department of Physics, Indian Institute of Technology, New Delhi 110016 (India); Kumar, Mahesh; Govind [Surface Physics and Nanostructure Group, National Physical Laboratory, New Delhi 110012 (India); Mehta, B.R. [Department of Physics, Indian Institute of Technology, New Delhi 110016 (India); Shivaprasad, S.M., E-mail: smsprasad@jncasr.ac.in [Jawaharlal Nehru Centre for Advanced Scientific Research, International Center for Material Science and Chemistry and Physics Materials Unit, Jakkur, 560064 Bangalore, Karnataka (India)

    2009-10-30

    For the advance of GaN based optoelectronic devices, one of the major barriers has been the high defect density in GaN thin films, due to lattice parameter and thermal expansion incompatibility with conventional substrates. Of late, efforts are focused in fine tuning epitaxial growth and in search for a low temperature method of forming low defect GaN with zincblende structure, by a method compatible to the molecular beam epitaxy process. In principle, to grow zincblende GaN the substrate should have four-fold symmetry and thus zincblende GaN has been prepared on several substrates including Si, 3C-SiC, GaP, MgO, and on GaAs(0 0 1). The iso-structure and a common shared element make the epitaxial growth of GaN on GaAs(0 0 1) feasible and useful. In this study ion-induced conversion of GaAs(0 0 1) surface into GaN at room temperature is optimized. At the outset a Ga-rich surface is formed by Ar{sup +} ion bombardment. Nitrogen ion bombardment of the Ga-rich GaAs surface is performed by using 2-4 keV energy and fluence ranging from 3 x 10{sup 13} ions/cm{sup 2} to 1 x 10{sup 18} ions/cm{sup 2}. Formation of surface GaN is manifested as chemical shift. In situ core level and true secondary electron emission spectra by X-ray photoelectron spectroscopy are monitored to observe the chemical and electronic property changes. Using XPS line shape analysis by deconvolution into chemical state, we report that 3 keV N{sub 2}{sup +} ions and 7.2 x 10{sup 17} ions/cm{sup 2} are the optimal energy and fluence, respectively, for the nitridation of GaAs(0 0 1) surface at room temperature. The measurement of electron emission of the interface shows the dependence of work function to the chemical composition of the interface. Depth profile study by using Ar{sup +} ion sputtering, shows that a stoichiometric GaN of 1 nm thickness forms on the surface. This, room temperature and molecular beam epitaxy compatible, method of forming GaN temperature can serve as an excellent template for

  16. Laser-induced down-conversion and infrared phosphorescence emissivity of novel ligand-free perovskite nanomaterials

    Science.gov (United States)

    Ahmed, M. A.; Khafagy, Rasha M.; El-sayed, O.

    2014-03-01

    For the first time, standalone and ligand-free series of novel rare-earth-based perovskite nanomaterials are used as near infrared (NIR) and mid infrared (MIR) emitters. Nano-sized La0.7Sr0.3M0.1Fe0.9O3; where M = 0, Mn2+, Co2+ or Ni2+ were synthesized using the flash auto-combustion method and characterized using FTIR, FT-Raman, SEM and EDX. Photoluminescence spectra were spontaneously recorded during pumping the samples with 0.5 mW of green laser emitting continuously at 532 nm. La0.7Sr0.3FeO3 (where M = 0) did not result in any infrared emissivity, while intense near and mid infrared down-converted phosphorescence was released from the M-doped samples. The released phosphorescence greatly shifted among the infrared spectral region with changing the doping cation. Ni2+-doped perovskite emitted at the short-wavelength near-infrared region, while Mn2+ and Co2+-doped perovskites emitted at the mid-wavelength infrared region. The detected laser-induced spontaneous parametric down-conversion phosphorescence (SPDC) occurred through a two-photon process by emitting two NIR or MIR photons among a cooperative energy transfer between the La3+ cations and the M2+ cations. Combining SrFeO3 ceramic with both a rare earth cation (RE3+) and a transition metal cation (Mn2+, Co2+ or Ni2+), rather than introducing merely RE3+ cations, greatly improved and controlled the infrared emissivity properties of synthesized perovskites through destroying their crystal symmetry and giving rise to asymmetrical lattice vibration and the nonlinear optical character. The existence of SPDC in the M2+-doped samples verifies their nonlinear character after the absence of this character in La0.7Sr0.3FeO3. Obtained results verify that, for the first time, perovskite nanomaterials are considered as nonlinear optical crystals with intense infrared emissivity at low pumping power of visible wavelengths, which nominates them for photonic applications and requires further studies regarding their lasing

  17. Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum

    DEFF Research Database (Denmark)

    Grønlund, Mette; Olsen, Anne; Johansen, Elisabeth

    2010-01-01

    Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However, the avail......Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However......, the available PEBV-VIGS protocols are inadequate for studying genes involved in the symbiosis with arbuscular mycorrhizal fungi (AMF). Here we describe a PEBV-VIGS protocol suitable for reverse genetics studies in pea of genes involved in the symbiosis with AMF and show its effectiveness in silencing genes...... involved in the early and late stages of AMF symbiosis....

  18. Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions.

    Science.gov (United States)

    Tsai, Ju-Ying; Chen, Fang-Hsin; Hsieh, Tsung-Yu; Hsiao, Ya-Yun

    2015-07-01

    Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.

  19. Paralogous gene conversion, allelic divergence of attacin genes and its expression profile in response to BmNPV infection in silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    G Lekha

    2015-08-01

    Full Text Available The genomic organization, structure and polymorphism of attacin gene within the mulberry silkworm Bombyx mori strains have been analyzed. Genomic contig (AADK01007556 of B. mori attacin gene contains locus with two transcribed basic attacin genes, which were designated as attacin I and attacin II. Survey of the naturally occurring genetic variation in different strains of silkworm B. mori at the promoter and coding regions of two attacin genes revealed high levels of silent nucleotide variations (1- 4 % per nucleotide heterozygosity without polymorphism at the amino acid level (nonSynonymous substitution. We also investigated variations in gene expression of attacin I and attacin II in silkworm B. mori infected with nucleopolyhedrovirus (BmNPV. Two B. mori strains, Sarupat, CSR-2 which were resistant and susceptible to BmNPV infection respectively were used in this study. Expression profiles of B. mori genes were analyzed using microarray technique and results revealed that the immune response genes including attacin were selectively up regulated in virus invaded midguts of both races. Microarray data and real-time qPCR results revealed that attacin I gene was significantly up-regulated in the midgut of Sarupat following BmNPV infection, indicating its specific role in the anti-viral response. Our results imply that these up-regulated attacin genes were not only involved in anti-bacterial mechanism, but are also involved in B. mori immune response against BmNPV infection.

  20. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    Science.gov (United States)

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  1. Cyclic AMP-inducible genes respond uniformly to seasonal lighting conditions in the rat pineal gland.

    Science.gov (United States)

    Spessert, R; Gupta, B B P; Rohleder, N; Gerhold, S; Engel, L

    2006-12-01

    The encoding of photoperiodic information ensues in terms of the daily profile in the expression of cyclic AMP (cAMP)-inducible genes such as the arylalkylamine N-acetyltransferase (AA-NAT) gene that encodes the rate-limiting enzyme in melatonin formation. In the present study, we compared the influence of the photoperiodic history on the cAMP-inducible genes AA-NAT, inducible cyclic AMP early repressor (ICER), fos-related antigen-2 (FRA-2), mitogen-activated protein kinase phosphatase-1 (MKP-1), nerve growth factor inducible gene-A (NGFI-A) and nerve growth factor inducible gene-B (NGFI-B) in the pineal gland of rats. For this purpose, we monitored the daily profiles of each gene in the same pineal gland under a long (light/dark 16:8) and a short (light/dark 8:16) photoperiod by measuring the respective mRNA amounts by real-time polymerase chain reaction analysis. We found that, for all genes under investigation, the duration of increased nocturnal expression is lengthened and, in relation to light onset, the nocturnal rise is earlier under the long photoperiod (light/dark 16:8). Furthermore, with the exception of ICER, all other cAMP-inducible genes tend to display higher maximum expression under light/dark 8:16 than under light/dark 16:8. Photoperiod-dependent changes persist for all of the cAMP-inducible genes when the rats are kept for two cycles under constant darkness. Therefore, all cAMP-inducible genes are also influenced by the photoperiod of prior entrained cycles. Our study indicates that, despite differences regarding the expressional control and the temporal phasing of the daily profile, cAMP-inducible genes are uniformly influenced by photoperiodic history in the rat pineal gland.

  2. Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Hilary Collver; Katherine Kinken

    2004-01-01

    Genomic research has made a large number of sequences of novel genes or expressed sequence tags available. To investigate functions of these genes, a system for conditional control of gene expression would be a useful tool. Inducible transgene expression that uses green fluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines of cotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir (NOR; Abies nordmanniana Lk.), and rice (RIC; Oryza sativa L. Cv. Radon). Transgenic cell lines were used to test the function of the chemical inducer dexamethasone. Inducible transgene expression was observed with fluorescence and confocal microscopy, and was confirmed by northern blot analyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h after treatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgenic cells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducer for optimum inducible gene expression system varied among transgenic cell lines. The inducible gene expression system described here was very effective and could be valuable in evaluating the function of novel gene.

  3. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

    Directory of Open Access Journals (Sweden)

    Sara Tengvall

    Full Text Available Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA. Targeting the expression of the collagen type II (CII to antigen presenting cells (APCs induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1 increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

  4. Conversational Narcissism.

    Science.gov (United States)

    Vangelisti, Anita L.; And Others

    1990-01-01

    Examines narcissistic communication and the ways it is exhibited in everyday conversation. Identifies the following behavioral referents: boasting, refocusing the topic of conversation on the self, exaggerating hand and body movements, using a loud tone of voice, and "glazing over" when others speak. Suggests that conversational…

  5. Contentious Conversations

    Science.gov (United States)

    Zuidema, Leah A.

    2011-01-01

    The idea of joining a conversation through reading and writing is not new; in his 1941 book "The Philosophy of Literary Form: Studies in Symbolic Action," Kenneth Burke suggests that the acts of reading and writing are like entering a parlor where others are already conversing. The author explores the place of professional debate within NCTE and…

  6. Appropriateness of reference genes for normalizing messenger RNA in mouse 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis using quantitative real time PCR

    Science.gov (United States)

    Eissa, Nour; Kermarrec, Laëtitia; Hussein, Hayam; Bernstein, Charles N.; Ghia, Jean-Eric

    2017-01-01

    2,4-Dinitrobenzene sulfonic acid (DNBS)-induced colitis is an experimental model that mimics Crohn’s disease. Appropriateness of reference genes is crucial for RT-qPCR. This is the first study to determine the stability of reference gene expression (RGE) in mice treated with DNBS. DNBS experimental Colitis was induced in male C57BL/6 mice. RNA was extracted from colon tissue and comprehensive analysis of 13 RGE was performed according to predefined criteria. Relative colonic TNF-α and IL-1β mRNA levels were calculated. Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), or β2-microglobulin (β2m) showed the highest fluctuation within the inflamed and control groups. Conversely, ribosomal protein large P0 (Rplp0), non-POU domain containing (Nono), TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. TNF-α and IL-1β mRNA levels was dependent on the reference gene used and varied from significant when the most stable genes were used to non-significant when the least stable genes were used. The appropriate choice of RGE is critical to guarantee satisfactory normalization of RT-qPCR data when using DNBS-Model. We recommend using Rplp0, Nono, Tbp, Hprt and Eef2 instead of common reference genes. PMID:28186172

  7. Coordinate gene regulation by fimbriae-induced signal transduction

    DEFF Research Database (Denmark)

    Schembri, Mark; Klemm, Per

    2001-01-01

    whether fimbriae expression can affect expression of other genes, Analysis of gene expression in two E.coli strains, differing in the fim locus, indicated the flu gene to be affected. The flu gene encodes the antigen 43 (Ag43) surface protein, specifically involved in bacterial aggregation...... of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated...

  8. Glomerulonephritis-induced changes in kidney gene expression in rats

    Directory of Open Access Journals (Sweden)

    Mira Pavkovic

    2015-12-01

    Full Text Available We investigated a glomerulonephritis (GN model in rats induced by nephrotoxic serum (NTS which contains antibodies against the glomerular basement membrane (GBM. The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003 [2]. Male Wistar Kyoto (WKY and Sprague–Dawley (SD rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265. The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The β-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb and complement component 6 (C6 were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]. Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6.

  9. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene.

    Science.gov (United States)

    van der Kop, D A; Schuyer, M; Pinas, J E; van der Zaal, B J; Hooykaas, P J

    1999-03-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.

  10. Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study.

    Science.gov (United States)

    McGuire, Lynne I; Poleggi, Anna; Poggiolini, Ilaria; Suardi, Silvia; Grznarova, Katarina; Shi, Song; de Vil, Bart; Sarros, Shannon; Satoh, Katsuya; Cheng, Keding; Cramm, Maria; Fairfoul, Graham; Schmitz, Matthias; Zerr, Inga; Cras, Patrick; Equestre, Michele; Tagliavini, Fabrizio; Atarashi, Ryuichiro; Knox, David; Collins, Steven; Haïk, Stéphane; Parchi, Piero; Pocchiari, Maurizio; Green, Alison

    2016-07-01

    Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt-Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160-165.

  11. Cerebrospinal fluid real‐time quaking‐induced conversion is a robust and reliable test for sporadic creutzfeldt–jakob disease: An international study

    Science.gov (United States)

    McGuire, Lynne I.; Poleggi, Anna; Poggiolini, Ilaria; Suardi, Silvia; Grznarova, Katarina; Shi, Song; de Vil, Bart; Sarros, Shannon; Satoh, Katsuya; Cheng, Keding; Cramm, Maria; Fairfoul, Graham; Schmitz, Matthias; Zerr, Inga; Cras, Patrick; Equestre, Michele; Tagliavini, Fabrizio; Atarashi, Ryuichiro; Knox, David; Collins, Steven; Haïk, Stéphane; Parchi, Piero; Pocchiari, Maurizio

    2016-01-01

    Real‐time quaking‐induced conversion (RT‐QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt–Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT‐QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160–165 PMID:27130376

  12. RNA splicing regulates the temporal order of TNF-induced gene expression.

    Science.gov (United States)

    Hao, Shengli; Baltimore, David

    2013-07-16

    When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

  13. Relationship between gene responses and symptoms induced by Rice grassy stunt virus

    Directory of Open Access Journals (Sweden)

    Kouji eSatoh

    2013-10-01

    Full Text Available Rice grassy stunt virus (RGSV is a serious threat to rice production in Southeast Asia. RGSV is a member of the genus Tenuivirus, and it induces leaf yellowing, stunting, and excess tillering on rice plants. Here we examined gene responses of rice to RGSV infection to gain insight into the gene responses which might be associated with the disease symptoms. The results indicated that 1 many genes related to cell wall synthesis and chlorophyll synthesis were predominantly suppressed by RGSV infection; 2 RGSV infection induced genes associated with tillering process; 3 RGSV activated genes involved in inactivation of gibberellic acid and indole-3-acetic acid ; and 4 the genes for strigolactone signaling were suppressed by RGSV. These results suggest that these gene responses to RGSV infection account for the excess tillering specific to RGSV infection as well as other symptoms by RGSV, such as stunting and leaf chlorosis.

  14. Rationale for developing new virus vectors to analyze gene function in grasses through virus-induced gene silencing.

    Science.gov (United States)

    Ramanna, Hema; Ding, Xin Shun; Nelson, Richard S

    2013-01-01

    The exploding availability of genome and EST-based sequences from grasses requires a technology that allows rapid functional analysis of the multitude of genes that these resources provide. There are several techniques available to determine a gene's function. For gene knockdown studies, silencing through RNAi is a powerful tool. Gene silencing can be accomplished through stable transformation or transient expression of a fragment of a target gene sequence. Stable transformation in rice, maize, and a few other species, although routine, remains a relatively low-throughput process. Transformation in other grass species is difficult and labor-intensive. Therefore, transient gene silencing methods including Agrobacterium-mediated and virus-induced gene silencing (VIGS) have great potential for researchers studying gene function in grasses. VIGS in grasses already has been used to determine the function of genes during pathogen challenge and plant development. It also can be used in moderate-throughput reverse genetics screens to determine gene function. However, the number of viruses modified to serve as silencing vectors in grasses is limited, and the silencing phenotype induced by these vectors is not optimal: the phenotype being transient and with moderate penetration throughout the tissue. Here, we review the most recent information available for VIGS in grasses and summarize the strengths and weaknesses in current virus-grass host systems. We describe ways to improve current virus vectors and the potential of other grass-infecting viruses for VIGS studies. This work is necessary because VIGS for the foreseeable future remains a higher throughput and more rapid system to evaluate gene function than stable transformation.

  15. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

    Science.gov (United States)

    Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens. PMID:26248204

  16. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

    Science.gov (United States)

    Zhang, Hanbang; Ehrenkaufer, Gretchen M; Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  17. Conversion Disorder

    Science.gov (United States)

    ... Recent significant stress or emotional trauma Being female — women are much more likely to develop conversion disorder Having a mental health condition, such as mood or anxiety disorders, dissociative disorder or certain personality disorders Having ...

  18. Conversation Analysis.

    Science.gov (United States)

    Schiffrin, Deborah

    1990-01-01

    Summarizes the current state of research in conversation analysis, referring primarily to six different perspectives that have developed from the philosophy, sociology, anthropology, and linguistics disciplines. These include pragmatics; speech act theory; interactional sociolinguistics; ethnomethodology; ethnography of communication; and…

  19. Conversation Analysis.

    Science.gov (United States)

    Schiffrin, Deborah

    1990-01-01

    Summarizes the current state of research in conversation analysis, referring primarily to six different perspectives that have developed from the philosophy, sociology, anthropology, and linguistics disciplines. These include pragmatics; speech act theory; interactional sociolinguistics; ethnomethodology; ethnography of communication; and…

  20. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

  1. Strategic conversation

    Directory of Open Access Journals (Sweden)

    Nicholas Asher

    2013-08-01

    Full Text Available Models of conversation that rely on a strong notion of cooperation don’t apply to strategic conversation — that is, to conversation where the agents’ motives don’t align, such as courtroom cross examination and political debate. We provide a game-theoretic framework that provides an analysis of both cooperative and strategic conversation. Our analysis features a new notion of safety that applies to implicatures: an implicature is safe when it can be reliably treated as a matter of public record. We explore the safety of implicatures within cooperative and non cooperative settings. We then provide a symbolic model enabling us (i to prove a correspondence result between a characterisation of conversation in terms of an alignment of players’ preferences and one where Gricean principles of cooperative conversation like Sincerity hold, and (ii to show when an implicature is safe and when it is not. http://dx.doi.org/10.3765/sp.6.2 BibTeX info

  2. CTCF-mediated chromatin loops enclose inducible gene regulatory domains

    NARCIS (Netherlands)

    Oti, M.O.; Falck, J.; Huynen, M.A.; Zhou, Huiqing

    2016-01-01

    BACKGROUND: The CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to

  3. Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

    DEFF Research Database (Denmark)

    Pinto, Rita; Hansen, Lars; Hintze, John Birger Hjalmar

    2017-01-01

    Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven...... to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii......) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide...

  4. Identification of novel light-induced genes in the suprachiasmatic nucleus

    Directory of Open Access Journals (Sweden)

    Piontkivska Helen

    2007-11-01

    Full Text Available Abstract Background The transmission of information about the photic environment to the circadian clock involves a complex array of neurotransmitters, receptors, and second messenger systems. Exposure of an animal to light during the subjective night initiates rapid transcription of a number of immediate-early genes in the suprachiasmatic nucleus of the hypothalamus. Some of these genes have known roles in entraining the circadian clock, while others have unknown functions. Using laser capture microscopy, microarray analysis, and quantitative real-time PCR, we performed a comprehensive screen for changes in gene expression immediately following a 30 minute light pulse in suprachiasmatic nucleus of mice. Results The results of the microarray screen successfully identified previously known light-induced genes as well as several novel genes that may be important in the circadian clock. Newly identified light-induced genes include early growth response 2, proviral integration site 3, growth-arrest and DNA-damage-inducible 45 beta, and TCDD-inducible poly(ADP-ribose polymerase. Comparative analysis of promoter sequences revealed the presence of evolutionarily conserved CRE and associated TATA box elements in most of the light-induced genes, while other core clock genes generally lack this combination of promoter elements. Conclusion The photic signalling cascade in the suprachiasmatic nucleus activates an array of immediate-early genes, most of which have unknown functions in the circadian clock. Detected evolutionary conservation of CRE and TATA box elements in promoters of light-induced genes suggest that the functional role of these elements has likely remained the same over evolutionary time across mammalian orders.

  5. Transposon-induced nuclear mutations that alter chloroplast gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, A.

    1992-01-01

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  6. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  7. Direct Conversion of Energy

    Energy Technology Data Exchange (ETDEWEB)

    Corliss, William R

    1964-01-01

    Topics include: direct versus dynamic energy conversion; laws governing energy conversion; thermoelectricity; thermionic conversion; magnetohydrodynamic conversion; chemical batteries; the fuel cell; solar cells; nuclear batteries; and advanced concepts including ferroelectric conversion and thermomagnetic conversion.

  8. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  9. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis

    DEFF Research Database (Denmark)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior

    2017-01-01

    of Gene Ontology processes and protein networks. RESULTS: A total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDASimproved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed......BACKGROUND: Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy...... women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDASimproved) overlap substantially with changes observed among healthy women and differ from changes...

  10. Elicitor and fusarium-induced expression of NPR-1 like genes in banana

    CSIR Research Space (South Africa)

    Endah, R

    2008-11-01

    Full Text Available NPR1 is an essential positive regulator of salicylic acid-induced PR gene expression and systemic acquired resistance. Two novel full-length NPR1-like genes; MNPR1A and MNPR1B, were isolated by application of the PCR and RACE techniques. The two...

  11. Alcohol-induced histone acetylation reveals a gene network involved in alcohol tolerance.

    Directory of Open Access Journals (Sweden)

    Alfredo Ghezzi

    Full Text Available Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

  12. Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology

    National Research Council Canada - National Science Library

    Guo Yan Xie Xiaojia Guo Changqing Wang Zhaoyang Liu Qingguo

    2015-01-01

    OBJECTIVE:To explore electro-acupuncture's(EA's)effect on gene expression in heart of rats with stress-induced pre-hypertension and try to reveal its biological mechanism based on gene chip...

  13. An insulin-induced DNA-binding protein for the human growth hormone gene.

    OpenAIRE

    Prager, D; Gebremedhin, S; Melmed, S

    1990-01-01

    The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced D...

  14. A virus-induced gene silencing method to study soybean cyst nematode parasitism in Glycine max

    OpenAIRE

    Kandoth, Pramod K; Heinz, Robert; Yeckel, Greg; Gross, Nathan W; Juvale, Parijat S; Hill, John; Whitham, Steven A.; Baum, Thomas J.; Mitchum, Melissa G.

    2013-01-01

    Background Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. Findings Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for anal...

  15. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    OpenAIRE

    Sang-Ho Park; Hoseong Choi; Semin Kim; Won Kyong Cho; Kook-Hyung Kim

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the n...

  16. Probiotic bacteria change Echherichia coli-induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria.METHODS: A 19200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E.coli, Lactobacillus plantarum, and a combination of the two.RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were upregulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group.CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination,and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

  17. Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efifciency During the Conversion of Lycopene-to-Provitamin A

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-cheng; ZHOU Wen-jing; XU Qiang; TAO Neng-guo; YE Jun-li; GUO Fei; XU Juan; DENG Xiu-xin

    2013-01-01

    Citrus fruits are rich in carotenoids. In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYb, EC:1.14.-.-) is a key regulatory enzyme in the catalysis of lycopene to β-carotene, an important dietary precursor of vitamin A for human nutrition. Two closely related lycopeneβ-cyclase cDNAs, designated CsLCYb1 and CsLCYb2, were isolated from the pulp of orange fruits (Citrus sinensis). The expression level of CsLCYb genes is lower in the lfavedo and juice sacs of a lycopene-accumulating genotype Cara Cara than that in common genotype Washington, and this might be correlated with lycopene accumulation in Cara Cara fruit. The CsLCYb1 efifciently converted lycopene into the bicyclicβ-carotene in an Escherichia coli expression system, but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into theβ-carotene and the monocyclic γ-carotene. In tomato transformation studies, expression of CsLCYb1 under the control of the caulilfower mosaic virus (CaMV) 35S constitutive promoter resulted in a virtually complete conversion of lycopene intoβ-carotene, and the ripe fruits displayed a bright orange colour. However, the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation. In fruits of the CsLCYb1 transgenic plants, most of the lycopene was converted intoβ-carotene with provitamin A levels reaching about 700 µg g-1 DW. Unexpectedly, most transgenic tomatoes showed a reduction in total carotenoid accumulation, and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits. Collectively, these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efifciency, and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

  18. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen

    2010-01-01

    cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  19. RNA-Seq analysis of high NaCl-induced gene expression.

    Science.gov (United States)

    Izumi, Yuichiro; Yang, Wenjing; Zhu, Jun; Burg, Maurice B; Ferraris, Joan D

    2015-10-01

    High extracellular NaCl is known to change expression of numerous genes, many of which are regulated by the osmoprotective transcription factor nuclear factor of activated T cells-5 (NFAT5). In the present study we employed RNA-Seq to provide a comprehensive, unbiased account of genes regulated by high NaCl in mouse embryonic fibroblast cells (MEFs). To identify genes regulated by NFAT5 we compared wild-type MEFs (WT-MEFs) to MEFs in which mutation of the NFAT5 gene inhibits its transcriptional activity (Null-MEFs). In WT-MEFs adding NaCl to raise osmolality from 300 to 500 mosmol/kg for 24 h increases expression of 167 genes and reduces expression of 412. Raising osmolality through multiple passages (adapted cells) increases expression of 196 genes and reduces expression of 528. In Null-MEFs, after 24 h of high NaCl, expression of 217 genes increase and 428 decrease, while in adapted Null-MEFs 143 increase and 622 decrease. Fewer than 10% of genes are regulated in common between WT- and null-MEFs, indicating a profound difference in regulation of high-NaCl induced genes induced by NFAT5 compared with those induced in the absence of NFAT5. Based on our findings we suggest a mechanism for this phenomenon, which had previously been unexplained. The NFAT5 DNA-binding motif (osmotic response element) is overrepresented in the vicinity of genes that NFAT5 upregulates, but not genes that it downregulates. We used Gene Ontology and manual curation to determine the function of the genes targeted by NFAT5, revealing many novel consequences of NFAT5 transcriptional activity.

  20. Lipopolysaccharide triggers nuclear import of Lpcat1 to regulate inducible gene expression in lung epithelia

    Institute of Scientific and Technical Information of China (English)

    Bryon; Ellis; Leah; Kaercher; Courtney; Snavely

    2012-01-01

    AIM:To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene tran-scription. METHODS:Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation. RESULTS:Lpcat1 translocates into the nucleus from thecytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli , two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overex-pressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.CONCLUSION:These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.

  1. Analysis of target genes induced by IL-13 cytotoxin in human glioblastoma cells.

    Science.gov (United States)

    Han, Jing; Yang, Liming; Puri, Raj K

    2005-03-01

    IL-13 cytotoxin comprised of IL-13 and a mutated form of Pseudomonas exotoxin (fusion protein termed IL-13-PE38QQR) has been shown to inhibit protein synthesis leading to necrotic and apoptotic cell death in glioblastoma cells that express high levels of interleukin-13 receptors (IL-13R). To identify target genes of cell death and other cellular genes with IL-13 receptors in glioblastoma cells, we utilized the cDNA microarrays to analyze global gene expression profiles after IL-13 cytotoxin and IL-13 treatment. IL-13 cytotoxin mediated cytotoxicity to U251 cells in a dose-dependent manner. Hierarchical cluster analysis of differentially expressed genes in U251 glioma cells at different time points after IL-13 cytotoxin treatment showed three major groups, each representing a specific expression pattern. Randomly selected differentially expressed genes from each group were confirmed by RT-PCR analysis. Most down-regulated genes belong to cell adhesion, motility, angiogenesis, DNA repair, and metabolic pathways. While up-regulated genes belong to cell cycle arrest, apoptosis, signaling and various metabolic pathways. Unexpectedly, at early time points, both IL-13 and IL-13 cytotoxin induced several genes belonging to different pathways most notably IL-8, DIO2, END1, and ALDH1A3 indicating that these genes are early response genes and their products may be associated with IL-13R. In addition, IL-13 cytotoxin induced IL-13Ralpha2 mRNA expression during the treatment in glioma cells. Our results indicate that novel cellular genes are involved with IL-13 receptors and that IL-13 cytotoxin induced cell death involves various target genes in human glioblastoma cells. On going studies will determine the role of associated genes and their products in the IL-13R functions in glioma cells.

  2. Fast and sensitive detection of indels induced by precise gene targeting

    DEFF Research Database (Denmark)

    Yang, Zhang; Steentoft, Catharina; Hauge, Camilla

    2015-01-01

    The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect...... and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect...

  3. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    Science.gov (United States)

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  4. Copper induces the expression of cholesterogenic genes in human macrophages.

    Science.gov (United States)

    Svensson, Per Arne; Englund, Mikael C O; Markström, Emilia; Ohlsson, Bertil G; Jernås, Margareta; Billig, Håkan; Torgerson, Jarl S; Wiklund, Olov; Carlsson, Lena M S; Carlsson, Björn

    2003-07-01

    Accumulation of lipids and cholesterol by macrophages and subsequent transformation into foam cells are key features in development of atherosclerosis. Serum copper concentrations have been shown to be associated with cardiovascular disease. However, the mechanism behind the proatherogenic effect of copper is not clear. We used DNA microarrays to define the changes in gene expression profile in response to copper exposure of human macrophages. Expression monitoring by DNA microarray revealed 91 genes that were regulated. Copper increased the expression of seven cholesterogenic genes (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, IPP isomerase, squalene synthase, squalene epoxidase, methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase) and low-density lipoprotein receptor (LDL-R), and decreased the expression of CD36 and lipid binding proteins. The expression of LDL-R and HMG CoA reductase was also investigated using real time PCR. The expression of both of these genes was increased after copper treatment of macrophages (Pmechanism for the association between copper and atherosclerosis. The effect of copper on cholesterogenic genes may also have implications for liver steatosis in early stages of Wilson's disease.

  5. Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis.

    Science.gov (United States)

    Guan, Chunfeng; Ji, Jing; Zhang, Xuqiang; Li, Xiaozhou; Jin, Chao; Guan, Wenzhu; Wang, Gang

    2015-03-01

    Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Fluoroquinolone-induced gene transfer in multidrug-resistant Salmonella

    Science.gov (United States)

    Fluoroquinolones are broad spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity. Bacterial exposure to fluoroquinolones can cause DNA damage and induce a bacterial SOS response to stimulate repair of damaged DNA. Certain prophages (integrated in bacterial chromosomes) ...

  7. Radiation-induced gene expression in the nematode caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, G.A.; Jones, T.A.; Chesnut, A.; Smith, A.L. [Loma Linda Univ., CA (United States)

    2002-12-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by reverse transcription polymerase chain reaction (RT-PCR) differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density. (author)

  8. Radiation-induced gene expression in the nematode Caenorhabditis elegans

    Science.gov (United States)

    Nelson, Gregory A.; Jones, Tamako A.; Chesnut, Aaron; Smith, Anna L.

    2002-01-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by RT-PCR differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density.

  9. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  10. Modulation of Gene Expression Networks underlying Realgar-Induced Differentiation of Acute Promyelocytic Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西

    2002-01-01

    Objective: To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. Methods: The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. Results: The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. Conclusion: The ubiquitin-proteasome degradation pathway may play an important role in the degradation of PML/RAR α fusion protein and the differentiation of NB4 cells.

  11. Conversational Telugu.

    Science.gov (United States)

    Beinstein, Judith; And Others

    The purpose of this text is to develop elementary conversational skills in Telugu. The language materials consist of four types of language learning activities. The first, and most predominant, is the unit microwave cycle. These cycles divide the learning process into two basic phases, the first of which involves mimicry, memorization, and…

  12. Conversion Disorder

    National Research Council Canada - National Science Library

    Fisher, Robert S; Stonnington, Cynthia M; Barry, John J

    2006-01-01

    ... to proceed after establishing a diagnosis of conversion disorder. Case Presentation "Ms. A," a 53-year-old left-handed woman, was admitted to our epilepsy monitoring unit for evaluation of a 4-month history of tremors, head bobbing, and episodic loss of awareness. The onset of these symptoms was 1 week after she had visited an emergency department...

  13. Tunable fringe magnetic fields induced by converse magnetoelectric coupling in a FeGa/PMN-PT multiferroic heterostructure

    Science.gov (United States)

    Fitchorov, Trifon; Chen, Yajie; Hu, Bolin; Gillette, Scott M.; Geiler, Anton; Vittoria, Carmine; Harris, Vincent G.

    2011-12-01

    The fringe magnetic field, induced by magnetoelectric coupling in a bilayer Fe-Ga/Pb(Mg1/3Nb2/3)O3_PbTiO3 (PMN-PT) multifunctional composite, was investigated. The induced external field is characterized as having a butterfly hysteresis loop when tuned by an applied electric field. A tuning coefficient of the electrically induced fringe magnetic field is derived from the piezoelectric and magnetostrictive properties of the composite. A measured maximum tuning coefficient, 4.5 Oe/(kV cm-1), is found to agree well with theoretical prediction. This work establishes a foundation in the design of transducers based on the magnetoelectric effect.

  14. Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.

    Directory of Open Access Journals (Sweden)

    Mykaella Andrade de Araújo

    Full Text Available Real-time quantitative RT-PCR (qPCR is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO model of mesial temporal lobe epilepsy (MTLE, we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA. In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.

  15. Differentially Gene Expression Profile Related to Inflammation in Endometrial Cells Induce by Lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Li-hua QIN; Ruo-guang WANG; Sheng LI; Chun-mei LI

    2009-01-01

    Objective To investigate differentially expressed genes related to inflammation in endometrial cells induced by Lipopolysaccharide(LPS).Methods Normal endometrium in the proliferative phase of specimen from 3 cases for the experiment was collected. The LPS group were treated with 50 μg/ml LPS. Total RNA was extracted using Trizol reagent from cells. RNA quality was assessed by determining the OD260/280 ratio by agarose gel electrophoresis, the chip was scanned by laser scanner. The acquired was analyzed. Results A total of differentially expressed genes were found, these genes were relative to many aspects. Among them, the expression of genes involved in inflammation were up-regulated by LPS, such as overexpression of lL-lβ, 8, etc.Conclusion The results indicates that inflammation-related genes may be one of the mechanisms of abnormal uterine bleeding by LPS-induced.

  16. Identification of in vivo-induced genes during infection of chickens with Salmonella enterica serovar Enteritidis.

    Science.gov (United States)

    Geng, Shizhong; Liu, Zhicheng; Lin, Zhijie; Barrow, Paul; Pan, Zhiming; Li, Qiuchun; Jiao, Xinan

    2015-06-01

    Chickens are an important source of food worldwide and are often infected with food-poisoning serovars of Salmonella enterica, frequently Salmonella Enteritidis (SE), without exhibiting clinical signs of disease. Ivi (in vivo induced) genes identified using in vivo-induced antigen technology (IVIAT) are expressed only during bacterial infection and have the potential value of identifying epidemic strains and antigens which can form the basis for sub-unit vaccine development. We applied IVIAT to SE strain 50041 and identified 42 ivi genes. Eight representative ivi genes were further confirmed by qRT-PCR as being expressed only in vivo within 48 h of infection compared with that of in vitro-cultured. Although our results indicated that the identified ivi genes are expressed only in vivo, further research is needed to elucidate the exact roles of these genes during infection and pathogenesis.

  17. Coordinate gene regulation by fimbriae-induced signal transduction

    DEFF Research Database (Denmark)

    Schembri, Mark; Klemm, Per

    2001-01-01

    of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated...

  18. Identification of genes induced by salt stress from Medicago ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-08

    Nov 8, 2010 ... Local Alignment Search Tool (BLAST) analysis of deduced protein sequences revealed that 35 .... uni-ESTs may be either new genes or segments unique to .... Yang QC, Wu MS, Wang QP, Kang JM (2005). Cloning ... Page 7 ...

  19. Early enriched environment induces an increased conversion of proBDNF to BDNF in the adult rat's hippocampus.

    Science.gov (United States)

    Cao, Wenyu; Duan, Juan; Wang, Xueqin; Zhong, Xiaolin; Hu, Zhaolan; Huang, Fulian; Wang, Hongtao; Zhang, Juan; Li, Fang; Zhang, Jianyi; Luo, Xuegang; Li, Chang-Qi

    2014-05-15

    An enriched environment has been shown to influence brain plasticity and function by involving the action of brain-derived neurotrophic factor (BDNF). BDNF, which is synthesized as a precursor molecule (proBDNF) that undergoes proteolytic cleavage, plays an important role in synaptic plasticity and contributes to several brain functions such as memory, learning, and behavior. The neurotrophins and proneurotrophins often play opposite roles in the brain, suggesting that proteolytic cleavage of proneurotrophins controls the action of neurotrophins. However, few studies have focused on the expression and cleavage of proBDNF after exposure to an enriched environment. Our study aimed to explore the effects of an early-enriched environment on the conversion of proBDNF to BDNF in the adult rats' hippocampus. We found that there was no difference in the expression of proBDNF in the hippocampus between the SE (standard environment) and EE (enriched environment) rats, but a significantly increased BDNF protein level was found in the EE rats. Thus, a remarkably enhanced ratio of BDNF to proBDNF (BDNF/proBDNF) was observed in the EE rats. In addition, the EE resulted in a remarkably up-regulated matrix metalloproteinase-9 (MMP-9) in the hippocampus, which played a key role in converting proBDNF to BDNF in the extracellular space. Furthermore, the expression of synapse-related proteins (NR1 and NR2A) was analyzed, and the results indicated that EE could significantly increase the expression of NR1 and NR2A in the hippocampus. In addition, the behavioral results showed that EE reduced anxiety-like behavior in the elevated-plus maze test and reduced immobility time in the forced swimming test. Moreover, the EE resulted in an increased preference for sucrose compared to the SE. These results suggested that the EE up-regulated MMP-9 levels within the hippocampus, which might facilitate the conversion of proBDNF to BDNF, thereby contributing to the long lasting alterations of

  20. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  1. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  2. Plasmon-induced selective carbon dioxide conversion on earth-abundant aluminum-cuprous oxide antenna-reactor nanoparticles.

    Science.gov (United States)

    Robatjazi, Hossein; Zhao, Hangqi; Swearer, Dayne F; Hogan, Nathaniel J; Zhou, Linan; Alabastri, Alessandro; McClain, Michael J; Nordlander, Peter; Halas, Naomi J

    2017-06-21

    The rational combination of plasmonic nanoantennas with active transition metal-based catalysts, known as 'antenna-reactor' nanostructures, holds promise to expand the scope of chemical reactions possible with plasmonic photocatalysis. Here, we report earth-abundant embedded aluminum in cuprous oxide antenna-reactor heterostructures that operate more effectively and selectively for the reverse water-gas shift reaction under milder illumination than in conventional thermal conditions. Through rigorous comparison of the spatial temperature profile, optical absorption, and integrated electric field enhancement of the catalyst, we have been able to distinguish between competing photothermal and hot-carrier driven mechanistic pathways. The antenna-reactor geometry efficiently harnesses the plasmon resonance of aluminum to supply energetic hot-carriers and increases optical absorption in cuprous oxide for selective carbon dioxide conversion to carbon monoxide with visible light. The transition from noble metals to aluminum based antenna-reactor heterostructures in plasmonic photocatalysis provides a sustainable route to high-value chemicals and reaffirms the practical potential of plasmon-mediated chemical transformations.Plasmon-enhanced photocatalysis holds promise for the control of chemical reactions. Here the authors report an Al@Cu2O heterostructure based on earth abundant materials to transform CO2 into CO at significantly milder conditions.

  3. Unraveling the Fundamental Mechanisms of Solvent-Additive-Induced Optimization of Power Conversion Efficiencies in Organic Photovoltaic Devices.

    Science.gov (United States)

    Herath, Nuradhika; Das, Sanjib; Zhu, Jiahua; Kumar, Rajeev; Chen, Jihua; Xiao, Kai; Gu, Gong; Browning, James F; Sumpter, Bobby G; Ivanov, Ilia N; Lauter, Valeria

    2016-08-10

    The realization of controllable morphologies of bulk heterojunctions (BHJ) in organic photovoltaics (OPVs) is one of the key factors enabling high-efficiency devices. We provide new insights into the fundamental mechanisms essential for the optimization of power conversion efficiencies (PCEs) with additive processing to PBDTTT-CF:PC71BM system. We have studied the underlying mechanisms by monitoring the 3D nanostructural modifications in BHJs and correlated the modifications with the optical analysis and theoretical modeling of charge transport. Our results demonstrate profound effects of diiodooctane (DIO) on morphology and charge transport in the active layers. For small amounts of DIO (3 vol %), DIO facilitates a loosely packed mixed morphology with large clusters of PC71BM, leading to deterioration in PCE. Theoretical modeling of charge transport reveals that DIO increases the mobility of electrons and holes (the charge carriers) by affecting the energetic disorder and electric field dependence of the mobility. Our findings show the implications of phase separation and carrier transport pathways to achieve optimal device performances.

  4. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  5. Changes in gene expression induced by Micro-Immunotherapy

    Directory of Open Access Journals (Sweden)

    Maurice Jeaner

    2012-09-01

    Full Text Available Background: Metabolic syndrome (MS is a metabolic disorder associated with obesity, type-II diabetes, and “low grade inflammation”, with the concomitant increased risk of cardiovascular events. As a chronic inflammatory process, MS results in a dysregulation of the cytokine profile. 2L®INFLAM, a Micro-immunotherapy (MI medication formulated with highly diluted cytokines, is currently prescribed in Belgium for inflammatory diseases and potentially may be helpful for MS patients. Aims: To investigate the impact of 2L®INFLAM on selected gene expression markers (mRNA in patients suffering from MS, in addition to biological and clinical parameters. Methodology: Four well characterized MS adult patients with stabilized body-weight were advised to take one capsule of 2L®INFLAM per day (by sublingual-oral route for 6 months (composition in table 1. Concomitantly to biological and clinical examination, genes expression status was assessed by a DNA microarray technology (Oxygen™ comprising 200 genes involved mainly in oxidative stress and inflammation. Whole blood collection was performed before and after treatment (3-6 months and mRNA levels measured. Gene expression was classified in 3 series (normally expressed, up or down-regulated and genes related to diabetes predisposition were scored by using a proprietary Diascore (Probiox. Results: Before MI medication, a significant percentage of dysregulated genes (median: 16.3% as well as a positive Diascore (median: 1.6 were noticed. Impressive correction of dysregulated genes (reaching 90% for one patient was observed after 3 months of treatment (median: 2.3% in addition to an improvement of Diascore in 3 MS patients out of 4 (median: 0.5. During the same period, both clinical and biological parameters remained unchanged. Conclusions: MS patients showing a high level of gene dysregulation efficiently normalized after 3 months of 2L

  6. Topical report on a preconceptual design for the Spallation-Induced Lithium Conversion (SILC) target for the accelerator production of tritium (APT)

    Energy Technology Data Exchange (ETDEWEB)

    Van Tuyle, G.J.; Cokinos, D.M.; Czajkowski, C.; Franz, E.M.; Kroeger, P.; Todosow, M.; Youngblood, R.; Zucker, M.

    1993-09-30

    The preconceptual design of the APT Li-Al target system, also referred to as the Spallation-Induced Lithium Conversion (SILC), target system, is summarized in this report. The system has been designed to produce a ``3/8 Goal`` quantity of tritium using the 200-mA, 1.0 GeV proton beam emerging from the LANL-designed LINAC. The SILC target system consists of a beam expander, a heavy-water-cooled lead spallation neutron source assembly surrounded by light-water-cooled Li-Al blankets, a target window, heat removal systems, and related safety systems. The preconceptual design of each of these major components is described. Descriptions are also provided for the target fabrication, tritium extraction, and waste-steam processes. Performance characteristics are presented and discussed.

  7. Molecular cloning and characterization of two inducible NAD⁺-adh genes encoding NAD⁺-dependent alcohol dehydrogenases from Acetobacter pasteurianus SKU1108.

    Science.gov (United States)

    Masud, Uraiwan; Matsushita, Kazunobu; Theeragool, Gunjana

    2011-11-01

    The cytosolic NAD⁺-dependent alcohol dehydrogenases (NAD⁺-ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The adhI and adhII genes, which encode NAD⁺-ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD⁺-ADHs. The NAD⁺-adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli, ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81-105 bp and 74-92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.

  8. Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5.

    Science.gov (United States)

    Buxadé, Maria; Lunazzi, Giulia; Minguillón, Jordi; Iborra, Salvador; Berga-Bolaños, Rosa; Del Val, Margarita; Aramburu, José; López-Rodríguez, Cristina

    2012-02-13

    Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

  9. System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens.

    Science.gov (United States)

    Kubo, Minoru; Imai, Akihiro; Nishiyama, Tomoaki; Ishikawa, Masaki; Sato, Yoshikatsu; Kurata, Tetsuya; Hiwatashi, Yuji; Reski, Ralf; Hasebe, Mitsuyasu

    2013-01-01

    Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

  10. Interferon induced IFIT family genes in host antiviral defense

    Science.gov (United States)

    Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IF stimulated ...

  11. Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo.

    Science.gov (United States)

    Dolan, Eimear B; Tallon, David; Cheung, Wing-Yee; Schaffler, Mitchell B; Kennedy, Oran D; McNamara, Laoise M

    2016-06-01

    Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague-Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes (Rankl, Opg and M-csf), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production (Cox2), vascularization (Vegf) and inflammatory (Il1a) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile.

  12. Virus-induced opposite effect on Bombyx mori gene transcriptions

    Directory of Open Access Journals (Sweden)

    Y Yin

    2016-09-01

    Full Text Available Bombyx mori bidensovirus (BmBDV and Bombyx mori nucleopolyhedrovirus (BmNPV are serious pathogens of Bombyx mori. In this study, we reported the changes of transcription level of several immune genes, including bmi, argo, dicer, cap1, cap3 and car, in Bombyx mori midgut after exposure to BmBDV or BmNPV. Silkworm strains 798 (anti-BmBDV and 306 (susceptible to BmBDV were subjected to BmBDV infection, and NB (anti-BmNPV and HUABA (35 (susceptible to BmNPV were subjected to BmNPV infection. The results showed that the transcription levels differ largely among different silkworm strains, and that the extent to which the gene transcriptions were affected by the viruses was different. However, both BmNPV and BmBDV viruses can reverse the transcription patterns of these genes when the silkworms were administered with the viruses compared with those control groups. The transcript levels of bmi and dicer were decreased in 798 and 306 strains that were inoculated with BmBDV compared with their respective controls, but were increased in NB and HUABA (35 inoculated with BmNPV. The transcript levels of argo and cap3 were risen in 798, 306 and NB strains when inoculated with their respective viruses, but were decreased in HUABA (35 strain. The transcript levels of cap1 were risen in all silkworm strains, while the levels of car were decreased in 798, 306 and HUABA (35 strains, and increased in NB strain when inoculated with their respective viruses. These findings may contribute to more in-depth understanding on functions of these genes in virus infection and proliferation.

  13. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  14. Exploring Mycobacterium tuberculosis infection-induced alterations in gene expression in macrophage by microarray hybridization

    Institute of Scientific and Technical Information of China (English)

    XIE; Jianping; (谢建平); LI; Yao; (李; 瑶); YUE; Jun; (乐; 军); XU; Yongzhong; (徐永忠); HUANG; Daqiang; (黄达蔷); LIANG; Li; (梁; 莉); WANG; Honghai; (王洪海)

    2003-01-01

    Tuberculosis remains a serious threat to public health. Its causative agent Mycobacte- rium tuberculosis is an intracellular pathogen which survives and replicates within cells of the host immune system, primarily macrophages. Knowledge of the bacteria-macrophage interaction can help to develop novel measures to combat the disease. The global gene expression of macro- phage following invasion by and growth of M. tuberculosis was studied by cDNA microarray. Of the 12800 human genes analyzed, totally 473 (3.7%) macrophage genes were differentially expressed after being infected by M. tuberculosis, among which, only 25 (5.2%, corresponding to less than 0.2% of the 12800 genes) genes were up-regulated, while others (94.8%) were down-regulated against the control. Of the 473 genes, 376 genes are registered in the GenBank, and 97 are novel genes. Expression of 5 up-regulated genes has been induced by more than 3-fold. 25 genes were down-regulated by more than 3-fold. Syndecan binding protein has been down-regu- lated up to 12.5-fold. The data gave an insight into the early gene expression in macrophage ensuing M. tuberculosis infection and a basis for further study.

  15. Myostatin gene mutated mice induced with tale nucleases.

    Science.gov (United States)

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  16. Conversion Disorder

    Directory of Open Access Journals (Sweden)

    Yacov Rofé

    2013-11-01

    Full Text Available Conversion disorder remains a mystery that has only become more complicated with the decline of the scientific status of psychoanalysis (e.g., Piper, Lillevik, & Kritzer, 2008; Rofé, 2008 and recent neurological findings suggest that this behavior is controlled by biological mechanisms (van Beilen, Vogt, & Leenders, 2010. Moreover, existing theories have difficulty explaining the efficacy of various interventions, such as psychoanalysis, behavior therapy, drug therapy and religious therapy. This article reviews research and clinical evidence pertaining to both the development and treatment of conversion disorder and shows that this seemingly incompatible evidence can be integrated within a new theory, the Rational-Choice Theory of Neurosis (RCTN; Rofé, 2010. Despite the striking differences, RCTN continues Freud's framework of thinking as it employs a new concept of repression and replaces the unconscious with self-deception. Moreover, it incorporates Freud's idea, implicitly expressed in his theory, that neurotic disorders are, in fact, rational behaviors.

  17. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    asy gene is a novel apoptosis-inducing gene,but its mechanism is unclear.To investigate the mechanism of asy inducing apoptosis,a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system.The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels.Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end,and share a common open reading frame encoding a polypeptide of 236 amino acids.Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence.Two highly hydrophobic regions encoding potentially two transmembrane domains are present.The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu).Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells.Compared with asy gene,overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

  18. Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

    Directory of Open Access Journals (Sweden)

    David Proud

    Full Text Available Human rhinovirus (HRV infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

  19. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

    Directory of Open Access Journals (Sweden)

    Hiroyasu Kamei

    Full Text Available BACKGROUND: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker. CONCLUSIONS/SIGNIFICANCE: These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic

  20. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  1. Conversational sensing

    Science.gov (United States)

    Preece, Alun; Gwilliams, Chris; Parizas, Christos; Pizzocaro, Diego; Bakdash, Jonathan Z.; Braines, Dave

    2014-05-01

    Recent developments in sensing technologies, mobile devices and context-aware user interfaces have made it pos- sible to represent information fusion and situational awareness for Intelligence, Surveillance and Reconnaissance (ISR) activities as a conversational process among actors at or near the tactical edges of a network. Motivated by use cases in the domain of Company Intelligence Support Team (CoIST) tasks, this paper presents an approach to information collection, fusion and sense-making based on the use of natural language (NL) and controlled nat- ural language (CNL) to support richer forms of human-machine interaction. The approach uses a conversational protocol to facilitate a ow of collaborative messages from NL to CNL and back again in support of interactions such as: turning eyewitness reports from human observers into actionable information (from both soldier and civilian sources); fusing information from humans and physical sensors (with associated quality metadata); and assisting human analysts to make the best use of available sensing assets in an area of interest (governed by man- agement and security policies). CNL is used as a common formal knowledge representation for both machine and human agents to support reasoning, semantic information fusion and generation of rationale for inferences, in ways that remain transparent to human users. Examples are provided of various alternative styles for user feedback, including NL, CNL and graphical feedback. A pilot experiment with human subjects shows that a prototype conversational agent is able to gather usable CNL information from untrained human subjects.

  2. Cytokines inducing bone marrow SCA+ cells migration into pancreatic islet and conversion into insulin-positive cells in vivo.

    Directory of Open Access Journals (Sweden)

    LuGuang Luo

    Full Text Available We hypothesize that specific bone marrow lineages and cytokine treatment may facilitate bone marrow migration into islets, leading to a conversion into insulin producing cells in vivo. In this study we focused on identifying which bone marrow subpopulations and cytokine treatments play a role in bone marrow supporting islet function in vivo by evaluating whether bone marrow is capable of migrating into islets as well as converting into insulin positive cells. We approached this aim by utilizing several bone marrow lineages and cytokine-treated bone marrow from green fluorescent protein (GFP positive bone marrow donors. Sorted lineages of Mac-1(+, Mac-1(-, Sca(+, Sca(-, Sca(-/Mac-1(+ and Sca(+/Mac-1(- from GFP positive mice were transplanted to irradiated C57BL6 GFP negative mice. Bone marrow from transgenic human ubiquitin C promoter GFP (uGFP, with strong signal C57BL6 mice was transplanted into GFP negative C57BL6 recipients. After eight weeks, migration of GFP positive donor' bone marrow to the recipient's pancreatic islets was evaluated as the percentage of positive GFP islets/total islets. The results show that the most effective migration comes from the Sca(+/Mac(- lineage and these cells, treated with cytokines for 48 hours, were found to have converted into insulin positive cells in pancreatic islets in vivo. This study suggests that bone marrow lineage positive cells and cytokine treatments are critical factors in determining whether bone marrow is able to migrate and form insulin producing cells in vivo. The mechanisms causing this facilitation as well as bone marrow converting to pancreatic beta cells still need to be investigated.

  3. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  4. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  5. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    Science.gov (United States)

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. Copyright © 2016

  6. Identification of antiviralrelevant genes in the cultured fish cells induced by UV-inactivated virus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antivi-ral-relevant genes. In this study, suppressive subtractive hy-bridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich ele-ment in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

  7. Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis.

    Science.gov (United States)

    van der Linde, Karina; Kastner, Christine; Kumlehn, Jochen; Kahmann, Regine; Doehlemann, Gunther

    2011-01-01

    Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.

  8. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Institute of Scientific and Technical Information of China (English)

    Yuan Li; Dan Luo; Hui-Fen Yue; Li-Sheng Zhang; Jian-Ren Gu; Da-Fang Wan; Jian-Jia Su; Ji Cao; Chao Ou; Xiao-Kun Qiu; Ke-Chen Ban; Chun Yang; Liu-Liang Qin

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1),to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism.METHODS: Tree shrews ( 7upaia belangeri chinensis)were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding noncancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared.RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC,genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC.CONCLUSION: A considerable number of genes could change

  9. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

    Science.gov (United States)

    Chandrakanthan, Vashe; Yeola, Avani; Kwan, Jair C.; Oliver, Rema A.; Qiao, Qiao; Kang, Young Chan; Zarzour, Peter; Beck, Dominik; Boelen, Lies; Unnikrishnan, Ashwin; Villanueva, Jeanette E.; Nunez, Andrea C.; Knezevic, Kathy; Palu, Cintia; Nasrallah, Rabab; Carnell, Michael; Macmillan, Alex; Whan, Renee; Yu, Yan; Hardy, Philip; Grey, Shane T.; Gladbach, Amadeus; Delerue, Fabien; Ittner, Lars; Mobbs, Ralph; Walkley, Carl R.; Purton, Louise E.; Ward, Robyn L.; Wong, Jason W. H.; Hesson, Luke B.; Walsh, William; Pimanda, John E.

    2016-01-01

    Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration. PMID:27044077

  10. Laser-induced breakdown and damage generation by nonlinear frequency conversion in ferroelectric crystals: Experiment and theory

    Energy Technology Data Exchange (ETDEWEB)

    Louchev, Oleg A.; Saito, Norihito; Wada, Satoshi [Center for Advanced Photonics, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Hatano, Hideki; Kitamura, Kenji [National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

    2013-11-28

    Using our experimental data for ns pulsed second harmonic generation (SHG) by periodically poled stoichiometric LiTaO{sub 3} (PPSLT) crystals, we consider in detail the mechanism underlying laser-induced damage in ferroelectric crystals. This mechanism involves generation and heating of free electrons, providing an effective kinetic pathway for electric breakdown and crystal damage in ns pulsed operation via combined two-photon absorption (TPA) and induced pyroelectric field. In particular, a temperature increase in the lattice of ≈1 K induced initially by ns SHG and TPA at the rear of operating PPSLT crystal is found to induce a gradient of spontaneous polarization generating a pyroelectric field of ≈10 kV/cm, accelerating free electrons generated by TPA to an energy of ≈10 eV, followed by impact ionization and crystal damage. Under the damage threshold for ns operation, the impact ionization does not lead to the avalanche-like increase of free electron density, in contrast to the case of shorter ps and fs pulses. However, the total number of collisions by free electrons, ≈10{sup 18} cm{sup −3} (generated during the pulse and accelerated to the energy of ≈10 eV), can produce widespread structural defects, which by entrapping electrons dramatically increase linear absorption for both harmonics in subsequent pulses, creating a positive feedback for crystal lattice heating, pyroelectric field and crystal damage. Under pulse repetition, defect generation starting from the rear of the crystal can propagate towards its center and front side producing damage tracks along the laser beam and stopping SHG. Theoretical analysis leads to numerical estimates and analytical approximation for the threshold laser fluence for onset of this damage mechanism, which agree well with our (i) experiments for the input 1064 nm radiation in 6.8 kHz pulsed SHG by PPSLT crystal, (ii) pulsed low frequency 532 nm radiation transmission experiments, and also (iii) with the data

  11. Gene expression in rat skin induced by irritating chemicals.

    Science.gov (United States)

    Rogers, James V; Garrett, Carol M; McDougal, James N

    2003-01-01

    Occupational skin disease is the second most significant cause of occupational disease, after accidents. Irritation from occupational chemicals such as solvents, hydrocarbons, and surfactants are one cause of this disease. Gene expression studies provide useful information about normal processes in the skin and responses of the skin to exogenous chemicals. We exposed rats, cutaneously, to sodium lauryl sulfate (SLS, 1% and 10% aqueous solution), m-xylene (pure liquid), and d-limonene (pure liquid) for 1 h and measured transcriptional responses at the end of the exposure and 3 h later for comparison with untreated skin samples. Total skin RNA was isolated and analyzed using the Affymetrix RatTox U34 array. Using the Affymetrix software, we found that 234 of approximately 850 genes were detected as present in at least 80% of the normal skin samples. The largest number of these genes was related to metabolism, oxidative/cellular stress, and signal transduction. Limonene caused the largest change in mRNA levels with a total of 34 increased transcripts and 4 decreased transcripts. Xylene treatment resulted in 6 increased transcripts and 14 decreased transcripts, while 10% SLS caused 5 transcripts to increase and 17 to decrease. Only two transcripts were observed to change in skin following a 1% SLS exposure. Sodium lauryl sulfate transcript changes increased with dose and were maximum at 4 h. Limonene transcript changes were more numerous at 1 h than at 4 h. The observed differences may reflect different mechanisms of irritation. Copyright 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:123-137, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10079

  12. Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

    Science.gov (United States)

    Bharti, Poonam; Jyoti, Poonam; Kapoor, Priya; Sharma, Vandana; Shanmugam, V; Yadav, Sudesh Kumar

    2017-08-01

    This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

  13. Identification of Candida albicans genes induced during thrush offers insight into pathogenesis.

    Science.gov (United States)

    Cheng, Shaoji; Clancy, Cornelius J; Checkley, Mary Ann; Handfield, Martin; Hillman, Jeffrey D; Progulske-Fox, Ann; Lewin, Alfred S; Fidel, Paul L; Nguyen, M Hong

    2003-06-01

    Candida albicans causes a wide spectrum of diseases, ranging from mucocutaneous infections like oral thrush to disseminated candidiasis. Screening for C. albicans genes expressed within infected hosts might advance understanding of candidal pathogenesis, but is impractical using existing techniques. In this study, we used an antibody-based strategy to identify C. albicans genes expressed during thrush. We adsorbed sera from HIV-infected patients with thrush against candidal cells grown in vitro and screened a C. albicans genomic expression library. We identified 10 genes encoding immunogenic antigens and used reverse transcription-polymerase chain reaction to verify that they were induced within thrush pseudomembranes recovered from a patient. The in vivo induced genes are involved in diverse functions, including regulation of yeast-hyphal morphogenesis, adhesion to host cells, nutrient uptake, phospholipid biosynthesis and amino acid catabolism. Four genes encode known virulence determinants (HWP1, CST20, CPP1 and RBF1). Another gene, LPD1, for which a role in candidal pathogenesis is unknown, encodes a protein homologous to a bacterial virulence determinant. Most importantly, disruption of CaNOT5, a newly identified gene, conferred defects in morphogenesis, decreased adherence to human buccal epithelial cells and attenuated mortality during murine disseminated candidiasis, proving that our strategy can identify genes encoding novel virulence determinants.

  14. Identification of Differently Expressed Genes in Chemical Carcinogen-induced Rat Bladder Cancers

    Institute of Scientific and Technical Information of China (English)

    Guangfu CHEN; Franky L. CHAN; Xu ZHANG; Peter S.F. CHAN

    2009-01-01

    Possible altered gene expression patterns in bladder turnout carcinogenesis in rat bladder cancers induced by BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine] was examined by cDNA microarray analysis of gene expression profiles.Thirty Sprague-Dawley rats were given drinking water containing 0.05% BBN ad libitum for 24 to 28-weeks.Equal numbers of control rats were given tap water without BBN.After treatment,the rat bladders were excised for RNA extraction and histopathological examinations.Total RNAs were extracted from rat transitional cell carcinoma (TCC) tissues and micro-dissected normal rat bladder epithelia.The atlas glass rat microarray was used,which included oligonucleotides of 1081 rat genes.Some of the up-regulated genes in rat bladder TCCs were further confirmed by Northern blotting.Our results showed that the transcriptions of 30 genes were significantly elevated in the rat bladder TCCs,and these included fly proto-oncogene,Lipocortin 2,COX Ⅳ,COX Ⅴ a,and cathepsin D.Also,15 genes were significantly down-regulated in the rat bladder TCCs and they included B7.1,TNFrl,APOAI and VHL.The resuits of cDNA microarray analysis demonstrated that normal rat bladder epithelia and bladder TCC exhibited different and specific gene statement profiles.The increased expressions of the identified genes may play an important role in the chemically induced bladder carcinogenesis.

  15. TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapy-induced apoptosis

    DEFF Research Database (Denmark)

    Davidsen, Marie Louise; Würts, S.Ø.; Rømer, Maria Unni Koefoed;

    2006-01-01

    in cancer. In this regard, several studies have demonstrated an antiapoptotic effect of TIMP-1 in a number of different cell types. Since chemotherapy works by inducing apoptosis in cancer cells, we raised the hypothesis that TIMP-1 promotes resistance against chemotherapeutic drugs. In order to investigate...... this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene...... deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells...

  16. The model of defense gene expression induced by signaling molecule β-ocimene

    Institute of Scientific and Technical Information of China (English)

    LIU Chunlin; RUAN Ying; GUAN Chunyun

    2004-01-01

    @@ β-ocimene, a kind of monoterpene, was found recently as a plant communication signal molecule[1]. It has two isomeric forms in nature: cis-β-ocimene and trans-β- ocimene. According to recent reports, all investigated plants, such as corn, cotton, lima bean, potato, tobacco, arabidopsis, and Mediterranean pine, could release the chemical component β-ocimene after fed by arthropod herbivores[2-5], suggesting thatβ-ocimene is an important functioal component in the herbivore-induced volatile. Nowadays, we know that β-ocimene can induce the expression of defense genes relative to salicylic acid in detatched leaves. But many problems of β-ocimene, for example, whether β-ocimene can induce the defense gene expression in intact plants, what role it can play in the expression model of defense genes, are elusive[1,6].

  17. Sleep deprivation attenuates endotoxin-induced cytokine gene expression independent of day length and circulating cortisol in male Siberian hamsters (Phodopus sungorus).

    Science.gov (United States)

    Ashley, Noah T; Walton, James C; Haim, Achikam; Zhang, Ning; Prince, Laura A; Fruchey, Allison M; Lieberman, Rebecca A; Weil, Zachary M; Magalang, Ulysses J; Nelson, Randy J

    2013-07-15

    Sleep is restorative, whereas reduced sleep leads to negative health outcomes, such as increased susceptibility to disease. Sleep deprivation tends to attenuate inflammatory responses triggered by infection or exposure to endotoxin, such as bacterial lipopolysaccharide (LPS). Previous studies have demonstrated that Siberian hamsters (Phodopus sungorus), photoperiodic rodents, attenuate LPS-induced fever, sickness behavior and upstream pro-inflammatory gene expression when adapted to short day lengths. Here, we tested whether manipulation of photoperiod alters the suppressive effects of sleep deprivation upon cytokine gene expression after LPS challenge. Male Siberian hamsters were adapted to long (16 h:8 h light:dark) or short (8 h:16 h light:dark) photoperiods for >10 weeks, and were deprived of sleep for 24 h using the multiple platform method or remained in their home cage. Hamsters received an intraperitoneal injection of LPS or saline (control) 18 h after starting the protocol, and were killed 6 h later. LPS increased liver and hypothalamic interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF) gene expression compared with vehicle. Among LPS-challenged hamsters, sleep deprivation reduced IL-1 mRNA levels in liver and hypothalamus, but not TNF. IL-1 attenuation was independent of circulating baseline cortisol, which did not increase after sleep deprivation. Conversely, photoperiod altered baseline cortisol, but not pro-inflammatory gene expression in sleep-deprived hamsters. These results suggest that neither photoperiod nor glucocorticoids influence the suppressive effect of sleep deprivation upon LPS-induced inflammation.

  18. Muscle Contraction Induces Acute Hydroxymethylation of the Exercise-Responsive Gene Nr4a3

    DEFF Research Database (Denmark)

    Pattamaprapanont, Pattarawan; Garde, Christian; Fabre, Odile

    2016-01-01

    promoters. Exercise induces dynamic DNA demethylation at gene promoters; however, the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a muscle contraction model that allows for the collection of samples that are repeatedly...... stimulated over time is required to determine whether contraction-induced demethylation is preceded by changes in the hydroxymethylcytosine level. Here, we established an acute skeletal muscle contraction model to mimic the effects of acute exercise on gene expression. We used this model to investigate...... the effect of muscle contraction on DNA demethylation and hydroxymethylation. First, we performed an acute exercise study in healthy humans to identify an exercise-responsive gene that we could study in culture. We identified the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene with the highest...

  19. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

    Science.gov (United States)

    Kato, Lucia; Begum, Nasim A; Burroughs, A Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-02-14

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

  20. µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1 into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Juan García Valero

    Full Text Available Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

  1. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  2. Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

    OpenAIRE

    Izumi, Kenichi; Mine, Keiichiro; Inoue, Yoshitaka; Teshima, Miho; Ogawa, Shuichiro; Kai, Yuji; Kurafuji, Toshinobu; Hirakawa, Kanako; Miyakawa, Daiki; Ikeda, Haruka; Inada, Akari; Hara, Manami; Yamada, Hisakata; Akashi, Koichi; Niho, Yoshiyuki

    2015-01-01

    Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 backgrou...

  3. Stable oncogenic transformation induced by microcell-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    吕有勇; Donald G.Blair

    1995-01-01

    Oncogenes have been identified using DNA-mediated transfection, but the size of the transferable and unrearranged DNA, gene rearrangement and amplification which occur during the transfection process limit the use of the techniques. We have evaluated microcell-mediated gene transfer techniques for the transfer and analysis of dominant oncogenes. MNNG-HOS, a transformed human cell line which contained the met oncogene mapping to human chromosome 7 was infected with retroviruses carrying drug resistance markers and used to optimize microcell preparation and transfer. Stable and drug-resistant hybrids containing single human chromosomes as well as the foci of the transformed cells containing the activated met oncogene and intact hitman chromosomes were obtained. Hybridization analysis with probes (i.e. collA2, pJ3.11) mapping up to 1 Mb away from met shows that the cells from the individual focr contain different amounts of apparently unrearranged human DNA associated with the oncogene, and the microcell-g

  4. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Science.gov (United States)

    Li, Yuan; Wan, Da-Fang; Su, Jian-Jia; Cao, Ji; Ou, Chao; Qiu, Xiao-Kun; Ban, Ke-Chen; Yang, Chun; Qin, Liu-Liang; Luo, Dan; Yue, Hui-Fen; Zhang, Li-Sheng; Gu, Jian-Ren

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could

  5. ohr, Encoding an Organic Hydroperoxide Reductase, Is an In Vivo-Induced Gene in Actinobacillus pleuropneumoniae

    OpenAIRE

    Shea, Robin J.; Mulks, Martha H.

    2002-01-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused in part by neutrophil degranulation. In an effort to understand the pathogenesis of this disease, we have developed an in vivo expression technology (IVET) system to identify genes that are specifically up-regulated during infection. One of the genes that we have identified as being induced in vivo is ohr, encoding organic hydroperoxide reducta...

  6. Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

    Science.gov (United States)

    Civitarese, Anthony E; Hesselink, Matthijs K C; Russell, Aaron P; Ravussin, Eric; Schrauwen, Patrick

    2005-12-01

    Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 +/- 0.6 yr; body mass index: 23.8 +/- 1.0 kg/m(2); maximal O(2) consumption: 3.85 +/- 0.21 l/min). Plasma FFA concentration increased during exercise (P glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P expression of pyruvate dehydrogenase kinase-4 (P glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-alpha(2); P glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.

  7. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  8. Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue.

    Science.gov (United States)

    Dai, Yumei; Toley, Bhushan J; Swofford, Charles A; Forbes, Neil S

    2013-06-01

    Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity. The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cell communication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the PBAD promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies.

  9. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    Science.gov (United States)

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  10. Gene expression in rats with Barrett's esophagus and esophageal adenocarcinoma induced by gastroduodenoesophageal reflux

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Jie Chen; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To study the different gene expression profiles in rats with Barrett's esophagus (BE) and esophageal adenocarcinoma (EA) induced by gastro-duodenoesophageal reflux.METHODS: Esophagoduodenostomy was performed in 8-wk old Sprague-Dawley rats to induce gastro-duodenoesophageal reflux, and a group of rats that received sham operation served as control. Esophageal epithelial pathological tissues were dissected and frozen in liquid nitrogen immediately. The expression profiles of 4 096genes in EA and BE tissues were compared to normal esophagus epithelium in normal control (NC) by cDNA microarray.RESULTS: Four hundred and forty-eight genes in BE were more than three times different from those in NC, including 312 upregulated and 136 downregulated genes. Three hundred and seventy-seven genes in EA were more than three times different from those in NC, including 255upregulated and 142 downregulated genes. Compared to BE, there were 122 upregulated and 156 downregulated genes in EA. In the present study, the interested genes were those involved in carcinogenesis. Among them, the upregulated genes included cathepsin C, aminopeptidase M, arachidonic acid epoxygenase, tryptophan-2,3-dioxygenase, ubiquitin-conjugating enzyme, cyclic GMP-stimulated phosphodiesterase, tissue inhibitor of metalloproteinase-1, betaine-homocysteine methyltransferase, lysozyme, complement 4b binding protein,complement 9 protein, insulin-like growth factor binding protein, UDP-glucuronosyltransferase, tissue inhibitor of metalloproteinase-3, aldolase B, retinoid X receptor gamma, carboxylesterase and testicular cell adhesion molecule 1. The downregulated genes included glutathione synthetase, lecithin-cholesterol acyltransferase, p55CDC,heart fatty acid binding protein, cell adhesion regulator and endothelial cell selectin ligand.CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux may develop into BE and even EA gradually. The gene

  11. Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology.

    Science.gov (United States)

    Guo, Yan; Xie, Xiaojia; Guo, Changqing; Wang, Zhaoyang; Liu, Qingguo

    2015-06-01

    To explore electro-acupuncture's (EA's) effect on gene expression in heart of rats with stress-induced pre-hypertension and try to reveal its biological mechanism based on gene chip technology. Twenty-seven Wistar male rats were randomly divided into 3 groups. The stress-induced hypertensive rat model was prepared by electric foot-shocks combined with generated noise. Molding cycle lasted for 14 days and EA intervene was applied,on rats in model + EA group during model preparation. Rat Gene 2.0 Sense Target Array technology was used for the determination of gene expression profiles and the screened key genes were verified by real-time quantitative polymerase chain reaction (RT-PCR) method. Compared with blank control group, 390 genes were changed in model group; compared with model control group, 330 genes were changed in model+EA group. Significance analysis of gene function showed that the differentially expressed genes are those involved in biological process, molecular function and cellular components. RT-PCR result of the screened key genes is consistent with that of gene chip test. EA could significantly lower blood pressure of stress-induced pre-hypertension rats and affect its gene expression profile in heart. Genes that related to the contraction of vascular smooth muscle may be involved in EA's anti-hypertensive mechanism.

  12. Functional connections and pathways of coenzyme Q10-inducible genes: an in-silico study.

    Science.gov (United States)

    Schmelzer, Constance; Lindner, Inka; Vock, Christina; Fujii, Kenji; Döring, Frank

    2007-10-01

    Coenzyme Q10 (CoQ10, ubiquinone) is an essential cofactor in the electron transport chain, serves as a potent antioxidant in mitochondria and lipid membranes, and is often used as a dietary supplement for a number of diseases including cardiovascular diseases. Recently, we obtained evidence that CoQ10 (Kaneka Q10) affects the expression of hundreds of human genes. To decipher the functional and regulatory connections of these genes, a literature search combined with transcription factor binding site analysis was performed using Genomatix BiblioSphere and MatInspector. This in-silico analysis revealed 17 CoQ10-inducible genes which are functionally connected by signalling pathways of G-protein coupled receptors, JAK/STAT, integrin, and beta-arrestin. Promoter analysis of these CoQ10-inducible genes showed one group of NF B-regulated genes, namely IL5, thrombin, vitronectin receptor and C-reactive protein (CRP). Furthermore, a common promoter framework containing binding sites of the transcription factor families EVI1, HOXF, HOXC, and CLOX was identified in the promoters of IL5, CRP, and vitronectin receptor. The identified CoQ10-inducible genes and pathways play an important role in inflammatory response. Since these effects are based on an in-vitro study, the effect of CoQ10 on vascular health in vivo needs to be addressed in further animal and/or human intervention studies.

  13. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    Science.gov (United States)

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    Science.gov (United States)

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  15. Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene.

    Science.gov (United States)

    Kim, Tae Hyun; Park, Jong Ho; Kim, Moon Chul; Cho, Sung Ho

    2008-01-01

    Treatment with the cutin monomer 16-hydroxypalmitic acid (HPA), a major component of cutin, elicited the synthesis of hydrogen peroxide (H2O2) in rice leaves and induced the expression of the lipid transfer protein gene OsLTP5. Treatment with HPA also induced expression of OsLTP1, OsLTP2, and the pathogen-related PR-10 genes to a lesser extent. The OsLTP5 transcript was expressed prominently in stems and flowers, but was barely detectable in leaves. Expression of OsLTP5 was induced in shoots in response to ABA and salicylic acid. It is proposed that HPA is perceived by rice as a signal, inducing defense reactions.

  16. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Science.gov (United States)

    Sandre, Olivier; Genevois, Coralie; Garaio, Eneko; Adumeau, Laurent; Mornet, Stéphane; Couillaud, Franck

    2017-01-01

    The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release. PMID:28208731

  17. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Directory of Open Access Journals (Sweden)

    Olivier Sandre

    2017-02-01

    Full Text Available The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

  18. Degranulation of rat cerebellum induces selective variations in gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Eliyahu, D.; Soreq, H.

    1982-02-01

    Selective variations in the composition of poly(A)-containing mRNA were found to be induced in the rat cerebellum by X-irradiation. mRNA populations prepared from normal and X-irradiated rat cerebella at different stages of their development displayed equal efficiencies when translated in vitro in reticulocyte lysates. Specific differences were revealed, however, when the labeled translation products of both mRNA preparations were subjected to two-dimensional gel electrophoresis followed by fluorography of the dried gels. Of more than 100 polypeptide products, several showed marked intensity differences, indicating changes in the abundance of their directing mRNA species. These differences appear both in developing and in mature cerebellar mRNA, and the extent of modification in mRNA is much higher than the consequent changes in the composition of proteins in the irradiated cerebellum. The degranulation-induced modifications in levels of specific cerebellar mRNA species can be used to identify proteins whose biosynthesis depends on the presence of interneurons.

  19. Molecular cloning and functional analyses of low-temperature induced genes from Ammopiptanthus mongolicus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene is...

  20. [Osmotic shock induces expression of Vibrio fischeri lux genes in Escherichia coli cells].

    Science.gov (United States)

    Zavil'gel'skiĭ, G B; Kotova, V Iu

    2003-04-01

    The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells of Escherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC-RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.

  1. Activation of the α7 nicotinic receptor promotes lipopolysaccharide-induced conversion of M1 microglia to M2

    Science.gov (United States)

    Zhang, Qichun; Lu, Ying; Bian, Huimin; Guo, Liwei; Zhu, Huaxu

    2017-01-01

    The α7 subtype of the nicotinic acetylcholine receptor (α7 nAChR) plays an essential role in the cholinergic anti-inflammatory pathway that regulates macrophage/microglia function in inflammation. Similar to M1 and M2 macrophages, M1 and M2 microglia exhibit pro-inflammation and anti-inflammation properties, respectively. In the present study, we analyzed function-associated phenotypes to detect the transformation of microglia with activation of α7 nAChRs. We used lentivirus-mediated shRNA to knockdown the expression of α7 nAChR in BV-2 microglia incubated with lipopolysaccharides (LPS, 0.1 μg/mL) and measured the acetylcholine (Ach, 1 μg/mL)-mediated release of cytokines, such as IL-1β, IL-4, IL-6, and IL-10, in the culture supernatant via radioimmunoassay. After stimulation with Ach, the expression of typical biomarkers for different microglia phenotypes, Iba-1 and Arg-1, was determined by cellular immunofluorescence. Furthermore, the expression of signaling molecules, including p38, JAK2/STAT3, PI3K/Akt and miR-124, was analyzed via western blotting and real-time PCR. We found that Ach inhibited LPS-induced IL-1β and IL-6 elevation and promoted IL-4 and IL-10 production and that knockdown of the α7 nAChR abolished these effects of Ach. In addition, Ach decreased LPS-induced Iba-1 expression and increased Arg-1 levels in an α7 nAChR-dependent manner. The LPS-inhibited activation of JAK2/STAT3 and PI3K/Akt was also rescued by Ach, an effect that was blocked by knockdown of the α7 nAChR. In contrast, Ach triggered the phosphorylation of JAK2 and STAT3 that was otherwise inactivated by LPS in BV-2 cells. Finally, the levels of miR-124 and downstream targets C/EBPα and PU.1 were significantly enhanced in LPS-treated BV-2 microglia, and the effect of Ach on this signaling pathway was blocked by α7 nAChR knockdown as expected. Overall, our data demonstrate that activation ofα7 nAChRs inhibits the transformation of M1 microglia and promotes the M2

  2. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.;

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death...... upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed, P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-04/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  3. Identification of genes regulated during mechanical load-induced cardiac hypertrophy

    Science.gov (United States)

    Johnatty, S. E.; Dyck, J. R.; Michael, L. H.; Olson, E. N.; Abdellatif, M.; Schneider, M. (Principal Investigator)

    2000-01-01

    Cardiac hypertrophy is associated with both adaptive and adverse changes in gene expression. To identify genes regulated by pressure overload, we performed suppressive subtractive hybridization between cDNA from the hearts of aortic-banded (7-day) and sham-operated mice. In parallel, we performed a subtraction between an adult and a neonatal heart, for the purpose of comparing different forms of cardiac hypertrophy. Sequencing more than 100 clones led to the identification of an array of functionally known (70%) and unknown genes (30%) that are upregulated during cardiac growth. At least nine of those genes were preferentially expressed in both the neonatal and pressure over-load hearts alike. Using Northern blot analysis to investigate whether some of the identified genes were upregulated in the load-independent calcineurin-induced cardiac hypertrophy mouse model, revealed its incomplete similarity with the former models of cardiac growth. Copyright 2000 Academic Press.

  4. Identification of genes regulated during mechanical load-induced cardiac hypertrophy

    Science.gov (United States)

    Johnatty, S. E.; Dyck, J. R.; Michael, L. H.; Olson, E. N.; Abdellatif, M.; Schneider, M. (Principal Investigator)

    2000-01-01

    Cardiac hypertrophy is associated with both adaptive and adverse changes in gene expression. To identify genes regulated by pressure overload, we performed suppressive subtractive hybridization between cDNA from the hearts of aortic-banded (7-day) and sham-operated mice. In parallel, we performed a subtraction between an adult and a neonatal heart, for the purpose of comparing different forms of cardiac hypertrophy. Sequencing more than 100 clones led to the identification of an array of functionally known (70%) and unknown genes (30%) that are upregulated during cardiac growth. At least nine of those genes were preferentially expressed in both the neonatal and pressure over-load hearts alike. Using Northern blot analysis to investigate whether some of the identified genes were upregulated in the load-independent calcineurin-induced cardiac hypertrophy mouse model, revealed its incomplete similarity with the former models of cardiac growth. Copyright 2000 Academic Press.

  5. Gene Analysis of Arsenic Trioxide—induced Apoptosis of Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGZidong; LIWeiyu; 等

    2002-01-01

    Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells.

  6. Linagliptin Limits High Glucose Induced Conversion of Latent to Active TGFß through Interaction with CIM6PR and Limits Renal Tubulointerstitial Fibronectin.

    Directory of Open Access Journals (Sweden)

    Muralikrishna Gangadharan Komala

    Full Text Available In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4 inhibitors have been shown to be antifibrotic. We have previously shown that cation independent mannose-6-phosphate receptor (CIM6PR facilitates the conversion of latent to active transforming growth factor β1 (GFß1 in renal proximal tubular cells (PTCs and linagliptin (a DPP4 inhibitor reduced this conversion with downstream reduction in fibronectin transcription.We wanted to demonstrate that linagliptin reduces high glucose induced interaction between membrane bound DPP4 and CIM6PR in vitro and demonstrate reduction in active TGFß mediated downstream effects in a rodent model of type 1 diabetic nephropathy independent of high glycaemic levels.We used human kidney 2 (HK2 cells and endothelial nitric oxide synthase knock out mice to explore the mechanism and antifibrotic potential of linagliptin independent of glucose lowering. Using a proximity ligation assay, we show that CIM6PR and DPP4 interaction was increased by high glucose and reduced by linagliptin and excess mannose-6-phosphate (M6P confirming that linagliptin is operating through an M6P-dependent mechanism. In vivo studies confirmed these TGFß1 pathway related changes and showed reduced fibronectin, phosphorylated smad2 and phosphorylated smad2/3 (pSmad2/3 with an associated trend towards reduction in tubular atrophy, which was independent of glucose lowering. No reduction in albuminuria, glomerulosclerotic index or cortical collagen deposition was observed.Linagliptin inhibits activation of TGFß1 through a M6P dependent mechanism. However this in isolation is not sufficient to reverse the multifactorial nature of diabetic nephropathy.

  7. Symmetry breaking and light-induced spin-state trapping in a mononuclear FeII complex with the two-step thermal conversion

    Science.gov (United States)

    Buron-Le Cointe, M.; Ould Moussa, N.; Trzop, E.; Moréac, A.; Molnar, G.; Toupet, L.; Bousseksou, A.; Létard, J. F.; Matouzenko, G. S.

    2010-12-01

    Crystallographic, magnetic, and Raman investigations of the mononuclear [FeII(Hpy-DAPP)](BF4)2 complex are presented. Its particular feature is a two-step thermal spin conversion in spite of a unique symmetry-independent iron site per unit cell. The plateau around 140 K is associated with a symmetry breaking visible by the appearance of weak (0k0) k odd Bragg peaks. Symmetries of the high-temperature high-spin state and of the low-temperature low-spin state are both monoclinic P21/c , so that the symmetry breaking on the plateau is associated with a reentrant phase transition. It is discussed in relation with Ising-type microscopic models. At the plateau level, the two symmetry-independent molecules differ both by their spin state and the conformation (chair versus twist-boat) of one metallocycle. At low-temperature photoinduced phenomena have been investigated: a partial phototransformation [light-induced excited spin-state trapping (LIESST) effect] is observed under visible red irradiation. Raman spectroscopy shows that the molecular photoinduced state is the high-spin one. Nevertheless, as no macroscopic symmetry breaking is observed, the unique average cationic [FeII(Hpy-DAPP)] state of the unit cell is intermediate between pure low-spin and high-spin states and presents a conformational disorder for one metallocycle. Reverse-LIESST has also been evidenced using near infrared excitation. Thus, the mononuclear [Fe(Hpy-DAPP)](BF4)2 compound offers the opportunity to discuss the interplay between spin conversion, molecular conformational change, and ordering processes.

  8. Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

    Directory of Open Access Journals (Sweden)

    Jens Durruthy-Durruthy

    Full Text Available Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP. Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

  9. Acute ozone-induced differential gene expression profiles in rat lung.

    Science.gov (United States)

    Nadadur, Srikanth S; Costa, Daniel L; Slade, Ralph; Silbjoris, Robert; Hatch, Gary E

    2005-12-01

    Ozone is an oxidant gas that can directly induce lung injury. Knowledge of the initial molecular events of the acute O3 response would be useful in developing biomarkers of exposure or response. Toward this goal, we exposed rats to toxic concentrations of O3 (2 and 5 ppm) for 2 hr and the molecular changes were assessed in lung tissue 2 hr postexposure using a rat cDNA expression array containing 588 characterized genes. Gene array analysis indicated differential expression in almost equal numbers of genes for the two exposure groups: 62 at 2 ppm and 57 at 5 ppm. Most of these genes were common to both exposure groups, suggesting common roles in the initial toxicity response. However, we also identified the induction of nine genes specific to 2-ppm (thyroid hormone-beta receptor c-erb-A-beta; and glutathione reductase) or 5-ppm exposure groups (c-jun, induced nitric oxide synthase, macrophage inflammatory protein-2, and heat shock protein 27). Injury markers in bronchoalveolar lavage fluid (BALF) were used to assess immediate toxicity and inflammation in rats similarly exposed. At 2 ppm, injury was marked by significant increases in BALF total protein, N-acetylglucosaminidase, and lavageable ciliated cells. Because infiltration of neutrophils was observed only at the higher 5 ppm concentration, the distinctive genes suggested a potential amplification role for inflammation in the gene profile. Although the specific gene interactions remain unclear, this is the first report indicating a dose-dependent direct and immediate induction of gene expression that may be separate from those genes involved in inflammation after acute O3 exposure.

  10. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  11. Regulation of mouse hepatic genes in response to diet induced obesity, insulin resistance and fasting induced weight reduction

    Directory of Open Access Journals (Sweden)

    Mantzoros Christos

    2005-06-01

    Full Text Available Abstract Background Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrys and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. Results Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/ weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced

  12. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen;

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing...... cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  13. Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian Beck; Wojtaszewski, Jørgen; Viollet, Benoit

    2005-01-01

    We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body a2- and a1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmi...

  14. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...

  15. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    Science.gov (United States)

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

  16. Abrogation of p53-induced apoptosis by the hepatitis B virus X gene.

    NARCIS (Netherlands)

    X.W. Wang (Xin Wei); M.K. Gibson (Michael); W. Vermeulen (Wim); H. Yeh; K. Forrester; H.-W. Stürzbecher; J.H.J. Hoeijmakers (Jan); C.C. Harris

    1996-01-01

    textabstractThe p53 tumor suppressor gene product is a transcriptional transactivator and a potent apoptotic inducer. The fact that many of the DNA tumor virus oncoproteins bind to p53 and affect these p53 functions indicates that this interaction is an important step in oncogenic transformation. We

  17. Gene expression profiling and pathway analysis of hepatotoxicity induced by triptolide in Wistar rats.

    Science.gov (United States)

    Wang, Jiaying; Jiang, Zhenzhou; Ji, Jinzi; Wang, Xinzhi; Wang, Tao; Zhang, Yun; Tai, Ting; Chen, Mi; Sun, Lixin; Li, Xia; Zhang, Luyong

    2013-08-01

    Triptolide (TP), a major component of TWHF, is widely used to treat rheumatoid arthritis, systemic lupus erythematosus, nephritis and leprosy. However, its clinical use is limited by hepatotoxicity. To further elucidate the underlying mechanism of its hepatotoxic effects, hepatic gene expression profiles were analyzed. TP (1000 and 300 μg/kg) was orally administered to Wistar rats for 14 days. Current study indicated that female rats were more sensitive to TP-induced hepatotoxicity than males. Genome-wide microarray analyses identified 3329 differentially expressed genes in liver of female rats. Analyses of these genes identified over-represented functions associated with insulin signaling pathway, glucose metabolism, cell cycle, oxidative stress and apoptosis, which were consistent with the results of significant increase of Caspase-3 activity and reduction of serum glucose, GSH/GSSG ratio, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities, liver glycogen. In addition, it was observed for the first time that glucocorticoids and IGF1 might get involved in TP-induced hepatotoxicity. These data suggest that TP treatment could alter the hepatic redox status, reduce serum glucose and induce hepatocyte apoptosis, consistent with the differential expression of genes involved in insulin signaling pathway, glucose metabolism pathway and cell stress pathway, all of which might contribute to the overall TP-induced hepatotoxicity.

  18. An in planta induced gene of Phytophthora infestans codes for ubiquitin

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Risseeuw, E.P.; Davidse, L.C.

    1991-01-01

    An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction

  19. An in planta induced gene of Phytophthora infestans codes for ubiquitin

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Risseeuw, E.P.; Davidse, L.C.

    1991-01-01

    An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was isolated from a genomic library by differential hybridization using labelled cDNA derived from poly(A)⁺ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P,

  20. Gene expression array analyses predict increased proto-oncogene expression in MMTV induced mammary tumors.

    Science.gov (United States)

    Popken-Harris, Pamela; Kirchhof, Nicole; Harrison, Ben; Harris, Lester F

    2006-08-01

    Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

  1. Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

    Science.gov (United States)

    Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

    2016-05-01

    Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

  2. Identification and characterization of the inducible murine mast cell gene, imc-415.

    Science.gov (United States)

    Cho, S H; Cho, J J; Kim, I S; Vliagoftis, H; Metcalfe, D D; Oh, C K

    1998-11-01

    Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.

  3. Cloning of murine BRI3 gene and study on its function for inducing cell death

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.

  4. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

    2004-01-01

    elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene......+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance....

  5. Identification of LPS-inducible genes downregulated by ubiquinone in human THP-1 monocytes.

    Science.gov (United States)

    Schmelzer, Constance; Döring, Frank

    2010-01-01

    Coenzyme Q(10) (CoQ(10)) is an obligatory element in the respiratory chain and functions as a potent antioxidant of lipid membranes. More recently, anti-inflammatory effects as well as an impact of CoQ(10) on gene expression have been observed. To reveal putative effects of Q(10) on LPS-induced gene expression, whole genome expression analysis was performed in the monocytic cell line THP-1. Thousand one hundred twenty-nine and 710 probe sets have been identified to be significantly (P genes revealed a functional connection in the NFkappaB pathway and confirmed our applied in vitro stimulation model. Moreover, 33 LPS-sensitive genes have been identified to be significantly downregulated by Q(10)-treatment between a factor of 1.32 and 1.85. GeneOntology (GO) analysis revealed for the Q(10)-sensitve genes a primary involvement in protein metabolism (e.g., HERC1 and EPS15), cell proliferation (e.g., CCDC100 and SMURF1), and transcriptional processes (e.g., CNOT4 and STK4). Three genes were either related to NFkappaB transcription factor activity (ERC1), cytokinesis (DIAPH2), or modulation of oxidative stress (MSRA). In conclusion, our data provide evidence that Q(10) downregulates LPS-inducible genes in the monocytic cell line THP-1. Thus, the previously described effects of Q(10) on the reduction of proinflammatory mediators might be due to its antioxidant impact on gene expression.

  6. Gene expression in Barrett's esophagus and reflux esophagitis induced by gastroduodenoesophageal reflux in rats

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Chen Jie; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To investigate the difference of gene expression profiles between Barrett's esophagus and reflux esophagitis induced by gastroduodenoesophageal reflux in rats.METHODS: Eight-week-old Sprague-Dawley rats were treated esophagoduodenostomy to produce gastroduodenoesophageal reflux, and another group received sham operation as control. Esophageal epithelial tissues were dissected and frozen in liquid nitrogen immediately for pathology 40 wk after surgery. The expression profiles of 4 096 genes in reflux esophagitis and Barrett's esophagus tissues were compared with normal esophageal epithelium by cDNA microarray.RESULTS: Four hundred and forty-eight genes in Barrett'sesophagus were more than three times different from those in normal esophageal epithelium, including 312 up regulated and 136 down-regulated genes. Two hundred and thirty-twogenes in RE were more than three times different from those in normal esophageal epithelium, 90up-regulated and 142 down-regulated genes. Compared to reflux esophagitis, there were 214 up-regulated and 142 down-regulated genes in Barrett's esophagus. CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux can develop esophagitis and Barrett's esophagus gradually.The gene expression level is different between reflux esophagitis and Barrett's esophagus and the differentially expressed genes might be related to the occurrence and development of Barrett's esophagus and the promotion or progression in adenocarcinoma.

  7. Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

    Science.gov (United States)

    Gebert, J; Schnölzer, M; Warnken, U; Kopitz, J

    2017-01-01

    Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified. © 2017 Elsevier Inc. All rights reserved.

  8. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus.

    Science.gov (United States)

    Liscombe, David K; O'Connor, Sarah E

    2011-11-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003-0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.

  9. Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.

    Science.gov (United States)

    Chen, Yuejun; Cao, Jingyuan; Xiong, Man; Petersen, Andrew J; Dong, Yi; Tao, Yunlong; Huang, Cindy Tzu-Ling; Du, Zhongwei; Zhang, Su-Chun

    2015-08-06

    Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

  10. UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B.

    Science.gov (United States)

    Yang, Kuan; Boswell, Mikki; Walter, Dylan J; Downs, Kevin P; Gaston-Pravia, Kimberly; Garcia, Tzintzuni; Shen, Yingjia; Mitchell, David L; Walter, Ronald B

    2014-06-01

    Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adjbasal expression level of each transcript for each skin sample, (2) the changes in expression levels for each gene in the transcriptome upon exposure to increasing doses of UVB, and (3) clusters of genes that exhibit similar patterns of change in expression upon UVB exposure. These data provide a foundation for understanding the molecular genetic response of fish skin to UVB exposure.

  11. Laparotomy in mice induces blood cell expression of inflammatory and stress genes.

    Science.gov (United States)

    Ko, Fred; Isoda, Fumiko; Mobbs, Charles

    2015-04-01

    Surgical trauma induces immune and stress responses although its effects on postsurgical inflammatory and stress gene expression remain poorly characterized. This study sought to improve current scientific knowledge by investigating the effects of laparotomy on mouse blood cell inflammatory and stress gene expression. Three-month-old male C57BL/6J mice were subjected to 2% isoflurane or 2% isoflurane with laparotomy and sacrificed 4 h postintervention. Blood was collected and blood cell expression of 158 genes central to inflammatory and stress responses was assayed using quantitative polymerase chain reaction arrays. Mice subjected to isoflurane with laparotomy, compared with mice receiving isoflurane alone, had >2-fold upregulation of genes in inflammation (Osm, IL1rn, IL1b, and Csf1), oxidative stress (Hmox1), heat shock (Hspa1b), growth arrest (Cdkn1a), and DNA repair (Ugt1a2). These genes demonstrated similar expression patterns by Pearson correlation and cluster analysis. Thus, laparotomy induces coordinated, postsurgical blood cell expression of unique inflammatory and stress genes whose roles in influencing surgical outcomes need further investigation.

  12. ADHESION INDUCES MATRIX METALLOPROTEINASE-9 GENE EXPRESSION IN OVARIAN CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    田方; 颜春洪; 薛红; 肖凤君

    2002-01-01

    Objective: To investigate the expression of matrix metalloproteinase-9 (MMP-9) gene in cancer cells induced by adhesion with fibronectin and the underlying mechanism of cell invasion. Methods: Following adhesion of ovarian cancer cells A2780 to fibronectin, MMP mRNA expression was assayed by using reverse transcription-polymerase chain reaction (RT-PCR). MMP-9 promoter was cloned from genomic DNA of HT1080 cells with PCR. The MMP-9-pGL2 reporter gene vector was constructed and then transiently transfected into A2780 cells. Results: Adhesion could induce the expression of MMP-9 gene in A2780 cells, but did not affect longer theexpression of MMP-2 or TIMP-1 gene. The induction was enhanced with longer adhesion time. When the transfected cells were allowed to adhere and spread on FN-coated surface, the promoter activity of MMP-9 gene was also enhanced dramatically. Conclusion: adhesion of cells with ECM may stimulate the expression of MMP-9 gene through stimulating the promoter activity, thereby enhancing cancer cell invasion and metastasis.

  13. Characterization of chemically induced liver injuries using gene co-expression modules.

    Directory of Open Access Journals (Sweden)

    Gregory J Tawa

    Full Text Available Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1 known biochemical pathways associated with liver injuries and 2 clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20% genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects.

  14. Characterization of Chemically Induced Liver Injuries Using Gene Co-Expression Modules

    Science.gov (United States)

    Tawa, Gregory J.; AbdulHameed, Mohamed Diwan M.; Yu, Xueping; Kumar, Kamal; Ippolito, Danielle L.; Lewis, John A.; Stallings, Jonathan D.; Wallqvist, Anders

    2014-01-01

    Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules) specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1) known biochemical pathways associated with liver injuries and 2) clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20%) genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects. PMID:25226513

  15. Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule

    Directory of Open Access Journals (Sweden)

    Esquerré Diane

    2007-11-01

    Full Text Available Abstract Background Recovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones. Results Significance analysis of microarrays (SAM and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery. Conclusion Our study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth

  16. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  17. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  18. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  19. Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

    Directory of Open Access Journals (Sweden)

    Anna Sliva

    2016-04-01

    Full Text Available The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS, and barcode analysis by sequencing (Bar-Seq. Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported, including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.

  20. A Novel NADPH-Dependent Aldehyde Reductase Gene from Saccharomyces cerevisiae NRRL Y-12632 Involved in the Detoxification of Aldehyde Inhibitors Derived from Lignocellulosic Biomass Conversion

    Science.gov (United States)

    Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural (HMF), anisaldehyde, benzaldehyde, cinnamaldehyde, and phenylaldehyde are commonly generated during lignocellulosic biomass conversion process for low-cost cellulosic ethanol production that interferes with subsequent microbial growth and...

  1. Therapeutic angiogenesis induced by human hepatocyte growth factor (HGF) gene in rat myocardial ischemia models

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the feasibility of myocardial ischemia gene therapy, we cloned human hepatocyte growth factor gene from human placenta cDNA library by the RT-PCR method. Recombination adenovirus Ad-HGF was constructed by the method of co-transfection and homologous recombination of plasmids in 293 cells. Ad-HGF was amplified in 293 cells and purified through CsCl density gradient centrifugation. Ad-HGF could be expressed in rat primary myocardial cells and HGF secreted into the culture media, which was tested by ELISA. The distribution and persistence of adenovirus in rat were investigated by green fluorescence protein as a report gene. In vivo we found that intramyocardial administration of Ad-HGF could induce angiogenesis in rat myocardium after ligation of coronary artery. The results suggested that Ad-HGF was effective in vitro and in vivo, and the data for designing human trial of gene therapy-- mediated cardiac angiogenesis were provided.

  2. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  3. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    Science.gov (United States)

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  4. Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato.

    Science.gov (United States)

    Yi, Jung Yoon; Seo, Hyo Won; Yang, Moon Sik; Robb, E Jane; Nazar, Ross N; Lee, Shin Woo

    2004-11-01

    PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

  5. Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

    Science.gov (United States)

    Cheng, Yo Ching; Hannaoui, Samia; John, Theodore R.; Dudas, Sandor; Czub, Stefanie; Gilch, Sabine

    2016-01-01

    Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding. PMID:27829062

  6. Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion.

    Science.gov (United States)

    Masujin, Kentaro; Orrú, Christina D; Miyazawa, Kohtaro; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Caughey, Byron

    2016-03-01

    Prion diseases of cattle include the classical bovine spongiform encephalopathy (C-BSE) and the atypical H-type BSE (H-BSE) and L-type BSE (L-BSE) strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays, no such test has yet been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. Here, we demonstrate an RT-QuIC assay for H-BSE that can detect as little as 10(-9) dilutions of brain tissue and neat cerebrospinal fluid samples from clinically affected cattle. Moreover, comparisons of the reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L-, and C-BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

  7. Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.

    Science.gov (United States)

    Orrú, Christina D; Favole, Alessandra; Corona, Cristiano; Mazza, Maria; Manca, Matteo; Groveman, Bradley R; Hughson, Andrew G; Acutis, Pier Luigi; Caramelli, Maria; Zanusso, Gianluigi; Casalone, Cristina; Caughey, Byron

    2015-04-01

    Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrPSen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.

  8. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sárvári, Anitta K., E-mail: anittasarvari@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Veréb, Zoltán, E-mail: jzvereb@gmail.com [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Uray, Iván P., E-mail: ipuray@mdanderson.org [Clinical Cancer Prevention Department, The University of Texas, MD Anderson Cancer Center, Houston, TX (United States); Fésüs, László, E-mail: fesus@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); MTA DE Apoptosis, Genomics and Stem Cell Research Group of the Hungarian Academy of Sciences (Hungary); Balajthy, Zoltán, E-mail: balajthy@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary)

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  9. Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

    Institute of Scientific and Technical Information of China (English)

    许东晖; 李宝健; 刘煜; 黄志纾; 古练权

    1996-01-01

    Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4’-trihydroxy-3’, 5’-dimethoxy-flavone (named tricin) and 5, 4’ -dihydroxy-3’, 5’ -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous plants

  10. Regulated gene insertion by steroid-induced PhiC31 integrase.

    Science.gov (United States)

    Sharma, Nynne; Moldt, Brian; Dalsgaard, Trine; Jensen, Thomas G; Mikkelsen, Jacob Giehm

    2008-06-01

    Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.

  11. Inducible gene silencing in podocytes: a new tool for studying glomerular function.

    Science.gov (United States)

    Bugeon, Laurence; Danou, Aliki; Carpentier, David; Langridge, Paul; Syed, Nelofer; Dallman, Margaret J

    2003-03-01

    Glomerular filtration is one of the primary functions of the kidney. Podocytes, a highly specialized cell type found in glomeruli, are believed to play a critical role in that function. Null mutations of genes expressed in podocytes like WT1, nephrin, and NEPH1 result in an embryo and perinatal lethal phenotype and therefore do not allow the functional analysis of these genes in the adult kidney. Here is describes the generation of a model that will allow such studies. We have engineered transgenic mice in which the disruption of targeted genes can be induced in a temporally controlled fashion in podocytes. For this, a transgene encoding the mutated estrogen receptor-Cre recombinase fusion protein was introduced into the mouse genome. Animals were crossed with Z/AP reporter mice to test for efficient and inducible recombination. We found that, after injection of inducer drug tamoxifen, Cre fusion protein translocates to the nuclei of podocytes, where it becomes active and mediates recombination of DNA carrying loxP target sequences. These animals provide for the first time a tool for silencing genes selectively in podocytes of adult animals.

  12. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Directory of Open Access Journals (Sweden)

    Jun-Chao Guo

    Full Text Available The extremely dismal prognosis of pancreatic cancer (PC is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  13. Effects of L-Theanine on Posttraumatic Stress Disorder Induced Changes in Rat Brain Gene Expression

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    Tomás Eduardo Ceremuga

    2014-01-01

    Full Text Available Posttraumatic stress disorder (PTSD is characterized by the occurrence of a traumatic event that is beyond the normal range of human experience. The future of PTSD treatment may specifically target the molecular mechanisms of PTSD. In the US, approximately 20% of adults report taking herbal products to treat medical illnesses. L-theanine is the amino acid in green tea primarily responsible for relaxation effects. No studies have evaluated the potential therapeutic properties of herbal medications on gene expression in PTSD. We evaluated gene expression in PTSD-induced changes in the amygdala and hippocampus of Sprague-Dawley rats. The rats were assigned to PTSD-stressed and nonstressed groups that received either saline, midazolam, L-theanine, or L-theanine + midazolam. Amygdala and hippocampus tissue samples were analyzed for changes in gene expression. One-way ANOVA was used to detect significant difference between groups in the amygdala and hippocampus. Of 88 genes examined, 17 had a large effect size greater than 0.138. Of these, 3 genes in the hippocampus and 5 genes in the amygdala were considered significant (P<0.05 between the groups. RT-PCR analysis revealed significant changes between groups in several genes implicated in a variety of disorders ranging from PTSD, anxiety, mood disorders, and substance dependence.

  14. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  15. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

    Full Text Available Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs, human dermal fibroblasts (hDFs, and hOF-derived induced pluripotent stem cells (hOF-iPSCs, linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  16. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Science.gov (United States)

    Guo, Jun-Chao; Li, Jian; Yang, Ying-Chi; Zhou, Li; Zhang, Tai-Ping; Zhao, Yu-Pei

    2013-01-01

    The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  17. RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding.

    Science.gov (United States)

    Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

    2014-01-01

    RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties.

  18. MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATION IN EXON 7/8 OF RAT HPRT GENE

    Institute of Scientific and Technical Information of China (English)

    任晓庆; 黄定九; 黄钢; 王利民

    2003-01-01

    Objective To investigate the relationship between the radiation dose and the HPRT gene locus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA).MethodsThe smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolated by 6 thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single strand conformation polymorphism.ResultsThe HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5×10-6 to 13×10-6. Of 91 HPRT gene mutation colonies, 13(14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation. The exon 7/8 mutation frequency was 30.8%. There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8.ConclusionThe DNA damage and gene mutation induced by radiation has positive relationship with radiation dose, and is a basis of proliferation inhibition and apoptosis of smooth muscle cells.

  19. Oligonucleotide microarray analysis of dietary-induced hyperlipidemia gene expression profiles in miniature pigs.

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    Junko Takahashi

    Full Text Available BACKGROUND: Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. METHODOLOGY: Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD. Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. PRINCIPAL FINDINGS: Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. CONCLUSIONS: No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in

  20. Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

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    Nuria Troyano-Suárez

    2015-01-01

    Full Text Available Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK, a scaffold protein at cell-extracellular matrix (ECM adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

  1. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐兵; 齐义鹏; Masuo; Yutsudo; 刘青珍

    2000-01-01

    asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of

  2. Isolation and Analysis of Drought-Induced Genes in Maize Roots

    Institute of Scientific and Technical Information of China (English)

    LI Hui-yong; HUANG Shu-hua; SHI Yun-su; SONG Yan-chun; ZHONG Zhong-bao; WANG Guo-ying; WANG Tian-yu; LI Yu

    2009-01-01

    Maize roots are important component for plant adaptation to soil water deficits because they are supposed to take up water and necessary solutes from the soil. In the present study, the drought-induced genes were isolated in maize roots. A suppression subtractive hybridization protocol was applied to construct a forward subtractive cDNA library from CN165 for drought-stressed maize roots and a number of drought-induced genes were isolated. Totally, 126 uniESTs (containing 82 singlets and 44 contigs) were obtained from 503 available ESTs sequences after macroarray hybridization. UniESTs were analyzed using BLASTN and BLASTX and the results showed that 92% of the uniESTs had homolgous sequences in maize nr database by BLASTN. About 89% of uniESTs appeared the homlogous amino acid sequences in rice protein database but not in maize protein database by BLASTX, implying that those genes are likely new functional genes in maize. Function analysis showed that those genes were involved in a broad spectrum of biological pathways, mainly in signaling and regulatory pathways related to stress tolerance.

  3. Characterization of Phototransduction Gene Knockouts Revealed Important Signaling Networks in the Light-Induced Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Jayalakshmi Krishnan

    2008-01-01

    Full Text Available Understanding the molecular pathways mediating neuronal function in retinas can be greatly facilitated by the identification of genes regulated in the retinas of different mutants under various light conditions. We attempted to conduct a gene chip analysis study on the genes regulated during rhodopsin kinase (Rhok-/- and arrestin (Sag-/- knockout and double knockouts in mice retina. Hence, mice were exposed to constant illumination of 450 lux or 6,000 lux on dilated pupils for indicated periods. The retinas were removed after the exposure and processed for microarray analysis. Double knockout was associated with immense changes in gene expression regulating a number of apoptosis inducing transcription factors. Subsequently, network analysis revealed that during early exposure the transcription factors, p53, c-MYC, c-FOS, JUN, and, in late phase, NF-B, appeared to be essential for the initiation of light-induced retinal rod loss, and some other classical pro- and antipoptotic genes appeared to be significantly important as well.

  4. Hydrogen peroxide-induced gene expression across kingdoms: a comparative analysis.

    Science.gov (United States)

    Vandenbroucke, Korneel; Robbens, Steven; Vandepoele, Klaas; Inzé, Dirk; Van de Peer, Yves; Van Breusegem, Frank

    2008-03-01

    Cells react to oxidative stress conditions by launching a defense response through the induction of nuclear gene expression. The advent of microarray technologies allowed monitoring of oxidative stress-dependent changes of transcript levels at a comprehensive and genome-wide scale, resulting in a series of inventories of differentially expressed genes in different organisms. We performed a meta-analysis on hydrogen peroxide (H(2)O(2))-induced gene expression in the cyanobacterium Synechocystis PCC 6803, the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, the land plant Arabidopsis thaliana, and the human HeLa cell line. The H(2)O(2)-induced gene expression in both yeast species was highly conserved and more similar to the A. thaliana response than that of the human cell line. Based on the expression characteristics of genuine antioxidant genes, we show that the antioxidant capacity of microorganisms and higher eukaryotes is differentially regulated. Four families of evolutionarily conserved eukaryotic proteins could be identified that were H(2)O(2) responsive across kingdoms: DNAJ domain-containing heat shock proteins, small guanine triphosphate-binding proteins, Ca(2+)-dependent protein kinases, and ubiquitin-conjugating enzymes.

  5. Enriched Environment-induced Maternal Weight Loss Reprograms Metabolic Gene Expression in Mouse Offspring*

    Science.gov (United States)

    Wei, Yanchang; Yang, Cai-Rong; Wei, Yan-Ping; Ge, Zhao-Jia; Zhao, Zhen-Ao; Zhang, Bing; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2015-01-01

    The global prevalence of weight loss is increasing, especially in young women. However, the extent and mechanisms by which maternal weight loss affects the offspring is still poorly understood. Here, using an enriched environment (EE)-induced weight loss model, we show that maternal weight loss improves general health and reprograms metabolic gene expression in mouse offspring, and the epigenetic alterations can be inherited for at least two generations. EE in mothers induced weight loss and its associated physiological and metabolic changes such as decreased adiposity and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, decreased plasma and hepatic lipid levels, and improved glucose tolerance and insulin sensitivity. Maternal weight loss altered gene expression patterns in the liver of offspring with coherent down-regulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling of offspring livers revealed numerous changes in cytosine methylation depending on maternal weight loss, including reproducible changes in promoter methylation over several key lipid biosynthesis genes, correlated with their expression patterns. Embryo transfer studies indicated that oocyte alteration in response to maternal metabolic conditions is a strong factor in determining metabolic and epigenetic changes in offspring. Several important lipid metabolism-related genes have been identified to partially inherit methylated alleles from oocytes. Our study reveals a molecular and mechanistic basis of how maternal lifestyle modification affects metabolic changes in the offspring. PMID:25555918

  6. ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

    Science.gov (United States)

    Rath, Nicola; Kalna, Gabriela; Clark, William; Olson, Michael F.

    2016-01-01

    The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer. PMID:27824338

  7. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    Science.gov (United States)

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  8. Jasmonate signal induced expression of cystatin genes for providing resistance against Karnal bunt in wheat.

    Science.gov (United States)

    Dutt, Shriparna; Pandey, Dinesh; Kumar, Anil

    2011-06-01

    Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pretreated with jasmonic acid (JA) or jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in reducing Karnal bunt (KB) infection by regulating cystatin gene expression. JA was found to improve the plant defense against KB as its exogenous application resulted in decrease in coefficient of infection (CI) in both susceptible and resistant varieties following pathogen inoculation. Transcript profiling of wheat cystatin genes at different days after inoculation (DAI) showed that JA pretreatment positively induced cystatin gene expression in both varieties with greater induction of expression in resistant variety than the susceptible one (Pcystatin genes, WC2, WC3 and WCMD was observed with their increased expression at 1DAI in the boot emergence stage which is most susceptible to KB and then slowly declined gradually at 3, 7 and 15 DAI in both the varieties. Except WC2, higher expression of other two cystatins viz. WC3 and WCMD at 1DAI showed higher response (Pcystatin by inhibitor assay were found to be consistent with those of transcript profiling. These findings suggest that jasmonic acid (JA) may act as a potential activator of induced resistance against Karnal bunt of wheat by upregulating cystatin gene expression.

  9. Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

    Science.gov (United States)

    Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun

    2016-11-29

    Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE(Ac)) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE(Ac)-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

  10. DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

    Science.gov (United States)

    Tse, Margaret J; Chu, Brian K; Roy, Mahua; Read, Elizabeth L

    2015-10-20

    Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

  11. Loss of function of the yellow-e gene causes dehydration-induced mortality of adult Tribolium castaneum.

    Science.gov (United States)

    Noh, Mi Young; Kramer, Karl J; Muthukrishnan, Subbaratnam; Beeman, Richard W; Kanost, Michael R; Arakane, Yasuyuki

    2015-03-15

    Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e (TcY-e), in the red flour beetle, Tribolium castaneum, we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (dsTcY-e) into late instar larvae had no effect on larval-pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4-5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining the dsTcY-e-treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued dsTcY-e-adults was slightly darker than that of

  12. Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

    Science.gov (United States)

    Hamilton, J W; Kaltreider, R C; Bajenova, O V; Ihnat, M A; McCaffrey, J; Turpie, B W; Rowell, E E; Oh, J; Nemeth, M J; Pesce, C A; Lariviere, J P

    1998-08-01

    Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure

  13. The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

    Institute of Scientific and Technical Information of China (English)

    YU; Cui; HU; Dongwei; DONG; Jiahong; CUI; Xiaofeng; WU; Jun

    2004-01-01

    Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.

  14. CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells.

    Science.gov (United States)

    Kim, Eun Ji; Kang, Ki Ho; Ju, Ji Hyeon

    2017-01-01

    Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology-and particularly clustered regularly interspaced short palindromic repeats (CRISPR)-will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.

  15. Parallel logic gates in synthetic gene networks induced by non-Gaussian noise.

    Science.gov (United States)

    Xu, Yong; Jin, Xiaoqin; Zhang, Huiqing

    2013-11-01

    The recent idea of logical stochastic resonance is verified in synthetic gene networks induced by non-Gaussian noise. We realize the switching between two kinds of logic gates under optimal moderate noise intensity by varying two different tunable parameters in a single gene network. Furthermore, in order to obtain more logic operations, thus providing additional information processing capacity, we obtain in a two-dimensional toggle switch model two complementary logic gates and realize the transformation between two logic gates via the methods of changing different parameters. These simulated results contribute to improve the computational power and functionality of the networks.

  16. Plant protein kinase genes induced by drought, high salt and cold stresses

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

  17. Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family.

    Science.gov (United States)

    Krenek, Sascha; Schlegel, Martin; Berendonk, Thomas U

    2013-02-21

    Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia

  18. Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD-inducible genes in human amniotic epithelial cells

    Directory of Open Access Journals (Sweden)

    Kokame Koichi

    2006-05-01

    Full Text Available Abstract Background Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD treated primary cultures of human amniotic epithelial cells. Methods Human amniotic epithelial cells were dispersed by trypsin from amniotic membranes and cultured in DME/Ham's F12 medium supplemented with 10% FBS. Two weeks after plating, cells were treated with 50 nM TCDD or DMSO (control, further incubated for 48 hrs, and the gene expression was analyzed by DNA microarray technology and quantitative real-time PCR. Results Thirty eight TCDD-inducible genes, including cytochromeP4501A1 and cytochromeP4501B1, were identified. One of the remarkable profiles of the gene expression was the prominent up-regulation of interferon-inducible genes. The genes involved in the interferon gene expression and interferon signaling pathways were also up-regulated. Furthermore, the expression of genes related to collagen synthesis or degradation was enhanced by TCDD. Conclusion Using DNA microarray and quantitative real-time PCR analyses, we identified TCDD-inducible genes, including interferon-inducible genes and genes related to collagen synthesis or degradation, in human amniotic epithelial cells.

  19. Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Zuo-lin JIN; Yong-kuan ZHANG; Hai-yan SUN; Zhu LIN; Ying-chun BI; Yin-zhong DUAN; Yin DING

    2008-01-01

    Aim:Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or os-teogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. Methods: Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. Results: Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregn-lated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-β superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. Conclusion: Our results demonstrated that hDFC dis-played osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

  20. Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling.

    Science.gov (United States)

    Liu, Cun-Fei; Zhang, Jia; Shen, Kai; Gao, Ping-Jin; Wang, Hai-Ya; Jin, Xin; Meng, Chao; Fang, Ning-Yuan

    2015-04-01

    Vascular adventitia and adventitia‑derived reactive oxygen species (ROS) contribute to vascular remodeling following vascular injury. A previous ex vivo study in adventitial fibroblasts showed that catalase, one of most important anti‑oxide enzymes, was downregulated by angiotensin II (AngII). The aim of the present study was to investigate whether adventitial gene transfer of catalase affects AngII‑induced vascular remodeling in vivo. Adenoviruses co‑expressing catalase and enhanced green fluorescent protein (eGFP) or expressing eGFP only were applied to the adventitial surface of common carotid arteries of Sprague‑Dawley rats. Alzet minipumps administering AngII (0.75 mg/kg/day) were then implanted subcutaneously for 14 days. Systolic blood pressure and biological parameters of vascular remodeling were measured in each group. Adventitial fibroblasts were cultured and p38 mitogen‑activated protein kinase (MAPK) phosphorylation was measured using western blot analysis. The results showed that adventitial gene transfer of catalase had no effect on AngII‑induced systolic blood pressure elevation. However, catalase adenovirus transfection significantly inhibited AngII‑induced media hypertrophy compared with that of the control virus (Pcatalase transfection significantly attenuated AngII‑induced ROS generation, macrophage infiltration, collagen deposition and adventitial α‑smooth muscle actin expression. Furthermore, catalase transfection significantly inhibited the AngII‑induced increase in p38MAPK phosphorylation. In conclusion, the results of the present study demonstrated that adventitial gene transfer of catalase significantly attenuated AngII‑induced vascular remodeling in rats via inhibition of adventitial p38MAPK phosphorylation.

  1. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    with variable risk of RIF (grouped into five classes from low to high risk) were irradiated with two different schemes: 1x3.5Gy with RNA isolated 2 and 24h after irradiation, and a fractionated scheme with 3x3.5Gy in intervals of 24h with RNA isolated 2h after the last dose. RNA was also isolated from non......BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals...

  2. Temporal gene expression profiling during rat femoral marrow ablation-induced intramembranous bone regeneration.

    Directory of Open Access Journals (Sweden)

    Joel K Wise

    -ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

  3. NR4A1 (Nur77 mediates thyrotropin-releasing hormone-induced stimulation of transcription of the thyrotropin β gene: analysis of TRH knockout mice.

    Directory of Open Access Journals (Sweden)

    Yasuyo Nakajima

    Full Text Available Thyrotropin-releasing hormone (TRH is a major stimulator of thyrotropin-stimulating hormone (TSH synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1 TRH is a highly specific regulator of the TSHβ gene, and 2 TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and

  4. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    Science.gov (United States)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  5. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

  6. Microarray analysis of genes differentially expressed in placentas of pregnancy-induced hypertension patients

    Institute of Scientific and Technical Information of China (English)

    李东红; 黄飞; 郑维国; 姜锋; 高平

    2003-01-01

    Objective: To uncover new clue for the research of the etiology of pregnancy-induced hypertension (PIH) by testing the gene expression difference between preeclamptic placentas and normal ones. Methods: mRNA level of 4 PIH placentas were examined using 4000 feature cDNA microarray in comparison with the pooled control consisting of total RNA from 4 cases of PIH placentas after the control cDNA and experimental cDNA were labeled by cy3 and cy5 respectively. Results: Fifty-eight to 131 genes were found down or up-regulated in 4 runs of hybridization. Among the differentially expressed genes, 22 genes, including genes encoding secreted protein ADRP, CYR61, EPI and HIF2, had the concordance in at least 2 cases were up-regulated or down-regulated. Conclusion: cDNA microarray is a high throughput and time-saving method to monitor the altered gene expression and the result could provide interesting clue and strategy for the etiological research of PIH.

  7. Exposure to ionizing radiation induced persistent gene expression changes in mouse mammary gland

    Directory of Open Access Journals (Sweden)

    Datta Kamal

    2012-12-01

    Full Text Available Abstract Background Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure. Methods Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data. Results Compared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p Conclusions Exposure to a clinically relevant radiation dose led to long-term activation of mammary gland genes involved in proliferative and metabolic pathways, which are known to have roles in carcinogenesis. When considered along with downregulation of a number of tumor suppressor genes, our study has implications for breast cancer initiation and progression after therapeutic radiation exposure.

  8. Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Ana M. Crane

    2015-04-01

    Full Text Available Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC lines. We then utilized zinc-finger nucleases (ZFNs, designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR. We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

  9. Gene Mutation as Biomarker of Radiation Induced Cell Injury and Genomic Instability

    Directory of Open Access Journals (Sweden)

    M Syaifudin

    2006-07-01

    Full Text Available Gene expression profiling and its mutation has become one of the most widely used approaches to identify genes and their functions in the context of identify and categorize genes to be used as radiation effect markers including cell and tissue sensitivities. Ionizing radiation produces genetic damage and changes in gene expression that may lead to cancer due to specific protein that controlling cell proliferation altered the function, its expression or both. P53 protein encoded by p53 gene plays an important role in protecting cell by inducing growth arrest and or cell suicide (apoptosis after deoxyribonucleic acid (DNA damage induced by mutagen such as ionizing radiation. The mutant and thereby dysfunctional of this gene was found in more than 50% of various human cancers, but it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests that alterations in the p53 gene might lead to oncogenic transformation. Current attractive model of carcinogenesis also showed that p53 gene is the major target of radiation. The majority of p53 mutations found so far is single base pairchanges (point mutations, which result in amino acid substitutionsor truncated forms of the P53 protein, and are widely distributedthroughout the evolutionarily conserved regions of the gene. Examination of p53 mutations in human cancer also shows an association between particular

  10. The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.

    Directory of Open Access Journals (Sweden)

    Mei Qiang

    Full Text Available BACKGROUND: The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B gene expression was persistently up-regulated following chronic intermittent ethanol (CIE treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5'-regulatory area following CIE treatment. METHODS: Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5'-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. RESULTS: Analysis of individual CpG methylation sites within the NR2B 5'regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced

  11. Kinetics of gene expression and bone remodelling in the clinical phase of collagen induced arthritis

    DEFF Research Database (Denmark)

    Denninger, Katja Caroline Marie; Litman, Thomas; Marstrand, Troels

    2015-01-01

    Introduction: Pathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time...... osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1...

  12. Molecular mapping of a new induced gene for nuclear male sterility in sunflower (Helianthus annuus L.)

    Science.gov (United States)

    A new NMS line, NMS HA89-872, induced by mitomycin C and streptomycin carries a single recessive male-sterile gene ms6. An F2 population of 88 plants was obtained from a cross between nuclear male-sterile mutant NMS HA89-872 (msms) and male-fertile line RHA271 (MsMs). 225 SSR primers and 9 RFLP-deri...

  13. Delivery of antioxidant enzyme genes to protect against ischemia/reperfusion-induced injury to retinal microvasculature.

    Science.gov (United States)

    Chen, Baihua; Caballero, Sergio; Seo, Soojung; Grant, Maria B; Lewin, Alfred S

    2009-12-01

    Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.

  14. Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells.

    Science.gov (United States)

    Geck, P; Szelei, J; Jimenez, J; Lin, T M; Sonnenschein, C; Soto, A M

    1997-01-01

    Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12-18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.

  15. Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion.

    Science.gov (United States)

    Dassanayake, Rohana P; Orrú, Christina D; Hughson, Andrew G; Caughey, Byron; Graça, Telmo; Zhuang, Dongyue; Madsen-Bouterse, Sally A; Knowles, Donald P; Schneider, David A

    2016-03-01

    Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200  mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15  h. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

  16. Antemortem detection of chronic wasting disease prions in nasal brush collections and rectal biopsies from white-tailed deer by real time quaking-induced conversion

    Science.gov (United States)

    Haley, Nicholas J.; Siepker, Chris; Walter, William D.; Thomsen, Bruce V.; Greenlee, Justin J.; Lehmkuhl, Aaron D.; Richt, Jürgen a.

    2016-01-01

    Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly 50 years ago in Colorado and Wyoming and has since spread to cervids in 23 states, two Canadian provinces, and the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of its spread; this is especially true in cases of relocation/reintroduction of farmed or free-ranging deer and elk or surveillance studies of private or protected herds, where depopulation is contraindicated. This study sought to evaluate the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay by using recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens and nasal brush samples collected antemortem from farmed white-tailed deer (n = 409). Antemortem findings were then compared to results from ante- and postmortem samples (RAMALT, brainstem, and medial retropharyngeal lymph nodes) evaluated by using the current gold standard in vitro assay, immunohistochemistry (IHC) analysis. We hypothesized that the sensitivity of RT-QuIC would be comparable to IHC analysis in antemortem tissues and would correlate with both the genotype and the stage of clinical disease. Our results showed that RAMALT testing by RT-QuIC assay had the highest sensitivity (69.8%) compared to that of postmortem testing, with a specificity of >93.9%. These data suggest that RT-QuIC, like IHC analysis, is an effective assay for detection of PrPCWD in rectal biopsy specimens and other antemortem samples and, with further research to identify more sensitive tissues, bodily fluids, or experimental conditions, has potential for large-scale and rapid automated testing for CWD diagnosis.

  17. Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth.

    Science.gov (United States)

    Liu, Ming; Hodish, Israel; Haataja, Leena; Lara-Lemus, Roberto; Rajpal, Gautam; Wright, Jordan; Arvan, Peter

    2010-11-01

    Type 1B diabetes (typically with early onset and without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed mutant proinsulins exerting dominant-negative (toxic gain of function) behavior in pancreatic beta cells. Here we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that, in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

  18. Analysis of human transforming growth factor β-induced gene mutation in corneal dystrophy

    Institute of Scientific and Technical Information of China (English)

    李杨; 孙旭光; 任慧媛; 董冰; 王智群; 孙秀英

    2004-01-01

    Background Corneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy. Methods Three families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor β-induced gene (BIGH3 gene) was performed. Results Five individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H. Conclusion Molecular genetic analysis can improve accurate diagnosis of corneal dystrophy.

  19. Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina.

    Science.gov (United States)

    Liu, Mao-Sen; Chien, Ching-Te; Lin, Tsan-Piao

    2008-04-01

    Selaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography. In this study, differences in gene expression and ABA contents were also analyzed during dehydration. The results revealed that trehalose (at >130 mg g(-1) DW) was the major soluble sugar, and low saturated fatty acid content in PLs (0.31) was maintained in both hydrated and desiccated tissues. In addition, the ABA content of S. tamariscina increased 3-fold, and genes involved in ABA signaling and cellular protection were up-regulated while photosystem-related genes were down-regulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.

  20. Stability of Barley stripe mosaic virus induced gene silencing in barley

    DEFF Research Database (Denmark)

    Bruun-Rasmussen, Marianne; Madsen, Christian Toft; Jessing, Stine

    2007-01-01

    Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector...... for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting...... inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector....

  1. Immediate-early Inducible Function in Upstream Region of junB Gene

    Institute of Scientific and Technical Information of China (English)

    HONG WAN; HIROSHI ISHIHARA; IZUMI TANAKA

    2006-01-01

    Objective To analyze the upstream region of radiation-induced junB gene. Methods Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/β-actin measured by quantitative Northern blot hybridization. Results CAT mRNA expression containing 900 bp and 1560 bpjunB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation. Conclusions 590~900 bp fragments located in the upstream region ofjunB gene play an important role in the early process of cells against radiation.

  2. Time delay induced transition of gene switch and stochastic resonance in a genetic transcriptional regulatory model

    Science.gov (United States)

    2012-01-01

    Background Noise, nonlinear interactions, positive and negative feedbacks within signaling pathways, time delays, protein oligomerization, and crosstalk between different pathways are main characters in the regulatory of gene expression. However, only a single noise source or only delay time in the deterministic model is considered in the gene transcriptional regulatory system in previous researches. The combined effects of correlated noise and time delays on the gene regulatory model still remain not to be fully understood. Results The roles of time delay on gene switch and stochastic resonance are systematically explored based on a famous gene transcriptional regulatory model subject to correlated noise. Two cases, including linear time delay appearing in the degradation process (case I) and nonlinear time delay appearing in the synthesis process (case II) are considered, respectively. For case I: Our theoretical results show that time delay can induce gene switch, i.e., the TF-A monomer concentration shifts from the high concentration state to the low concentration state ("on"→"off"). With increasing the time delay, the transition from "on" to "off" state can be further accelerated. Moreover, it is found that the stochastic resonance can be enhanced by both the time delay and correlated noise intensity. However, the additive noise original from the synthesis rate restrains the stochastic resonance. It is also very interesting that a resonance bi-peaks structure appears under large additive noise intensity. The theoretical results by using small-delay time-approximation approach are consistent well with our numerical simulation. For case II: Our numerical simulation results show that time delay can also induce the gene switch, however different with case I, the TF-A monomer concentration shifts from the low concentration state to the high concentration state ("off"→"on"). With increasing time delay, the transition from "on" to "off" state can be further

  3. Involvement of multiple transcription factors for metal-induced spy gene expression in Escherichia coli.

    Science.gov (United States)

    Yamamoto, Kaneyoshi; Ogasawara, Hiroshi; Ishihama, Akira

    2008-01-20

    Bacteria are directly exposed to metals in environment. To maintain the intracellular metal homeostasis, Escherichia coli contain a number of gene regulation systems, each for response to a specific metal. A periplasmic protein Spy of E. coli was found to be induced upon short-exposure to copper ion in CpxAR-dependent manner. Transcription of the spy gene was also induced by long-exposure to zinc ion. This induction, however, depended on another two-component system BaeSR. Using DNase-I footprinting assay, we identified two BaeR-binding regions on the spy promoter with a direct repeat of the BaeR-box sequence, TCTNCANAA. The zinc-responsive BaeR-binding sites were separated from copper-responsive CpxR-binding site, implying that the spy promoter responds to two species of metal independently through different using sensor-response regulator systems. Since BaeSR-dependent zinc response requires longer time, the induction of spy gene transcription by external zinc may include multiple steps such as through sensing the zinc-induced envelope disorder by BaeSR.

  4. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  5. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Kunkel Thomas A

    2005-06-01

    Full Text Available Abstract Background Antibody genes are diversified by somatic hypermutation (SHM, gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID. According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base excision repair. The DNA sequence context of mutation hotspots at G-C pairs during SHM is DGYW/WRCH (G-C is a hotspot position, R = A/G, Y = T/C, W = A/T, D = A/G/T. Results To investigate the mechanisms of AID-induced mutagenesis in a model system, we studied the genetic consequences of AID expression in yeast. We constructed a yeast vector with an artificially synthesized human AID gene insert using codons common to highly expressed yeast genes. We found that expression of the artificial hAIDSc gene was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain. A majority of mutations were at G-C pairs. In the ung1 strain, C-G to T-A transitions were found almost exclusively, while a mixture of transitions with 12% transversions was characteristic in the wild-type strain. In the ung1 strain mutations that could have originated from deamination of the transcribed stand were found more frequently. In the wild-type strain, the strand bias was reversed. DGYW/WRCH motifs were preferential sites of mutations. Conclusion The results are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. The sequence contexts of mutations in yeast induced by AID and those of somatic mutations at G-C pairs in immunoglobulin genes are significantly similar. This indicates that the intrinsic substrate specificity of AID itself is a primary determinant of mutational hotspots at G

  6. Comparison of gene expression changes induced by biguanides in db/db mice liver.

    Science.gov (United States)

    Heishi, Masayuki; Hayashi, Koji; Ichihara, Junji; Ishikawa, Hironori; Kawamura, Takao; Kanaoka, Masaharu; Taiji, Mutsuo; Kimura, Toru

    2008-08-01

    Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.

  7. Arabidopsis FLOWERING LOCUS D influences systemic-acquiredresistance-induced expression and histone modifications of WRKY genes

    Indian Academy of Sciences (India)

    Vijayata Singh; Shweta Roy; Deepjyoti Singh; Ashis Kumar Nandi

    2014-03-01

    A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.

  8. Insulin-induced gene 2 expression correlates with colorectal cancer metastasis and disease outcome.

    Science.gov (United States)

    Sun, Shengjie; Zhang, Guoqing; Sun, Qiong; Wu, Zhiyong; Shi, Weiwei; Yang, Bo; Li, Ying

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide accounting for ∼9% of cancer-related deaths, 90% of which are due to metastasis resulting from resistance to chemotherapeutic agents. Hence, it is imperative to develop novel biomarkers of CRC. Insulin-induced gene 2 (INSIG2) has been previously reported to be a negative regulator of cholesterol synthesis and was recently identified as a putative-positive prognostic biomarker for colon and pancreatic cancer prognosis. Even though it has been suggested as a colon cancer biomarker and as an inhibitor of Bax-mediated apoptosis, the role of INSIG2 in CRC is elusive. We initially validated that INSIG2 is a gene with univariate-negative prognostic capacity to discriminate human colon cancer survivorship and that if present along with adenomatous polyposis coli (APC) gene mutations further decrease overall survival. Gain- and loss-of-function studies of INSIG2 showed that the gene product is responsible for inducing migration and invasion and maintenance of the mesenchymal phenotype in vitro and metastasis in vivo. Interestingly, loss of INSIG2 did not affect tumorigenic potential per se, but affected hepatic invasion in a xenograft assay. Our findings reinforce that INSIG2 is a novel colon cancer biomarker, and suggest, for the first time, an exclusive connection between INSIG2 and metastatic dissemination without any effect on tumorigenesis. © 2015 IUBMB Life, 68(1):65-71, 2016.

  9. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    Science.gov (United States)

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-01-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

  10. Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina.

    Directory of Open Access Journals (Sweden)

    Martine Emery

    Full Text Available Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining "normal" GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.

  11. Gene expression microarray analysis of early oxygen-induced retinopathy in the rat.

    Science.gov (United States)

    Tea, Melinda; Fogarty, Rhys; Brereton, Helen M; Michael, Michael Z; Van der Hoek, Mark B; Tsykin, Anna; Coster, Douglas J; Williams, Keryn A

    2009-12-12

    Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

  12. Production of transgenic chickens expressing a tetracycline-inducible GFP gene.

    Science.gov (United States)

    Kwon, Mo Sun; Koo, Bon Chul; Roh, Ji Yeol; Kim, Minjee; Kim, Jin-Hoi; Kim, Teoan

    2011-07-15

    There is much interest in using farm animals as 'bioreactors' to produce large quantities of biopharmaceuticals. However, uncontrolled constitutive expression of foreign genes have been known to cause serious physiological disturbances in transgenic animals. The objective of this study was to test the feasibility of the controllable expression of an exogenous gene in the chicken. A retrovirus vector was designed to express GFP (green fluorescent protein) and rtTA (reverse tetracycline-controlled transactivator) under the control of the tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter, respectively. G0 founder chickens were produced by infecting the blastoderm of freshly laid eggs with concentrated retrovirus vector. Feeding the chickens obtained with doxycycline, a tetracycline derivative, resulted in emission of green body color under fluorescent light, and no apparent significant physiological dysfunctions. Successful germline transmission of the exogenous gene was also confirmed. Expression of the GFP gene reverted to the pre-induction levels when doxycycline was removed from the diet. The results showed that a tetracycline-inducible expression system in transgenic animals might be a promising solution to minimize physiological disturbances caused by the transgene. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts

    Directory of Open Access Journals (Sweden)

    Tui Neri

    2015-10-01

    Full Text Available Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41+ cells gradually gave rise to CD45+ cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.

  14. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  15. RNA-Seq reveals infection-induced gene expression changes in the snail intermediate host of the carcinogenic liver fluke, Opisthorchis viverrini.

    Directory of Open Access Journals (Sweden)

    Sattrachai Prasopdee

    2014-03-01

    Full Text Available BACKGROUND: Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay. METHODOLOGY/PRINCIPAL FINDINGS: Using high-throughput (Illumina sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs, associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts. CONCLUSIONS/SIGNIFICANCE: The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail

  16. Contribution of gene expression to metabolic fluxes in hypermetabolic livers induced through burn injury and cecal ligation and puncture in rats.

    Science.gov (United States)

    Banta, Scott; Vemula, Murali; Yokoyama, Tadaaki; Jayaraman, Arul; Berthiaume, François; Yarmush, Martin L

    2007-05-01

    Severe injury activates many stress-related and inflammatory pathways that can lead to a systemic hypermetabolic state. Prior studies using perfused hypermetabolic rat livers have identified intrinsic metabolic flux changes that were not dependent upon the continual presence of elevated stress hormones and substrate loads. We investigated the hypothesis that such changes may be due to persistent alterations in gene expression. A systemic hypermetabolic response was induced in rats by applying a moderate burn injury followed 2 days later by cecum ligation and puncture (CLP) to produce sepsis. Control animals received a sham-burn followed by CLP, or a sham-burn followed by sham-CLP. Two days after CLP, livers were analyzed for gene expression changes using DNA microarrays and for metabolism alterations by ex vivo perfusion coupled with Metabolic Flux Analysis. Burn injury prior to CLP increased fluxes while decreases in gene expression levels were observed. Conversely, CLP alone significantly increased metabolic gene expression, but decreased many of the corresponding metabolic fluxes. Burn injury combined with CLP led to the most dramatic changes, where concurrent changes in fluxes and gene expression levels occurred in about 1/3 of the reactions. The data are consistent with the notion that in this model, burn injury prior to CLP increased fluxes through post-translational mechanisms with little contribution of gene expression, while CLP treatment up-regulated the metabolic machinery by transcriptional mechanisms. Overall, these data show that mRNA changes measured at a single time point by DNA microarray analysis do not reliably predict metabolic flux changes in perfused livers.

  17. Schisming: The Collaborative Transformation from a Single Conversation to Multiple Conversations

    DEFF Research Database (Denmark)

    Egbert, Maria

    1997-01-01

    This article examines schisming, a phenomenon that relates to how the number of members in a group is related to the interaction of the group. More specifically, schisming is the observation that in a conversation with at least four participants, the conversation may systematically split into two...... or more smaller conversations. In the data examined here, schisming is induced when, during a conversation, one person introduces a turn oriented to a subset of coparticipants who break away from the ongoing conversation and establish a new conversation while the rest of the group continues the original...

  18. Gene expression patterns underlying parasite-induced alterations in host behaviour and life history.

    Science.gov (United States)

    Feldmeyer, Barbara; Mazur, Johanna; Beros, Sara; Lerp, Hannes; Binder, Harald; Foitzik, Susanne

    2016-01-01

    Many parasites manipulate their hosts' phenotype. In particular, parasites with complex life cycles take control of their intermediate hosts' behaviour and life history to increase transmission to their definitive host. The proximate mechanisms underlying these parasite-induced alterations are poorly understood. The cestode Anomotaenia brevis affects the behaviour, life history and morphology of parasitized Temnothorax nylanderi ants and indirectly of their unparasitized nestmates. To gain insights on how parasites alter host phenotypes, we contrast brain gene expression patterns of T. nylanderi workers parasitized with the cestode, their unparasitized nestmates and unparasitized workers from unparasitized colonies. Over 400 differentially expressed genes between the three groups were identified, with most uniquely expressed genes detected in parasitized workers. Among these are genes that can be linked to the increased lifespan of parasitized workers. Furthermore, many muscle (functionality) genes are downregulated in these workers, potentially causing the observed muscular deformations and their inactive behaviour. Alterations in lifespan and activity could be adaptive for the parasite by increasing the likelihood that infected workers residing in acorns are eaten by their definitive host, a woodpecker. Our transcriptome analysis reveals numerous gene expression changes in parasitized workers and their uninfected nestmates and indicates possible routes of parasite manipulation. Although causality still needs to be established, parasite-induced alterations in lifespan and host behaviour appear to be partly explained by morphological muscle atrophy instead of central nervous system interference, which is often the core of behavioural regulation. Results of this study will shed light upon the molecular basis of antagonistic species interactions.

  19. Transgene-induced gene silencing is not affected by a change in ploidy level.

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    Daniela Pignatta

    Full Text Available BACKGROUND: Whole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Diploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS inverted repeat (hairpin constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA. CONCLUSIONS/SIGNIFICANCE: Our results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

  20. Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

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    Takashi Maejima

    Full Text Available Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3 and thrombomodulin (THBD, were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2 family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET, and real time chromosome conformation capture (3C assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.

  1. An efficient virus-induced gene silencing vector for maize functional genomics research.

    Science.gov (United States)

    Wang, Rong; Yang, Xinxin; Wang, Nian; Liu, Xuedong; Nelson, Richard S; Li, Weimin; Fan, Zaifeng; Zhou, Tao

    2016-04-01

    Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  2. Inducible removal of UV-induced pyrimidine dimers from transcriptionally active and inactive genes of Saccharomyces cerevisiae.

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    Waters, R; Zhang, R; Jones, N J

    1993-05-01

    The prior UV irradiation of alpha haploid Saccharomyces cerevisiae with a UV dose of 25 J/m2 substantially increases the repairability of damage subsequently induced by a UV dose of 70 J/m2 given 1 h after the first irradiation. This enhancement of repair is seen at both the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. The presence in the medium of the protein synthesis inhibitor, cycloheximide in the period between the two irradiations eliminated this effect. Enhanced repair still occurred if cycloheximide was present only after the final UV irradiation. This indicated that the first result is not due to cycloheximide merely blocking the synthesis of repair enzymes associated with a hypothetical rapid turnover of such molecules. The enhanced repairability is not the result of changes in chromatin accessibility without protein synthesis, merely caused by the repair of the damage induced by the prior irradiation. The data clearly show that a UV-inducible removal of pyrimidine dimers has occurred which involves the synthesis of new proteins. The genes known to possess inducible promoters, and which are involved in excision are RAD2, RAD7, RAD16 and RAD23. Studies with the rad7 and rad16 mutants which are defective in the ability to repair HML alpha and proficient in the repair of MAT alpha showed that in rad7, preirradiation enhanced the repair at MAT alpha, whereas in rad16 this increased repai