Science.gov

Sample records for promoter tissue specificity

  1. Tissue Specific Promoters in Colorectal Cancer

    PubMed Central

    Rama, A. R.; Aguilera, A.; Melguizo, C.; Caba, O.; Prados, J.

    2015-01-01

    Colorectal carcinoma is the third most prevalent cancer in the world. In the most advanced stages, the use of chemotherapy induces a poor response and is usually accompanied by other tissue damage. Significant progress based on suicide gene therapy has demonstrated that it may potentiate the classical cytotoxic effects in colorectal cancer. The inconvenience still rests with the targeting and the specificity efficiency. The main target of gene therapy is to achieve an effective vehicle to hand over therapeutic genes safely into specific cells. One possibility is the use of tumor-specific promoters overexpressed in cancers. They could induce a specific expression of therapeutic genes in a given tumor, increasing their localized activity. Several promoters have been assayed into direct suicide genes to cancer cells. This review discusses the current status of specific tumor-promoters and their great potential in colorectal carcinoma treatment. PMID:26648599

  2. Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice

    PubMed Central

    Wang, Rui; Zhu, Menglin; Ye, Rongjian; Liu, Zuoxiong; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2015-01-01

    As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice. PMID:26655679

  3. Evaluation of a novel promoter from Populus trichocarpa for mature xylem tissue specific gene delivery.

    PubMed

    Nguyen, Van Phap; Cho, Jin-Seong; Choi, Young-Im; Lee, Sang-Won; Han, Kyung-Hwan; Ko, Jae-Heung

    2016-07-01

    Wood (i.e., secondary xylem) is an important raw material for many industrial applications. Mature xylem (MX) tissue-specific genetic modification offers an effective means to improve the chemical and physical properties of the wood. Here, we describe a promoter that drives strong gene expression in a MX tissue-specific manner. Using whole-transcriptome genechip analyses of different tissue types of poplar, we identified five candidate genes that had strong expression in the MX tissue. The putative promoter sequences of the five MX-specific genes were evaluated for their promoter activity in both transgenic Arabidopsis and poplar. Among them, we found the promoter of Potri.013G007900.1 (called the PtrMX3 promoter) had the strongest activity in MX and thus was further characterized. In the stem and root tissues of transgenic Arabidopsis plants, the PtrMX3 promoter activity was found exclusively in MX tissue. MX-specific activity of the promoter was reproduced in the stem tissue of transgenic poplar plants. The PtrMX3 promoter activity was not influenced by abiotic stresses or exogenously applied growth regulators, indicating the PtrMX3 promoter is bona fide MX tissue-specific. Our study provides a strong MX-specific promoter for MX-specific modifications of woody biomass. PMID:27038601

  4. Rosa26 Locus Supports Tissue-Specific Promoter Driving Transgene Expression Specifically in Pig

    PubMed Central

    Ma, Jing; Huang, Tianqing; Jiang, Dandan; Xie, Bingteng; Wu, Meiling; Wang, Jiaqiang; Song, Yuran; Wang, Ying; He, Yilong; Sun, Jialu; Hu, Kui; Guo, Runfa; Wang, Liu; Zhou, Qi; Mu, Yanshuang; Liu, Zhonghua

    2014-01-01

    Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus. PMID:25232950

  5. Cloning and characterization of a novel Athspr promoter specifically active in vascular tissue.

    PubMed

    Zhang, Liang; Yang, Tao; Li, Xiaoying; Hao, Hongyan; Xu, Shengtao; Cheng, Wei; Sun, Yingli; Wang, Chongying

    2014-05-01

    The vascular system--xylem, phloem and the cambium--is essential for water supply, nutrient transport, and physical support in higher plants. Although it is known that vascular-specific gene expression is regulated by cis-acting regulatory sequences in promoters, it is largely unknown how many regulatory elements exist and what their roles are in promoters. To understand the regulatory elements of vascular-specific promoters and their roles in vascular development, a T-DNA insertion mutant showing delayed growth and diminished resistance to environmental stress was isolated using promoter trap strategy. The novel gene, Arabidopsis thaliana heat shock protein-related (Athspr), was cloned from Arabidopsis ecotype C24. Strong GUS (β-glucuronidase) staining in the original promoter trap line was found in the vascular tissues of all organs in the mutant. The Athspr promoter was cloned and fused with GUS and eGFP (enhanced green fluorescent protein) reporter genes to verify its vascular-specific expression in Arabidopsis. Further histochemical analysis in transgenic plants demonstrated a similar GUS expression pattern in the vascular tissues. In addition, ATHSPR-eGFP driven by Athspr promoter was observed in vascular bundles of the transgenic seedling roots. Finally, comparative analysis with promoter motifs from 37 genes involved in vascular development revealed that Athspr and all other promoters active in vascular tissues contained regulatory elements responding to phytohormones, light, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrated that the Athspr promoter has a vascular tissue-specific activity and Athspr may have multiple functions in vascular development and resistance against various stresses. PMID:24675528

  6. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori

    SciTech Connect

    Liu Yan; Yu Lian; Guo Xiuyang; Guo Tingqing; Wang Shengpeng; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-31

    The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

  7. Acquired Tissue-Specific Promoter Bivalency Is a Basis for PRC2 Necessity in Adult Cells.

    PubMed

    Jadhav, Unmesh; Nalapareddy, Kodandaramireddy; Saxena, Madhurima; O'Neill, Nicholas K; Pinello, Luca; Yuan, Guo-Cheng; Orkin, Stuart H; Shivdasani, Ramesh A

    2016-06-01

    Bivalent promoters in embryonic stem cells (ESCs) carry methylation marks on two lysine residues, K4 and K27, in histone3 (H3). K4me2/3 is generally considered to promote transcription, and Polycomb Repressive Complex 2 (PRC2) places K27me3, which is erased at lineage-restricted genes when ESCs differentiate in culture. Molecular defects in various PRC2 null adult tissues lack a unifying explanation. We found that epigenomes in adult mouse intestine and other self-renewing tissues show fewer and distinct bivalent promoters compared to ESCs. Groups of tissue-specific genes that carry bivalent marks are repressed, despite the presence of promoter H3K4me2/3. These are the predominant genes de-repressed in PRC2-deficient adult cells, where aberrant expression is proportional to the H3K4me2/3 levels observed at their promoters in wild-type cells. Thus, in adult animals, PRC2 specifically represses genes with acquired, tissue-restricted promoter bivalency. These findings provide new insights into specificity in chromatin-based gene regulation. PMID:27212235

  8. Novel strong tissue specific promoter for gene expression in human germ cells

    PubMed Central

    2010-01-01

    Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12), and an important role - in the rest two cell lines. PMID:20716342

  9. Taproot promoters cause tissue specific gene expression within the storage root of sugar beet.

    PubMed

    Oltmanns, Heiko; Kloos, Dorothee U; Briess, Waltraud; Pflugmacher, Maike; Stahl, Dietmar J; Hehl, Reinhard

    2006-08-01

    The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots. PMID:16482437

  10. Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter

    PubMed Central

    Williams, Scott S.; Cobo-Stark, Patricia; Hajarnis, Sachin; Aboudehen, Karam; Shao, Xinli; Richardson, James A.; Patel, Vishal

    2014-01-01

    Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues. PMID:24899057

  11. Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

    PubMed Central

    Heard, J M; Herbomel, P; Ott, M O; Mottura-Rollier, A; Weiss, M; Yaniv, M

    1987-01-01

    The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription. Images PMID:3475566

  12. Screening of Tissue-Specific Genes and Promoters in Tomato by Comparing Genome Wide Expression Profiles of Arabidopsis Orthologues

    PubMed Central

    Lim, Chan Ju; Lee, Ha Yeon; Kim, Woong Bom; Lee, Bok-Sim; Kim, Jungeun; Ahmad, Raza; Kim, Hyun A; Yi, So Young; Hur, Cheol-Goo; Kwon, Suk-Yoon

    2012-01-01

    Constitutive overexpression of transgenes occasionally interferes with normal growth and developmental processes in plants. Thus, the development of tissue-specific promoters that drive transgene expression has become agriculturally important. To identify tomato tissue-specific promoters, tissue-specific genes were screened using a series of in silico-based and experimental procedures, including genome-wide orthologue searches of tomato and Arabidopsis databases, isolation of tissue-specific candidates using an Arabidopsis microarray database, and validation of tissue specificity by reverse transcription-polymerase chain reaction (RT-PCR) analysis and promoter assay. Using these procedures, we found 311 tissue-specific candidate genes and validated 10 tissue-specific genes by RT-PCR. Among these identified genes, histochemical analysis of five isolated promoter::GUS transgenic tomato and Arabidopsis plants revealed that their promoters have different but distinct tissue-specific activities in anther, fruit, and root, respectively. Therefore, it appears these in silico-based screening approaches in addition to the identification of new tissue-specific genes and promoters will be helpful for the further development of tailored crop development. PMID:22699756

  13. Characterization of tissue-specific transcription by the human synapsin I gene promoter

    SciTech Connect

    Thiel, G. Univ. of Texas, Dallas ); Greengard, P. ); Suedhof, T.C. )

    1991-04-15

    Synapsin Ia and synapsin Ib are abundant synaptic vesicle proteins that are derived by differential splicing from a single gene. To identify control elements directing the neuronal expression of synapsins Ia/b, the authors functionally analyzed the promoter region of the human synapsin I gene. A hybrid gene was constructed containing 2 kilobases of 5{prime} flanking sequence from the synapsin I gene fused to the bacterial gene chloramphenicol acetyltransferase and transfected into 12 different neuronal and nonneuronal cell lines. In general, expression of the chimeric reporter gene showed excellent correlation with endogenous expression of synapsin I in different neuronal cell lines, whereas transcription was low in all nonneuronal cell lines examined. The addition of the simian virus 40 enhancer promoted non-tissue-specific expression. Deletion mutagenesis of the synapsin I promoter revealed the presence of positive and negative sequence elements. A basal (constitutive) promoter that directs reporter gene expression in neuronal and nonneuronal cell lines was mapped to the region {minus}115 to +47. The promoter region from {minus}422 to {minus}22 contains positive elements that upon fusion with the herpes simplex virus thymidine kinase promoter potentiate its transcription in PC12 and neuroblastoma cells but not in Chinese hamster ovary cells.

  14. Tissue-specific activity of two manganese superoxide dismutase promoters in transgenic tobacco.

    PubMed Central

    Van Camp, W; Hérouart, D; Willekens, H; Takahashi, H; Saito, K; Van Montagu, M; Inzé, D

    1996-01-01

    In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity. PMID:8883376

  15. [shRNAs driven by K14 promoter induce tissue-specific RNA interference].

    PubMed

    Dai, Rong; Shen, Si-Jun; Wan, Peng-Cheng; Shi, Guo-Qing; Meng, Qing-Yong; Liu, Shou-Ren

    2011-07-01

    RNA interference is an efficient method for exploring gene function. Accumulating evidence suggests that RNA Pol II promoters can direct cell- or tissue-specific gene silencing. A eGFP-shRNA fusion construct transcribed from an RNA Pol II promoter (K14 promoter) was used to induce gene-specific shRNA silencing ofBMP4 gene expression. Recombinant vectors (pEGFP-C1-shRNA, psiCHECK-BMP4, and pEGFP-K14-shRNA) were constructed. Vectors pEGFP-C1-shRNA and psiCHECK-BMP4 were cotransfected into Hela cells (in vitro) and shRNA-induced inhibition efficiency was tested by a luciferase assay. The results showed that all the six interference sequences inhibited the expression of BMP4 with high efficiency (>60%), and the interference sequence 5# showed the highest efficiency. For in vivo screening of JB6-C41 cells transfected with vector pEGFP-K14-shRNA, the inhibition efficiency was assayed by quantitative RT-PCR and Western blotting analyses. The results showed that the mRNA and protein products of the exogenous BMP4 gene were efficiently and specifically inhibited. The efficiency of gene silencing was greater than 60%, except for sequence 3#. The declines in mRNA and protein expression levels were significantly correlated during gene silence by the shRNA. This system may be adapted for in vivo shRNA expression and gene silencing. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in the analysis of the mechanisms of hair follicle development in sheep. PMID:22049690

  16. Tissue-specific promoters active in CD44+CD24-/low breast cancer cells.

    PubMed

    Bauerschmitz, Gerd J; Ranki, Tuuli; Kangasniemi, Lotta; Ribacka, Camilla; Eriksson, Minna; Porten, Marius; Herrmann, Isabell; Ristimäki, Ari; Virkkunen, Pekka; Tarkkanen, Maija; Hakkarainen, Tanja; Kanerva, Anna; Rein, Daniel; Pesonen, Sari; Hemminki, Akseli

    2008-07-15

    It has been proposed that human tumors contain stem cells that have a central role in tumor initiation and posttreatment relapse. Putative breast cancer stem cells may reside in the CD44(+)CD24(-/low) population. Oncolytic adenoviruses are attractive for killing of these cells because they enter through infection and are therefore not susceptible to active and passive mechanisms that render stem cells resistant to many drugs. Although adenoviruses have been quite safe in cancer trials, preclinical work suggests that toxicity may eventually be possible with more active agents. Therefore, restriction of virus replication to target tissues with tissues-specific promoters is appealing for improving safety and can be achieved without loss of efficacy. We extracted CD44(+)CD24(-/low) cells from pleural effusions of breast cancer patients and found that modification of adenovirus type 5 tropism with the serotype 3 knob increased gene delivery to CD44(+)CD24(-/low) cells. alpha-Lactalbumin, cyclo-oxygenase 2, telomerase, and multidrug resistance protein promoters were studied for activity in CD44(+)CD24(-/low) cells, and a panel of oncolytic viruses was subsequently constructed. Each virus featured 5/3 chimerism of the fiber and a promoter controlling expression of E1A, which was also deleted in the Rb binding domain for additional tumor selectivity. Cell killing assays identified Ad5/3-cox2L-d24 and Ad5/3-mdr-d24 as the most active agents, and these viruses were able to completely eradicate CD44(+)CD24(-/low) cells in vitro. In vivo, these viruses had significant antitumor activity in CD44(+)CD24(-/low)-derived tumors. These findings may have relevance for elimination of cancer stem cells in humans. PMID:18632604

  17. Tissue-specific Leptin promoter DNA methylation is associated with maternal and infant perinatal factors.

    PubMed

    Lesseur, Corina; Armstrong, David A; Paquette, Alison G; Koestler, Devin C; Padbury, James F; Marsit, Carmen J

    2013-12-01

    Leptin a regulator of body weight is involved in reproductive and developmental functions. Leptin promoter DNA methylation (LEP) regulates gene expression in a tissue-specific manner and has been linked to adverse pregnancy outcomes. In non-pathologic human pregnancies, we assessed LEP methylation, genotyped the single nucleotide polymorphism (SNP) rs2167270 in placental (n=81), maternal and cord blood samples (n=60), and examined the association between methylation, genotype, and perinatal factors. Maternal blood LEP methylation was lower in pre-pregnancy obese women (P=0.01). Cord blood LEP methylation was higher in small for gestational age (SGA) (P=4.6×10(-3)) and A/A genotype (P=1.6×10(-4)), lower (-1.47, P=0.03) in infants born to pre-pregnancy obese mothers and correlated (P=0.01) with maternal blood LEP. Gender was associated with placental LEP methylation (P=0.05). These results suggest that LEP epigenetic control may be influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissue-specific manner and may have multigenerational implications. PMID:23911897

  18. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.

    PubMed

    Zheng, Xuelian; Deng, Wei; Luo, Keming; Duan, Hui; Chen, Yongqin; McAvoy, Richard; Song, Shuiqing; Pei, Yan; Li, Yi

    2007-08-01

    Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. PMID:17340093

  19. Lysine-specific demethylase 1 promotes brown adipose tissue thermogenesis via repressing glucocorticoid activation.

    PubMed

    Zeng, Xing; Jedrychowski, Mark P; Chen, Yi; Serag, Sara; Lavery, Gareth G; Gygi, Steve P; Spiegelman, Bruce M

    2016-08-15

    Brown adipocytes display phenotypic plasticity, as they can switch between the active states of fatty acid oxidation and energy dissipation versus a more dormant state. Cold exposure or β-adrenergic stimulation favors the active thermogenic state, whereas sympathetic denervation or glucocorticoid administration promotes more lipid accumulation. Our understanding of the molecular mechanisms underlying these switches is incomplete. Here we found that LSD1 (lysine-specific demethylase 1), a histone demethylase, regulates brown adipocyte metabolism in two ways. On the one hand, LSD1 associates with PRDM16 to repress expression of white fat-selective genes. On the other hand, LSD1 represses HSD11B1 (hydroxysteroid 11-β-dehydrogenase isozyme 1), a key glucocorticoid-activating enzyme, independently from PRDM16. Adipose-specific ablation of LSD1 impaired mitochondrial fatty acid oxidation capacity of the brown adipose tissue, reduced whole-body energy expenditure, and increased fat deposition, which can be significantly alleviated by simultaneously deleting HSD11B1. These findings establish a novel regulatory pathway connecting histone modification and hormone activation with mitochondrial oxidative capacity and whole-body energy homeostasis. PMID:27566776

  20. Mining tissue-specific contigs from peanut (Arachis hypogaea L.) for promoter cloning by deep transcriptome sequencing.

    PubMed

    Geng, Lili; Duan, Xiaohong; Liang, Chun; Shu, Changlong; Song, Fuping; Zhang, Jie

    2014-10-01

    Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters. PMID:25231965

  1. Manipulation of lignin composition in plants using a tissue-specific promoter

    DOEpatents

    Chapple, Clinton C. S.

    2003-08-26

    The present invention relates to methods and materials in the field of molecular biology, the manipulation of the phenylpropanoid pathway and the regulation of proteins synthesis through plant genetic engineering. More particularly, the invention relates to the introduction of a foreign nucleotide sequence into a plant genome, wherein the introduction of the nucleotide sequence effects an increase in the syringyl content of the plant's lignin. In one specific aspect, the invention relates to methods for modifying the plant lignin composition in a plant cell by the introduction there into of a foreign nucleotide sequence comprising at issue specific plant promoter sequence and a sequence encoding an active ferulate-5-hydroxylase (F5H) enzyme. Plant transformants harboring an inventive promoter-F5H construct demonstrate increased levels of syringyl monomer residues in their lignin, rendering the polymer more readily delignified and, thereby, rendering the plant more readily pulped or digested.

  2. Tissue-specific binding of testis nuclear proteins to a sequence element within the promoter of the testis-specific histone H1t gene.

    PubMed

    Grimes, S R; Wolfe, S A; Koppel, D A

    1992-08-01

    The rat histone H1t gene is transcribed only in testis germinal cells. This testis-specific chromosomal protein is first synthesized during spermatogenesis in pachytene spermatocytes and the entire complement of testis histones is replaced during the midspermatid stage of spermiogenesis by positively charged transition nuclear proteins TP1 and TP2. Mobility shift assays conducted using crude nuclear protein extracts from different tissues and an 18-bp DNA sequence element within the H1t promoter as a probe reveal binding only with nuclear proteins from testis. The binding is specifically competed with an excess of the same unlabeled DNA fragment but not with heterologous competitors. A larger oligonucleotide corresponding to the same sequence element plus 18 bp of the adjacent downstream H1/CCAAT element binds nuclear proteins from all tissues tested, but a unique low mobility band is formed only with testis extracts. Protein-DNA crosslinking experiments reveal that two major polypeptides with molecular weights of approximately 13 and 30 kDa bind to the 18-bp H1t promoter sequence element. This strong correlation between the tissue where the H1t gene is transcribed and the presence of testis-specific nuclear proteins that bind to a sequence element within the testis histone H1t promoter supports the possibility that these DNA-binding proteins may participate in formation of an active transcription initiation complex with the testis H1t promoter. PMID:1632632

  3. Characterization of the human CD4 gene promoter: transcription from the CD4 gene core promoter is tissue-specific and is activated by Ets proteins.

    PubMed Central

    Salmon, P; Giovane, A; Wasylyk, B; Klatzmann, D

    1993-01-01

    We analyzed the 5' transcription control sequences of the human CD4 gene. We located the transcription initiation site and showed that the CD4 core promoter (positions -40 to +16) lacks a classical "TATA" or initiator positioning consensus sequence but directs precise and efficient transcription when coupled to the ubiquitously active simian virus 40 enhancer. The transcriptional activity of the CD4 gene promoter correlated with CD4 expression in various cell types. Interestingly, the CD4 core promoter also displayed a tissue-specific transcriptional activity. Within this fragment, three nucleic acid sequences are completely conserved in the murine CD4 gene. One of these sequences contains a perfect ETS consensus sequence. Another ETS consensus sequence is located 1060 nt upstream. Electrophoretic-mobility-shift assays showed that the core promoter ETS motif binds an Ets-related protein specifically expressed at high levels in CD4+ cells. Moreover, in CD4- cells, overexpression of Ets-1 or Ets-2 efficiently and specifically activated transcription from the CD4 promoter and core promoter. These data indicate that Ets transcription factors play a central role in controlling CD4 gene expression, by binding to both a classical remote site and an unusual proximal activator sequence. Images Fig. 2 Fig. 4 PMID:8356078

  4. Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

    PubMed

    Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

    2013-01-01

    In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells. PMID:24391761

  5. Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

    PubMed Central

    Pierfelice, Tarran J.; Schreck, Karisa C.; Dang, Louis; Asnaghi, Laura; Gaiano, Nicholas; Eberhart, Charles G.

    2010-01-01

    While Notch signaling has been widely implicated in neoplastic growth, direct evidence for in vivo initiation of neoplasia by the pathway in murine models has been limited to tumors of lymphoid, breast, and choroid plexus cells. To examine tumorigenic potential in the eye and brain, we injected retroviruses encoding activated forms of Notch1, Notch2, or Notch3 into embryonic mice. Interestingly, the majority of animals infected with active Notch3 developed proliferative lesions comprised of pigmented ocular choroid cells, retinal and optic nerve glia, and lens epithelium. Notch3-induced lesions in the choroid, retina, and optic nerve were capable of invading adjacent tissues, suggesting that they were malignant tumors. While Notch3 activation induced choroidal tumors in up to 67% of eyes, Notch1 or Notch2 activation never resulted in such tumors. Active forms of Notch1 and Notch2 did generate a few small proliferative glial nodules in the retina and optic nerve, while Notch3 was ten-fold more efficient at generating growths, many of which were large invasive gliomas. Expression of active Notch1/Notch3 chimeric receptors implicated the RAM (RBPjk-association molecule) and transactivation domains (TAD) of Notch3 in generating choroidal and glial tumors, respectively. In contrast to our findings in the optic nerve and retina, introduction of active Notch receptors, including Notch3, into the brain never caused glial tumors. Our results highlight the differential ability of Notch receptor paralogs to initiate malignant tumor formation, and suggest that glial precursors of the optic nerve, but not the brain, are susceptible to transformation by Notch3. PMID:21245095

  6. Subtissue-Specific Evaluation of Promoter Efficiency by Quantitative Fluorometric Assay in Laser Microdissected Tissues of Rapeseed[W

    PubMed Central

    Jasik, Jan; Schiebold, Silke; Rolletschek, Hardy; Denolf, Peter; Van Adenhove, Katrien; Altmann, Thomas; Borisjuk, Ljudmilla

    2011-01-01

    β-Glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-β-d-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-β-d-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state. PMID:21825109

  7. Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs.

    PubMed

    Freitas, Rejane L; Carvalho, Claudine M; Fietto, Luciano G; Loureiro, Marcelo E; Almeida, Andrea M; Fontes, Elizabeth P B

    2007-11-01

    The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position -2000 to -700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (-1785/-1508), Frag III (-1507/-1237), Frag IV (-1236/-971) and Frag V (-970/-700), act independently to alter the constitutive pattern of -92pSBP2-mediated GUS expression in different organs. Frag V fused to -92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions -1485 to -1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive -92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter. PMID:17710554

  8. Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.

    PubMed

    Aikio, Mari; Elamaa, Harri; Vicente, David; Izzi, Valerio; Kaur, Inderjeet; Seppinen, Lotta; Speedy, Helen E; Kaminska, Dorota; Kuusisto, Sanna; Sormunen, Raija; Heljasvaara, Ritva; Jones, Emma L; Muilu, Mikko; Jauhiainen, Matti; Pihlajamäki, Jussi; Savolainen, Markku J; Shoulders, Carol C; Pihlajaniemi, Taina

    2014-07-29

    Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼ 50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat

  9. Human cytomegalovirus (HCMV) immediate-early enhancer/promoter specificity during embryogenesis defines target tissues of congenital HCMV infection.

    PubMed Central

    Koedood, M; Fichtel, A; Meier, P; Mitchell, P J

    1995-01-01

    Congenital human cytomegalovirus (HCMV) infection is a common cause of deafness and neurological disabilities. Many aspects of this prenatal infection, including which cell types are infected and how infection proceeds, are poorly understood. Transcription of HCMV immediate-early (IE) genes is required for expression of all other HCMV genes and is dependent on host cell transcription factors. Cell type-specific differences in levels of IE transcription are believed to underlie differences in infection permissivity. However, DNA transfection experiments have paradoxically suggested that the HCMV major IE enhancer/promoter is a broadly active transcriptional element with little cell type specificity. In contrast, we show here that expression of a lacZ gene driven by the HCMV major IE enhancer/promoter -524 to +13 segment is restricted in transgenic mouse embryos to sites that correlate with known sites of congenital HCMV infection in human fetuses. This finding suggests that the IE enhancer/promoter is a major determinant of HCMV infection sites in humans and that transcription factors responsible for its regulation are cell type-specifically conserved between humans and mice. The lacZ expression patterns of these transgenic embryos yield insight into congenital HCMV pathogenesis by providing a spatiotemporal map of the sets of vascular, neural, and epithelial cells that are likely targets of infection. These transgenic mice may constitute a useful model system for investigating IE enhancer/promoter regulation in vivo and for identifying factors that modulate active and latent HCMV infections in humans. PMID:7884867

  10. A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato

    PubMed Central

    Bassett, Carole L; Callahan, Ann M; Artlip, Timothy S; Scorza, Ralph; Srinivasan, Chinnathambi

    2007-01-01

    Background Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized. Results A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (β-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots. Conclusion The minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of

  11. Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T(1) seedlings of tomato exposed to metal ions.

    PubMed

    Kamaladini, Hossein; Nor Akmar Abdullah, Siti; Aziz, Maheran Abdul; Ismail, Ismanizan Bin; Haddadi, Fatemeh

    2013-02-15

    Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 μM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions. PMID:23290536

  12. Tissue-specific response of the human platelet-activating factor receptor gene to retinoic acid and thyroid hormone by alternative promoter usage.

    PubMed Central

    Mutoh, H; Fukuda, T; Kitamaoto, T; Masushige, S; Sasaki, H; Shimizu, T; Kato, S

    1996-01-01

    We have studied the effects of retinoic acid (RA) and thyroid hormone (3,3',5-triiodothyronine; T3) on platelet-activating factor receptor (PAFR) gene expression in intact rats and the ability of two human PAFR gene promoters (PAFR promoters 1 and 2) to generate two transcripts (PAFR transcripts 1 and 2). Northern blotting showed that RA and T3 regulated PAFR gene expression only in rat tissues that express PAFR transcript 2. Functional analysis of the human PAFR promoter 2 revealed that responsiveness to RA and T3 was conferred through a 24-bp element [PAFR-hormone response element (HRE) located from -67 to -44 bp of the transcription start site, whereas PAFR promoter 1 did not respond to these hormones. The PAFR-HRE is composed of three direct repeated TGACCT-like hexamer motifs with 2-and 4-bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T3. Thus, the PAF-PAFR pathway is regulated by the PAFR level altered by a tissue-specific response to RA and T3 through the PAFR-HRE of the PAFR promoter 2. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8570633

  13. Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems.

    PubMed

    Buzeli, Reginaldo A A; Cascardo, Júlio C M; Rodrigues, Leonardo A Z; Andrade, Maxuel O; Almeida, Raul S; Loureiro, Marcelo E; Otoni, Wagner C; Fontes, Elizabeth P B

    2002-11-01

    The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter. PMID:12374306

  14. Alternative promoters and repetitive DNA elements define the species-dependent tissue-specific expression of the FMO1 genes of human and mouse

    PubMed Central

    Shephard, Elizabeth A.; Chandan, Pritpal; Stevanovic-Walker, Milena; Edwards, Mina; Phillips, Ian R.

    2007-01-01

    In humans, expression of the FMO1 (flavin-containing mono-oxygenase 1) gene is silenced postnatally in liver, but not kidney. In adult mouse, however, the gene is active in both tissues. We investigated the basis of this species-dependent tissue-specific transcription of FMO1. Our results indicate the use of three alternative promoters. Transcription of the gene in fetal human and adult mouse liver is exclusively from the P0 promoter, whereas in extra-hepatic tissues of both species, P1 and P2 are active. Reporter gene assays showed that the proximal P0 promoters of human (hFMO1) and mouse (mFmo1) genes are equally effective. However, sequences upstream (−2955 to −506) of the proximal P0 of mFmo1 increased reporter gene activity 3-fold, whereas hFMO1 upstream sequences (−3027 to −541) decreased reporter gene activity by 75%. Replacement of the upstream sequence of human P0 with the upstream sequence of mouse P0 increased activity of the human proximal P0 8-fold. Species-specific repetitive elements are present immediately upstream of the proximal P0 promoters. The human gene contains five LINE (long-interspersed nuclear element)-1-like elements, whereas the mouse gene contains a poly A region, an 80-bp direct repeat, an LTR (long terminal repeat), a SINE (short-interspersed nuclear element) and a poly T tract. The rat and rabbit FMO1 genes, which are expressed in adult liver, lack some (rat) or all (rabbit) of the elements upstream of mouse P0. Thus silencing of FMO1 in adult human liver is due apparently to the presence upstream of the proximal P0 of L1 (LINE-1) elements rather than the absence of retrotransposons similar to those found in the mouse gene. PMID:17547558

  15. Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects

    PubMed Central

    Stimac, Monika; Dolinsek, Tanja; Lampreht, Ursa; Cemazar, Maja; Sersa, Gregor

    2015-01-01

    Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined. PMID:25909447

  16. Composing a Tumor Specific Bacterial Promoter

    PubMed Central

    Deyneko, Igor V.; Kasnitz, Nadine; Leschner, Sara; Weiss, Siegfried

    2016-01-01

    Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy. PMID:27171245

  17. Creation of Bt rice expressing a fusion protein of Cry1Ac and Cry1I-like using a green tissue-specific promoter.

    PubMed

    Yang, Yong-Yi; Mei, Feng; Zhang, Wei; Shen, Zhicheng; Fang, Jun

    2014-08-01

    The insecticidal genes from Bacillus thuringiensis Berliner (Bt) have long been successfully used for development of insect-resistant rice. However, commercial planting of Bt rice has been delayed by the concern over food safety, although no scientific evidence is ever found to justify the concern. To address this safety concern, we developed a transgenic insect-resistant rice line using a green tissue promoter to minimize the Bt protein expression in the rice seeds. The Bt protein expressed in the rice was a fusion protein of two different Bt toxins, Cry1Ac and Cry1I-like protein. The fusion of the two toxins may be helpful to delay the development of insect resistance to Bt rice. Laboratory and field bioassays demonstrated that the transgenic rice plants created by this study were highly active against the rice leaf folder Cnaphalocrocis medinalis (Guenée) and the striped stem borer Chilo suppressalis (Walker). Western analysis indicated that the fusion protein was specifically expressed in green tissues but not in seeds. Therefore, the transgenic rice created in this study should be useful to mitigate the food safety concern and to delay the development of insect resistance. PMID:25195461

  18. Highly interactive nature of flower-specific enhancers and promoters, and its potential impact on tissue-specific expression and engineering of multiple genes or agronomic traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular stacking via a single transgene enables multiple traits being engineered efficiently in crops. However, whether distinct plant promoters co-existed in the same transgene could, like their mammalian counterparts, mutually interact or interfere remains unknown. In this study, researchers d...

  19. Cloning and tissue-specific functional characterization of the promoter of the rat diazepam binding inhibitor, a peptide with multiple biological actions.

    PubMed Central

    Kolmer, M; Alho, H; Costa, E; Pani, L

    1993-01-01

    Diazepam binding inhibitor (DBI) is a 10-kDa polypeptide that regulates mitochondrial steroidogenesis, glucose-induced insulin secretion, metabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABAA receptors. To investigate the regulation of DBI gene expression, three positive clones were isolated from a rat genomic library. One of them contained a DBI genomic DNA fragment encompassing 4 kb of the 5' untranslated region, the first two exons, and part of the second intron of the DBI gene. Two other overlapping clones contained a processed DBI pseudogene. Several transcription initiation sites were detected by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription startpoint usage. Transient expression experiments using DNA fragments of different length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequence, whereas full activation was achieved with 423 bp of the 5' untranslated region. DNase I protection experiments with liver nuclear proteins demonstrated three protected regions at nt -387 to -333, -295 to -271, and -176 to -139 relative to the ATG initiation codon; in other tissues the pattern of protection was different. In gel shift assays the most proximal region (-176 to -139) was found to bind several general transcription factors as well as cell type-restricted nuclear proteins which may be related to specific regulatory patterns in different tissues. Thus, the DBI gene possesses some features of a housekeeping gene but also includes a variable regulation which appears to change with the function that it subserves in different cell types. Images Fig. 2 Fig. 4 Fig. 5 PMID:7690962

  20. An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

    PubMed Central

    Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

    2013-01-01

    On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

  1. Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene.

    PubMed Central

    Keplinger, B L; Guo, X; Quine, J; Feng, Y; Cavener, D R

    2001-01-01

    The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented. PMID:11156990

  2. Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

    PubMed

    Wang, Qing; Zhu, Yi; Sun, Lin; Li, Lebin; Jin, Shuangxia; Zhang, Xianlong

    2016-02-01

    A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (β-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology. PMID:26728504

  3. Characterization of the genomic structure and tissue-specific promoter of the human nuclear receptor NR5A2 (hB1F) gene.

    PubMed

    Zhang, C K; Lin, W; Cai, Y N; Xu, P L; Dong, H; Li, M; Kong, Y Y; Fu, G; Xie, Y H; Huang, G M; Wang, Y

    2001-08-01

    The human homologue of the Drosophila melanogaster orphan nuclear receptor fushi tarazu factor 1 (Ftz-F1), NR5A2 (hB1F), was initially identified as a regulatory factor that binds and activates enhancer II of hepatitis B virus. NR5A2 (hB1F) is expressed specifically in pancreas and liver, playing important roles in the regulation of several liver-specific genes. A detailed analysis on the genomic structure and promoter activity will greatly promote future studies on the function of the NR5A2 (hB1F) gene. In this report, a bacterial artificial chromosome clone and several phage clones covering the NR5A2 (hB1F) gene were isolated and the complete genomic sequence was obtained. Alignment of different cDNAs of the NR5A2 (hB1F) gene with the genomic sequence facilitated the delineation of its structural organization, which spans over 150 kb and consists of eight exons interrupted by seven introns. RT-PCR and 3'-RACE revealed that utilization of two polyadenylation signals results in the 3.8 and 5.2 kb transcripts that were observed previously. The transcription start site of the NR5A2 (hB1F) gene was mapped downstream of a canonical TATA box. An upstream fragment containing binding sites for several liver-specific and ubiquitous transcription factors exhibits hepatocyte-specific promoter activity. Transient transfections indicated that hepatocyte nuclear factors HNF1 and HNF3beta could activate NR5A2 (hB1F) promoter. PMID:11595170

  4. Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice

    SciTech Connect

    Andres, A.C.; Schoenenberger, C.A.; Groner, B.; Henninghausen, L.; LeMeur, M.; Gelinger, P.

    1987-03-01

    The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras - i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors.

  5. Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals

    PubMed Central

    2010-01-01

    Background Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals. Results Here we report that human PURA (hPURA) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated hPURA 5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds hPURA promoter sequences at TSS II in vivo. By co-transfecting hPURA reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate PURA transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of mPURA mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts. Conclusions Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of PURA promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts. PMID:21062477

  6. A tissue-specific repressor in the sea urchin embryo of Lytechinus pictus binds the distal G-string element in the LpS1-beta promoter.

    PubMed

    Seid, C A; Sater, A K; Falzone, R L; Tomlinson, C R

    1996-06-01

    LpS1 RNA transcripts and proteins are expressed exclusively in the aboral ectoderm of the embryo in the sea urchin Lytechinus pictus. We have characterized the LpS1-beta promoter to identify the cis-acting elements that may be involved in the aboral ectoderm-specific expression of the LpS1-beta gene. The distal G-string site, composed of six contiguous guanine deoxynucleotides located at -721 to -726, was analyzed. A mutation at the distal G-string caused over a two-fold increase in reporter chloramphenicol acetyltransferase gene activity and inappropriate expression of reporter green fluorescent protein in nonaboral ectoderm cells in L. pictus embryos. These results suggest that the proteins that bind the distal G-string act as a spatial repressor in the nonaboral ectoderm cells of the developing embryo. PMID:8672248

  7. Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

    NASA Technical Reports Server (NTRS)

    Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

    1997-01-01

    We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

  8. Tissue Engineering Chamber Promotes Adipose Tissue Regeneration in Adipose Tissue Engineering Models Through Induced Aseptic Inflammation

    PubMed Central

    Peng, Zhangsong; Dong, Ziqing; Chang, Qiang; Zhan, Weiqing; Zeng, Zhaowei; Zhang, Shengchang

    2014-01-01

    Tissue engineering chamber (TEC) makes it possible to generate significant amounts of mature, vascularized, stable, and transferable adipose tissue. However, little is known about the role of the chamber in tissue engineering. Therefore, to investigate the role of inflammatory response and the change in mechanotransduction started by TEC after implantation, we placed a unique TEC model on the surface of the groin fat pads in rats to study the expression of cytokines and tissue development in the TEC. The number of infiltrating cells was counted, and vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) expression levels in the chamber at multiple time points postimplantation were analyzed by enzyme-linked immunosorbent assay. Tissue samples were collected at various time points and labeled for specific cell populations. The result showed that new adipose tissue formed in the chamber at day 60. Also, the expression of MCP-1 and VEGF in the chamber decreased slightly from an early stage as well as the number of the infiltrating cells. A large number of CD34+/perilipin− perivascular cells could be detected at day 30. Also, the CD34+/perilipin+ adipose precursor cell numbers increased sharply by day 45 and then decreased by day 60. CD34−/perilipin+ mature adipocytes were hard to detect in the chamber content at day 30, but their number increased and then peaked at day 60. Ki67-positive cells could be found near blood vessels and their number decreased sharply over time. Masson's trichrome showed that collagen was the dominant component of the chamber content at early stage and was replaced by newly formed small adipocytes over time. Our findings suggested that the TEC implantation could promote the proliferation of adipose precursor cells derived from local adipose tissue, increase angiogenesis, and finally lead to spontaneous adipogenesis by inducing aseptic inflammation and changing local mechanotransduction. PMID:24559078

  9. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  10. Limited specificity of promoter constructs for gene therapy in osteosarcoma.

    PubMed

    Pollmann, Annika; Kabisch, Hartmut; Block, Andreas; Müller, Jürgen; Hellwinkel, Olaf J C

    2004-10-01

    Osteosarcoma (OS), a malignant bone neoplasia in childhood, has poor prognosis if metastases appear in the lung. A novel therapeutic approach could consist in a gene therapeutic treatment of OS metastases. However, if promiscuous viral vectors are applied for the delivery of potentially toxic transgenes, their misdelivery into normal tissues could cause severe complications. This problem could be circumvented by application of OS-specific promoters for transgene expression control. We analysed the function of promoters described to be tumour-, osteosarcoma- or osteoblast-specific. Expression rates driven by osteoblast- specific fragments from the collagen1A1-promoter, the human Osteocalcin-promoter, the bone-sialoprotein promoter and the beta-catenin promoter depending on vitamin supplementation were analysed in five OS cell lines, in normal lung fibroblasts and in a non-osteoblastic prostate cancer cell line (LNCaP) by dual luciferase assays. In addition, an unspecific but doxycyclin-repressible promoter construct (pAd.3r-luc) was examined. We found that all constructs were active in OS cell lines to varying extents. The complete human Osteocalcin promoter and the bone-sialoprotein promoter were partially induced by vitamin D3 or C respectively while the pAd.3r-luc activity could be shut down by doxycyclin. In contrast, the human Osteocalcin-promoter was not activated by vitamin D3 in LNCaP cells; its action remained relatively low. Interestingly, excepting the beta-catenin promoter, we measured strong activities of all promoters in lung fibroblast cells. Our study demonstrates that promoter activity should be evaluated not only for the target cells of the gene therapeutic approaches, but also for neighbouring normal tissues. Unspecific but repressible promoters could represent an alternative. PMID:15375610

  11. Selective estrogen receptor modulators: tissue specificity and clinical utility

    PubMed Central

    Martinkovich, Stephen; Shah, Darshan; Planey, Sonia Lobo; Arnott, John A

    2014-01-01

    Selective estrogen receptor modulators (SERMs) are a diverse group of nonsteroidal compounds that function as agonists or antagonists for estrogen receptors (ERs) in a target gene-specific and tissue-specific fashion. SERM specificity involves tissue-specific expression of ER subtypes, differential expression of co-regulatory proteins in various tissues, and varying ER conformational changes induced by ligand binding. To date, the major clinical applications of SERMs are their use in the prevention and treatment of breast cancer, the prevention of osteoporosis, and the maintenance of beneficial serum lipid profiles in postmenopausal women. However, SERMs have also been found to promote adverse effects, including thromboembolic events and, in some cases, carcinogenesis, that have proven to be obstacles in their clinical utility. In this review, we discuss the mechanisms of SERM tissue specificity and highlight the therapeutic application of well-known and emergent SERMs. PMID:25210448

  12. Tissue-specific and steroid-dependent interaction of transcription factors with the oestrogen-inducible apoVLDL II promoter in vivo.

    PubMed Central

    Wijnholds, J; Philipsen, J N; Ab, G

    1988-01-01

    Using in vivo dimethylsulphate footprinting, we have analysed protein--DNA interactions within the promoter region of the oestrogen-inducible gene encoding chicken apo very low density lipoprotein II (apoVLDL II). Most of the guanosine--protein contacts found, are located within the 230-bp DNA 5' flanking the gene and can be grouped into separate protein-binding sites. Two of these sites resemble the oestrogen-responsive element (ERE) which is the target site for the oestrogen receptor. A third site has some features in common with the chicken ovalbumin upstream promoter element binding the COUP transcription factor. All protein contacts identified are present in the apoVLDL-II-expressing liver exclusively, and are not found in the hormone-naive liver, in erythrocytes or the oviduct tubular gland. Our results demonstrate the binding in vivo of a protein, presumably the oestrogen receptor, to the ERE and suggest that the hormone activates transcription by establishing a transcription complex comprising several factors at the apoVLDL II promoter. Images PMID:3181140

  13. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  14. A Tissue-Specific Scaffold for Tissue Engineering-Based Ureteral Reconstruction

    PubMed Central

    Xu, Yongde; Fu, Weijun; Wang, Zhongxin; Li, Gang; Zhang, Xu

    2015-01-01

    Terminally differentiated somatic cells can rapidly change phenotypes when they are isolated from their native tissue and cultured in vitro. This problem may become a barrier to tissue engineering-based organ reconstruction, which utilizes somatic cells. The present study was designed to validate the feasibility of maintaining the urothelial cell phenotype in a tissue-specific ureteral scaffold. The tissue-specific scaffold was fabricated by blending poly (L-lactic acid) (PLLA) and ureteral extracellular matrix (UECM) using electrostatic spinning technology. PLLA was used to enhance the mechanical properties, and UECM was used to mimic the natural components of the ureter. Primary urothelial cells (UCs), derived from ureteral mucosa, were seeded onto the tissue-specific scaffold to assess cell adhesion, proliferation and phenotypes at designated time points. The results showed that UCs in the tissue-specific scaffold exhibited better proliferation compared to cells in pure PLLA or a PLLA-small intestinal submucosa (PLLA-SIS) scaffold (p<0.05). At different time points, the expression of a UC-specific marker (UroplakinⅢ) in the tissue-specific scaffold was significantly higher than its expression in pure PLLA or a PLLA-SIS scaffold (p<0.05). Therefore, the tissue-specific scaffold appears to be an ideal substrate for promoting UC survival and phenotype maintenance. PMID:25775033

  15. Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro

    PubMed Central

    Mo, Cuiping; Mu, Shuhua; Jiang, Xiaogang; Li, Xiaoyun; Zhong, Shizhen; Zhao, Zhenfu; Zhou, Guangqian

    2015-01-01

    The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentiation-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin-expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellular iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The proliferation rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differentiation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs. The use of MRI and neural-differentiation-inducible ferritin expression is a viable method for monitoring the neural differentiation of h

  16. Tissue engineering strategies for promoting vascularized bone regeneration.

    PubMed

    Almubarak, Sarah; Nethercott, Hubert; Freeberg, Marie; Beaudon, Caroline; Jha, Amit; Jackson, Wesley; Marcucio, Ralph; Miclau, Theodore; Healy, Kevin; Bahney, Chelsea

    2016-02-01

    This review focuses on current tissue engineering strategies for promoting vascularized bone regeneration. We review the role of angiogenic growth factors in promoting vascularized bone regeneration and discuss the different therapeutic strategies for controlled/sustained growth factor delivery. Next, we address the therapeutic uses of stem cells in vascularized bone regeneration. Specifically, this review addresses the concept of co-culture using osteogenic and vasculogenic stem cells, and how adipose derived stem cells compare to bone marrow derived mesenchymal stem cells in the promotion of angiogenesis. We conclude this review with a discussion of a novel approach to bone regeneration through a cartilage intermediate, and discuss why it has the potential to be more effective than traditional bone grafting methods. PMID:26608518

  17. The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues.

    PubMed

    Medberry, S L; Lockhart, B E; Olszewski, N E

    1992-02-01

    Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells. PMID:1633493

  18. Predicting tissue-specific enhancers in the human genome

    PubMed Central

    Pennacchio, Len A.; Loots, Gabriela G.; Nobrega, Marcelo A.; Ovcharenko, Ivan

    2007-01-01

    Determining how transcriptional regulatory signals are encoded in vertebrate genomes is essential for understanding the origins of multicellular complexity; yet the genetic code of vertebrate gene regulation remains poorly understood. In an attempt to elucidate this code, we synergistically combined genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to define sequence signatures characteristic of candidate tissue-specific enhancers in the human genome. We applied this strategy to microarray-based gene expression profiles from 79 human tissues and identified 7187 candidate enhancers that defined their flanking gene expression, the majority of which were located outside of known promoters. We cross-validated this method for its ability to de novo predict tissue-specific gene expression and confirmed its reliability in 57 of the 79 available human tissues, with an average precision in enhancer recognition ranging from 32% to 63% and a sensitivity of 47%. We used the sequence signatures identified by this approach to successfully assign tissue-specific predictions to ∼328,000 human–mouse conserved noncoding elements in the human genome. By overlapping these genome-wide predictions with a data set of enhancers validated in vivo, in transgenic mice, we were able to confirm our results with a 28% sensitivity and 50% precision. These results indicate the power of combining complementary genomic data sets as an initial computational foray into a global view of tissue-specific gene regulation in vertebrates. PMID:17210927

  19. A seed coat-specific promoter for canola.

    PubMed

    El-Mezawy, Aliaa; Wu, Limin; Shah, Saleh

    2009-12-01

    The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The beta-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola. PMID:19690805

  20. Tissue-specific promoter usage and diverse splicing variants of found in neurons, an ancestral Hu/ELAV-like RNA-binding protein gene of insects, in the direct-developing insect Gryllus bimaculatus.

    PubMed

    Watanabe, T; Aonuma, H

    2014-02-01

    Hu/ELAV-like RNA-binding proteins (RBPs) are involved in the post-transcriptional regulation of RNA metabolism including splicing, transport, translational control and turnover. The Hu/ELAV-like RBP genes are predominantly expressed in neurons, and are therefore used as common neuronal markers in many animals. Although the expression patterns and functions of the Hu/ELAV-like RBP genes have been extensively studied in the model insect Drosophila melanogaster, little is known in basal direct-developing insects. In the present study, we performed an identification and expression analysis of the found in neurons (fne) gene, an ancestral insect Hu/ELAV-like RBP gene, in the cricket Gryllus bimaculatus. Contrary to expectation that the Gryllus fne transcript would be predominantly expressed in the nervous system, expression analysis revealed that the Gryllus fne gene is expressed broadly. In addition, we discovered that alternative promoter usage directs tissue-specific and embryonic stage-dependent regulation of fne expression, and that alternative splicing contributes to the generation of diverse sets of fne transcripts. Our data provide novel insights into the evolutionary diversification of the Hu/ELAV-like RBP gene family in insects. PMID:24382152

  1. Adipose tissue extract promotes adipose tissue regeneration in an adipose tissue engineering chamber model.

    PubMed

    Lu, Zijing; Yuan, Yi; Gao, Jianhua; Lu, Feng

    2016-05-01

    An adipose tissue engineering chamber model of spontaneous adipose tissue generation from an existing fat flap has been described. However, the chamber does not completely fill with adipose tissue in this model. Here, the effect of adipose tissue extract (ATE) on adipose tissue regeneration was investigated. In vitro, the adipogenic and angiogenic capacities of ATE were evaluated using Oil Red O and tube formation assays on adipose-derived stem cells (ASCs) and rat aortic endothelial cells (RAECs), respectively. In vivo, saline or ATE was injected into the adipose tissue engineering chamber 1 week after its implantation. At different time points post-injection, the contents were morphometrically, histologically, and immunohistochemically evaluated, and the expression of growth factors and adipogenic genes was analyzed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. With the exception of the baseline control group, in which fat flaps were not inserted into a chamber, the total volume of fat flap tissue increased significantly in all groups, especially in the ATE group. Better morphology and structure, a thinner capsule, and more vessels were observed in the ATE group than in the control group. Expression of angiogenic growth factors and adipogenic markers were significantly higher in the ATE group. ATE therefore significantly promoted adipose tissue regeneration and reduced capsule formation in an adipose tissue engineering chamber model. These data suggest that ATE provides a more angiogenic and adipogenic microenvironment for adipose tissue formation by releasing various cytokines and growth factors that also inhibit capsule formation. PMID:26678825

  2. Ribosomopathies: Global process, tissue specific defects.

    PubMed

    Yelick, Pamela C; Trainor, Paul A

    2015-01-01

    Disruptions in ribosomal biogenesis would be expected to have global and in fact lethal effects on a developing organism. However, mutations in ribosomal protein genes have been shown in to exhibit tissue specific defects. This seemingly contradictory finding - that globally expressed genes thought to play fundamental housekeeping functions can in fact exhibit tissue and cell type specific functions - provides new insight into roles for ribosomes, the protein translational machinery of the cell, in regulating normal development and disease. Furthermore it illustrates the surprisingly dynamic nature of processes regulating cell type specific protein translation. In this review, we discuss our current knowledge of a variety of ribosomal protein mutations associated with human disease, and models to better understand the molecular mechanisms associated with each. We use specific examples to emphasize both the similarities and differences between the effects of various human ribosomal protein mutations. Finally, we discuss areas of future study that are needed to further our understanding of the role of ribosome biogenesis in normal development, and possible approaches that can be used to treat debilitating ribosomopathy diseases. PMID:26442198

  3. Ribosomopathies: Global process, tissue specific defects

    PubMed Central

    Yelick, Pamela C; Trainor, Paul A

    2015-01-01

    Disruptions in ribosomal biogenesis would be expected to have global and in fact lethal effects on a developing organism. However, mutations in ribosomal protein genes have been shown in to exhibit tissue specific defects. This seemingly contradictory finding - that globally expressed genes thought to play fundamental housekeeping functions can in fact exhibit tissue and cell type specific functions ‐ provides new insight into roles for ribosomes, the protein translational machinery of the cell, in regulating normal development and disease. Furthermore it illustrates the surprisingly dynamic nature of processes regulating cell type specific protein translation. In this review, we discuss our current knowledge of a variety of ribosomal protein mutations associated with human disease, and models to better understand the molecular mechanisms associated with each. We use specific examples to emphasize both the similarities and differences between the effects of various human ribosomal protein mutations. Finally, we discuss areas of future study that are needed to further our understanding of the role of ribosome biogenesis in normal development, and possible approaches that can be used to treat debilitating ribosomopathy diseases. PMID:26442198

  4. Induction of tissue-specific stem cells by reprogramming factors, and tissue-specific selection.

    PubMed

    Noguchi, H; Saitoh, I; Tsugata, T; Kataoka, H; Watanabe, M; Noguchi, Y

    2015-01-01

    Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells, there are still several unresolved issues related to the use of iPS cells for clinical applications, such as teratoma formation. In this study, we were able to generate tissue-specific stem (induced tissue-specific stem; iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by transient overexpression of reprogramming factors, combined with tissue-specific selection. The generation of iTS cells was easier than that of iPS cells. The iTS-P/iTS-L cells express genetic markers of endoderm and pancreatic/hepatic progenitors and were able to differentiate into insulin-producing cells/hepatocytes more efficiently than ES cells. Subcutaneous transplantation of both types of iTS cells into immunodeficient mice resulted in no teratoma formation. The technology used for the transient overexpression of reprogramming factors and tissue-specific selection may be useful for the generation of other tissue-specific stem cells, and the generation of iTS cells could have important implications for the clinical application of stem cells. PMID:25190146

  5. Induction of tissue-specific stem cells by reprogramming factors, and tissue-specific selection

    PubMed Central

    Noguchi, H; Saitoh, I; Tsugata, T; Kataoka, H; Watanabe, M; Noguchi, Y

    2015-01-01

    Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells, there are still several unresolved issues related to the use of iPS cells for clinical applications, such as teratoma formation. In this study, we were able to generate tissue-specific stem (induced tissue-specific stem; iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by transient overexpression of reprogramming factors, combined with tissue-specific selection. The generation of iTS cells was easier than that of iPS cells. The iTS-P/iTS-L cells express genetic markers of endoderm and pancreatic/hepatic progenitors and were able to differentiate into insulin-producing cells/hepatocytes more efficiently than ES cells. Subcutaneous transplantation of both types of iTS cells into immunodeficient mice resulted in no teratoma formation. The technology used for the transient overexpression of reprogramming factors and tissue-specific selection may be useful for the generation of other tissue-specific stem cells, and the generation of iTS cells could have important implications for the clinical application of stem cells. PMID:25190146

  6. KSHV Rta Promoter Specification and Viral Reactivation.

    PubMed

    Guito, Jonathan; Lukac, David M

    2012-01-01

    Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi's sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic "CANT DNA repeats" in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta's role as the switch is inefficient. Many factors modulate K-Rta's function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV

  7. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  8. Nuclear membrane diversity: underlying tissue-specific pathologies in disease?

    PubMed Central

    Worman, Howard J.; Schirmer, Eric C.

    2015-01-01

    Human ‘laminopathy’ diseases result from mutations in genes encoding nuclear lamins or nuclear envelope (NE) transmembrane proteins (NETs). These diseases present a seeming paradox: the mutated proteins are widely expressed yet pathology is limited to specific tissues. New findings suggest tissue-specific pathologies arise because these widely expressed proteins act in various complexes that include tissue-specific components. Diverse mechanisms to achieve NE tissue-specificity include tissue-specific regulation of the expression, mRNA splicing, signaling, NE-localization and interactions of potentially hundreds of tissue-specific NETs. New findings suggest these NETs underlie tissue-specific NE roles in cytoskeletal mechanics, cell-cycle regulation, signaling, gene expression and genome organization. This view of the NE as ‘specialized’ in each cell type is important to understand the tissue-specific pathology of NE-linked diseases. PMID:26115475

  9. ERAP140, a conserved tissue-specific nuclear receptor coactivator.

    PubMed

    Shao, Wenlin; Halachmi, Shlomit; Brown, Myles

    2002-05-01

    We report here the identification and characterization of a novel nuclear receptor coactivator, ERAP140. ERAP140 was isolated in a screen for ER alpha-interacting proteins using the ER alpha ligand binding domain as a probe. The ERAP140 protein shares no sequence and has little structural homology with other nuclear receptor cofactors. However, homologues of ERAP140 have been identified in mouse, Drosophila, and Caenorhabditis elegans. The expression of ERAP140 is cell and tissue type specific and is most abundant in the brain, where its expression is restricted to neurons. In addition to interacting with ER alpha, ERAP140 also binds ER beta, TR beta, PPAR gamma, and RAR alpha. ERAP140 interacts with ER alpha via a noncanonical interaction motif. The ER alpha-ERAP140 association can be competed by coactivator NR boxes, indicating ERAP140 binds ER alpha on a surface similar to that of other coactivators. ERAP140 can enhance the transcriptional activities of nuclear receptors with which it interacts. In vivo, ERAP140 is recruited by estrogen-bound ER alpha to the promoter region of endogenous ER alpha target genes. Furthermore, the E(2)-induced recruitment of ERAP140 to the promoter follows a cyclic pattern similar to that of other coactivators. Our results suggest that ERAP140 represents a distinct class of nuclear receptor coactivators that mediates receptor signaling in specific target tissues. PMID:11971969

  10. Tissue-specific control elements of the Thy-1 gene.

    PubMed Central

    Vidal, M; Morris, R; Grosveld, F; Spanopoulou, E

    1990-01-01

    We have exploited the structural homology, but different patterns of expression of the murine and human Thy-1 genes to map a number of tissue-specific enhancer elements in the genes. All of these are located downstream from the site of transcriptional initiation. The human gene contains separate elements which direct expression to the kidney or spleen epithelium. The murine gene lacks these elements but instead contains a thymocyte specific enhancer in the third intron. Developmentally-regulated expression in nerve cells is directed (at least in part) by an atypical element in the first intron. The latter is active on heterologous promoters, but is position and distance dependent. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:1968831

  11. GUS expression driven by constitutive and vascular specific promoters in citrus hybrid US-802

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus, and a critical component of the transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters...

  12. GUS reporter gene expression from Beta vulgaris root-specific promoters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To develop transgenic sugar beet with specialized agronomic traits for insect and disease tolerance and enhanced sugar accumulation and storage, a larger arsenal of constitutive, tissue-specific and temporal promoters is required. In the present study, a series of sugar beet promoters were tested f...

  13. TERT promoter mutations are rare in bone and soft tissue sarcomas of Japanese patients

    PubMed Central

    SAITO, TSUYOSHI; AKAIKE, KEISUKE; KURISAKI-ARAKAWA, AIKO; TODA-ISHII, MIDORI; MUKAIHARA, KENTA; SUEHARA, YOSHIYUKI; TAKAGI, TATSUYA; KANEKO, KAZUO; YAO, TAKASHI

    2016-01-01

    Recurrent hot-spot mutations in the telomerase reverse transcriptase (TERT) promoter have been reported in various types of tumor. In several tumor types, TERT promoter mutations are associated with poor clinical outcomes. TERT promoter mutations are reported to be rare in soft tissue tumors, with the exception of myxoid liposarcoma (MLS). Our previous study reported that TERT promoter mutations occurred in a subset of solitary fibrous tumors (SFTs) and were associated with adverse clinical outcomes in Japanese individuals. The site-specific frequency (e.g. central nervous or soft tissue origin) of TERT promoter mutations in our SFT cases appeared to be different from previously reported values in a European population. These findings prompted the present study to elucidate the potential role of ethnic background in the different frequencies of TERT promoter mutations in bone and soft tissue sarcomas. In the present study, TERT promoter mutations were examined in 180 cases of bone and soft tissue sarcomas. TERT promoter region mutations were identified in 10 cases [5 SFTs, 3 MLSs, 1 undifferentiated pleomorphic sarcoma (UPS) and 1 malignant granular cell tumor]. All mutations were C228T. The frequencies of TERT promoter mutation in MLS and UPS were 23.1 (3/13) and 5% (1/20), respectively. Only 1/5 patients with TERT-mutated tumors experienced local recurrence or distant metastasis. The present study revealed the first case of a malignant granular cell tumor with a TERT promoter mutation and revealed that the frequency of TERT promoter mutations in MLSs of Japanese patients is lower compared with that reported in German patients, providing evidence of a possible ethnic difference in the frequency of TERT promoter mutations. PMID:26870359

  14. Combinatorial regulation modules on GmSBP2 promoter: a distal cis-regulatory domain confines the SBP2 promoter activity to the vascular tissue in vegetative organs.

    PubMed

    Waclawovsky, Alessandro J; Freitas, Rejane L; Rocha, Carolina S; Contim, Luis Antônio S; Fontes, Elizabeth P B

    2006-01-01

    The Glycine max sucrose binding protein (GmSBP2) promoter directs phloem-specific expression of reporter genes in transgenic tobacco. Here, we identified cis-regulatory domains (CRD) that contribute with positive and negative regulation for the tissue-specific pattern of the GmSPB2 promoter. Negative regulatory elements in the distal CRD-A (-2000 to -700) sequences suppressed expression from the GmSBP2 promoter in tissues other than seed tissues and vascular tissues of vegetative organs. Deletion of this region relieved repression resulting in a constitutive promoter highly active in all tissues analyzed. Further deletions from the strong constitutive -700GmSBP2 promoter delimited several intercalating enhancer-like and repressing domains that function in a context-dependent manner. Histochemical examination revealed that the CRD-C (-445 to -367) harbors both negative and positive elements. This region abolished promoter expression in roots and in all tissues of stems except for the inner phloem. In contrast, it restores root meristem expression when fused to the -132pSBP2-GUS construct, which contains root meristem expression-repressing determinants mapped to the 44-bp CRD-G (-136 to -92). Thus, the GmSBP2 promoter is functionally organized into a proximal region with the combinatorial modular configuration of plant promoters and a distal domain, which restricts gene expression to the vascular tissues in vegetative organs. PMID:16574256

  15. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  16. Identification and characterization of promoters specifically and strongly expressed in maize embryos.

    PubMed

    Liu, Xiaoqing; Tian, Jian; Zhou, Xiaojin; Chen, Rumei; Wang, Lei; Zhang, Chunyi; Zhao, Jun; Fan, Yunliu

    2014-12-01

    The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos. PMID:25052028

  17. BDNF promoter I methylation correlates between post-mortem human peripheral and brain tissues.

    PubMed

    Stenz, Ludwig; Zewdie, Seblewongel; Laforge-Escarra, Térèse; Prados, Julien; La Harpe, Romano; Dayer, Alexandre; Paoloni-Giacobino, Ariane; Perroud, Nader; Aubry, Jean-Michel

    2015-02-01

    Several psychiatric disorders have been associated with CpG methylation changes in CG rich promoters of the brain-derived neurotrophic factor (BDNF) mainly by extracting DNA from peripheral blood cells. Whether changes in peripheral DNA methylation can be used as a proxy for brain-specific alterations remains an open question. In this study we aimed to compare DNA methylation levels in BDNF promoter regions in human blood cells, muscle and brain regions using bisulfite-pyrosequencing. We found a significant correlation between the levels of BDNF promoter I methylation measured in quadriceps and vPFC tissues extracted from the same individuals (n = 98, Pearson, r = 0.48, p = 4.5 × 10(-7)). In the hippocampus, BDNF promoter I and IV methylation levels were strongly correlated (Pearson, n = 37, r = 0.74, p = 1.4 × 10(-7)). We found evidence for sex-dependent effect on BDNF promoter methylation levels in the various tissues and blood samples. Taken together, these data indicate a strong intra-individual correlation between peripheral and brain tissue. They also suggest that sex determines methylation patterns in BDNF promoter region across different types of tissue, including muscle, brain, and blood. PMID:25450314

  18. A novel tissue model for angiogenesis: evaluation of inhibitors or promoters in tissue level

    NASA Astrophysics Data System (ADS)

    Dai, Bingling; Zhang, Yanmin; Zhan, Yingzhuan; Zhang, Dongdong; Wang, Nan; He, Langchong

    2014-01-01

    A novel tissue model for angiogenesis (TMA) is established for effective evaluation of angiogenesis inhibitors or promoters in vitro. Lung tissues were cultured in fibrinogen ``sandwich'' structure which resembled the formation of neovessels in vivo. The cells and capillary-like structures grew from the lung tissues were identified as endothelial cells and neovessels. Both immunohistochemisty and western blot results indicated that autocrine VEGF bound to the KDR and induced KDR autophosphorylation that could induce the proliferation of endothelial cells and their migration as well as the formation of microvessels on the lung tissue edge. With addition of the TMA, the murine VEGF and cultured medium produced by A549 tumor cells apparently promoted the increase of neovessels. Sorafenib as a tumor angiogenesis inhibitor and Tongxinluo as an angiogenesis promoter were both used to evaluate the TMA performance and they exhibited a good effect on neovessels in the TMA. The model established imitated angiogenesis in vivo and could well serve as an effective method in evaluating the angiogenesis inhibitors or promoters, and could also be practical for screening small molecules that affect blood vessel formation.

  19. Tissue specificity in the nuclear envelope supports its functional complexity

    PubMed Central

    de las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair RW; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution. PMID:24213376

  20. Specific white matter tissue microstructure changes associated with obesity.

    PubMed

    Kullmann, Stephanie; Callaghan, Martina F; Heni, Martin; Weiskopf, Nikolaus; Scheffler, Klaus; Häring, Hans-Ulrich; Fritsche, Andreas; Veit, Ralf; Preissl, Hubert

    2016-01-15

    Obesity-related structural brain alterations point to a consistent reduction in gray matter with increasing body mass index (BMI) but changes in white matter have proven to be more complex and less conclusive. Hence, more recently diffusion tensor imaging (DTI) has been employed to investigate microstructural changes in white matter structure. Altogether, these studies have mostly shown a loss of white matter integrity with obesity-related factors in several brain regions. However, the variety of these obesity-related factors, including inflammation and dyslipidemia, resulted in competing influences on the DTI indices. To increase the specificity of DTI results, we explored specific brain tissue properties by combining DTI with quantitative multi-parameter mapping in lean, overweight and obese young adults. By means of multi-parameter mapping, white matter structures showed differences in MRI parameters consistent with reduced myelin, increased water and altered iron content with increasing BMI in the superior longitudinal fasciculus, anterior thalamic radiation, internal capsule and corpus callosum. BMI-related changes in DTI parameters revealed mainly alterations in mean and axial diffusivity with increasing BMI in the corticospinal tract, anterior thalamic radiation and superior longitudinal fasciculus. These alterations, including mainly fiber tracts linking limbic structures with prefrontal regions, could potentially promote accelerated aging in obese individuals leading to an increased risk for cognitive decline. PMID:26458514

  1. Evolution of a tissue-specific splicing network

    PubMed Central

    Taliaferro, J. Matthew; Alvarez, Nehemiah; Green, Richard E.; Blanchette, Marco; Rio, Donald C.

    2011-01-01

    Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner. PMID:21406555

  2. Specific white matter tissue microstructure changes associated with obesity

    PubMed Central

    Kullmann, Stephanie; Callaghan, Martina F.; Heni, Martin; Weiskopf, Nikolaus; Scheffler, Klaus; Häring, Hans-Ulrich; Fritsche, Andreas; Veit, Ralf; Preissl, Hubert

    2016-01-01

    Obesity-related structural brain alterations point to a consistent reduction in gray matter with increasing body mass index (BMI) but changes in white matter have proven to be more complex and less conclusive. Hence, more recently diffusion tensor imaging (DTI) has been employed to investigate microstructural changes in white matter structure. Altogether, these studies have mostly shown a loss of white matter integrity with obesity-related factors in several brain regions. However, the variety of these obesity-related factors, including inflammation and dyslipidemia, resulted in competing influences on the DTI indices. To increase the specificity of DTI results, we explored specific brain tissue properties by combining DTI with quantitative multi-parameter mapping in lean, overweight and obese young adults. By means of multi-parameter mapping, white matter structures showed differences in MRI parameters consistent with reduced myelin, increased water and altered iron content with increasing BMI in the superior longitudinal fasciculus, anterior thalamic radiation, internal capsule and corpus callosum. BMI-related changes in DTI parameters revealed mainly alterations in mean and axial diffusivity with increasing BMI in the corticospinal tract, anterior thalamic radiation and superior longitudinal fasciculus. These alterations, including mainly fiber tracts linking limbic structures with prefrontal regions, could potentially promote accelerated aging in obese individuals leading to an increased risk for cognitive decline. PMID:26458514

  3. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo

    PubMed Central

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V.S.

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner. PMID:27446487

  4. Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases.

    PubMed

    Marbach, Daniel; Lamparter, David; Quon, Gerald; Kellis, Manolis; Kutalik, Zoltán; Bergmann, Sven

    2016-04-01

    Mapping perturbed molecular circuits that underlie complex diseases remains a great challenge. We developed a comprehensive resource of 394 cell type- and tissue-specific gene regulatory networks for human, each specifying the genome-wide connectivity among transcription factors, enhancers, promoters and genes. Integration with 37 genome-wide association studies (GWASs) showed that disease-associated genetic variants-including variants that do not reach genome-wide significance-often perturb regulatory modules that are highly specific to disease-relevant cell types or tissues. Our resource opens the door to systematic analysis of regulatory programs across hundreds of human cell types and tissues (http://regulatorycircuits.org). PMID:26950747

  5. Scaffolding in tissue engineering: general approaches and tissue-specific considerations

    PubMed Central

    Leong, K. W.

    2008-01-01

    Scaffolds represent important components for tissue engineering. However, researchers often encounter an enormous variety of choices when selecting scaffolds for tissue engineering. This paper aims to review the functions of scaffolds and the major scaffolding approaches as important guidelines for selecting scaffolds and discuss the tissue-specific considerations for scaffolding, using intervertebral disc as an example. PMID:19005702

  6. Tissue Specific Heterogeneity in Effector Immune Cell Response

    PubMed Central

    Tufail, Saba; Badrealam, Khan Farheen; Sherwani, Asif; Gupta, Umesh D.; Owais, Mohammad

    2013-01-01

    Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct “homing codes” (adhesion molecules and chemokine receptors) during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A) and sunlight (vitamin D3) prime dendritic cells, imprinting them to play centre stage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue-tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues along with giving an overview of tissue tropism in B cells. PMID:23986763

  7. Modular genes with metazoan-specific domains have increased tissue specificity.

    PubMed

    Cohen-Gihon, Inbar; Lancet, Doron; Yanai, Itai

    2005-04-01

    We have systematically examined the domain composition across a comprehensive set of tissue-specific, midrange and housekeeping genes as defined by their mode of expression in 52 normal mouse tissues. We show a definite correlation between the number of domains and the degree of tissue specificity. This trend is further supported by a novel analysis involving the time of origin of each domain. Genes containing metazoan-specific domains are more prevalent in signal transduction and cell-communication pathways, and are depleted in primary metabolism. Our analyses suggest that highly modular gene products have been recruited for tissue-specific functions that are required in complex organisms. PMID:15797615

  8. Specific immunofluorescence staining of Treponema pallidum in smears and tissues.

    PubMed

    Ito, F; Hunter, E F; George, R W; Swisher, B L; Larsen, S A

    1991-03-01

    To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent-antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the

  9. Norwalk Virus–specific Binding to Oyster Digestive Tissues

    PubMed Central

    Loisy, Fabienne; Atmar, Robert L.; Hutson, Anne M.; Estes, Mary K.; Ruvoën-Clouet, Nathalie; Pommepuy, Monique; Le Pendu, Jacques

    2006-01-01

    The primary pathogens related to shellfishborne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an α linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue. PMID:16707048

  10. Tissue-specific tagging of endogenous loci in Drosophila melanogaster

    PubMed Central

    Koles, Kate; Yeh, Anna R.; Rodal, Avital A.

    2016-01-01

    ABSTRACT Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues. PMID:26700726

  11. Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink.

    PubMed

    Skardal, Aleksander; Devarasetty, Mahesh; Kang, Hyun-Wook; Seol, Young-Joon; Forsythe, Steven D; Bishop, Colin; Shupe, Thomas; Soker, Shay; Atala, Anthony

    2016-01-01

    Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types. PMID:27166839

  12. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    PubMed Central

    Shilpa, V.; Bhagat, Rahul; Premalata, C.S.; Pallavi, V.R.; Ramesh, G.; Krishnamoorthy, Lakshmi

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression. PMID:25579142

  13. Alternative promoters of Peg3 with maternal specificity

    PubMed Central

    Perera, Bambarendage P. U.; Kim, Joomyeong

    2016-01-01

    Peg3 (paternally expressed gene 3) is an imprinted gene localized within an evolutionarily conserved 500-kb domain in human chromosome 19q13.4 and mouse proximal chromosome 7. In the current study, we have identified three alternative promoters for mouse Peg3 and one alternative promoter for human PEG3. These alternative promoters are localized within the 200-kb upstream region of human and mouse PEG3, which is well conserved and thus predicted to harbor several cis-regulatory elements for the PEG3 domain. In the mouse, two of these alternative promoters drive maternal-specific expression of Peg3 specifically in the hypothalamus of the adult brain, while the remaining third promoter drives bi-allelic expression of Peg3 with a paternal bias only in the neonatal-stage brain. In human, an alternative transcript is also detected at relatively very low levels in adult brain and placenta. Overall, the identification of alternative promoters in both mouse and human models suggests that these alternative promoters may be functionally selected features for the PEG3 imprinted domain during mammalian evolution. PMID:27075691

  14. Alternative promoters of Peg3 with maternal specificity.

    PubMed

    Perera, Bambarendage P U; Kim, Joomyeong

    2016-01-01

    Peg3 (paternally expressed gene 3) is an imprinted gene localized within an evolutionarily conserved 500-kb domain in human chromosome 19q13.4 and mouse proximal chromosome 7. In the current study, we have identified three alternative promoters for mouse Peg3 and one alternative promoter for human PEG3. These alternative promoters are localized within the 200-kb upstream region of human and mouse PEG3, which is well conserved and thus predicted to harbor several cis-regulatory elements for the PEG3 domain. In the mouse, two of these alternative promoters drive maternal-specific expression of Peg3 specifically in the hypothalamus of the adult brain, while the remaining third promoter drives bi-allelic expression of Peg3 with a paternal bias only in the neonatal-stage brain. In human, an alternative transcript is also detected at relatively very low levels in adult brain and placenta. Overall, the identification of alternative promoters in both mouse and human models suggests that these alternative promoters may be functionally selected features for the PEG3 imprinted domain during mammalian evolution. PMID:27075691

  15. A seed coat outer integument-specific promoter for Brassica napus.

    PubMed

    Wu, Limin; El-Mezawy, Aliaa; Shah, Saleh

    2011-01-01

    In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines. PMID:21052676

  16. Predicting Tissue-Specific Enhancers in the Human Genome

    SciTech Connect

    Pennacchio, Len A.; Loots, Gabriela G.; Nobrega, Marcelo A.; Ovcharenko, Ivan

    2006-07-01

    Determining how transcriptional regulatory signals areencoded in vertebrate genomes is essential for understanding the originsof multi-cellular complexity; yet the genetic code of vertebrate generegulation remains poorly understood. In an attempt to elucidate thiscode, we synergistically combined genome-wide gene expression profiling,vertebrate genome comparisons, and transcription factor binding siteanalysis to define sequence signatures characteristic of candidatetissue-specific enhancers in the human genome. We applied this strategyto microarray-based gene expression profiles from 79 human tissues andidentified 7,187 candidate enhancers that defined their flanking geneexpression, the majority of which were located outside of knownpromoters. We cross-validated this method for its ability to de novopredict tissue-specific gene expression and confirmed its reliability in57 of the 79 available human tissues, with an average precision inenhancer recognition ranging from 32 percent to 63 percent, and asensitivity of 47 percent. We used the sequence signatures identified bythis approach to assign tissue-specific predictions to ~;328,000human-mouse conserved noncoding elements in the human genome. Byoverlapping these genome-wide predictions with a large in vivo dataset ofenhancers validated in transgenic mice, we confirmed our results with a28 percent sensitivity and 50 percent precision. These results indicatethe power of combining complementary genomic datasets as an initialcomputational foray into the global view of tissue-specific generegulation in vertebrates.

  17. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

    PubMed

    Hirai, Tadayoshi; Kim, You-Wang; Kato, Kazuhisa; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi

    2011-12-01

    The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use. PMID:21359850

  18. Identification and characterization of three novel hemocyte-specific promoters in silkworm Bombyx mori.

    PubMed

    Zhang, Kui; Yu, Shuang; Su, Jingjing; Xu, Man; Tan, Peng; Zhang, Yajun; Xiang, Zhonghuai; Cui, Hongjuan

    2015-05-22

    Insect hemocytes play essential roles in the metabolism, metamorphosis and immunity, which are closely related events of growth and development. Here, four novel hemocyte-specific genes were obtained and conformed in our study, namely, Bmintβ2, Bmintβ3, BmCatO, and BmSw04862, respectively. Subsequently, their promoter sequences were cloned, and their activity in hemocytes, fat body, and silk gland were analyzed using recombinant AcNPV vector system in vivo. Our results showed that Bmintβ2, Bmintβ3, and BmCatO were hemocyte-specific promoters in the silkworm, Bombyx mori. Interestingly, Bmintβ2, and Bmintβ3 promoter regions were both located in their first intron. Further analysis of a series of BmCatO promoter truncations showed that a 254 bp region could function as a promoter element in the tissue-specificity expression. In summary, the results of this study revealed that we have identified three hemocyte-specific promoters in silkworm that will not only great significance for better understanding of hemocyte-specific gene, but also has potential applications in insect hematopoiesis and innate immunity research. PMID:25862374

  19. Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.)

    PubMed Central

    Koramutla, Murali Krishna; Bhatt, Deepa; Negi, Manisha; Venkatachalam, Perumal; Jain, Pradeep K.; Bhattacharya, Ramcharan

    2016-01-01

    Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters. PMID:27148290

  20. Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.).

    PubMed

    Koramutla, Murali Krishna; Bhatt, Deepa; Negi, Manisha; Venkatachalam, Perumal; Jain, Pradeep K; Bhattacharya, Ramcharan

    2016-01-01

    Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters. PMID:27148290

  1. MGMT promoter methylation in serum and cerebrospinal fluid as a tumor-specific biomarker of glioma

    PubMed Central

    WANG, ZHENG; JIANG, WEI; WANG, YAHONG; GUO, YANG; CONG, ZHENG; DU, FANGFANG; SONG, BIN

    2015-01-01

    O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is a conventional technique to predict the prognosis or individualized treatment of glioma in tumor tissue following surgery or biopsy. However, the technique cannot be applied in those glioma patients with concomitant neurological dysfunctions or advanced age. The present study aimed to find a new minimally invasive and efficient alternative method for the detection of MGMT promoter methylation. The expression of MGMT promoter methylation was assessed in peripheral blood and cerebrospinal fluid (CSF), and compared to the corresponding tumor tissue from glioma patients. The 89 patients in the study [32 World Health Organization (WHO) grade II, 19 WHO grade III and 38 WHO grade IV) were pathologically-diagnosed glioma and received radiation therapy following sample collection. The resected glioma tumor tissue (89), corresponding serum (89) and CSF (78) samples were collected for the detection of MGMT promoter methylation using methylation-specific polymerase chain reaction. The sensitivity and specificity of detecting MGMT promoter methylation in CSF and serum were compared. Among the tumor tissue samples, 51/89 (57.3%) showed MGMT promoter methylation. The specificity of the detection in the CSF and serum samples reached 100%. The sensitivity of MGMT promoter methylation detection in CSF and serum were 26/40 (65.0%) and 19/51 (37.3%), respectively (P<0.05). In the WHO II, III and IV subgroups, the sensitivities of MGMT promoter methylation detection using CSF were 8/12 (66.7%), 11/18 (61.1%) and 7/10 (70.0%), respectively, which were significantly higher than the sensitivities using serum (7/21, 33.3%; 7/19, 36.8%; and 5/11, 45.5%, respectively P<0.05). Among patients with residual postoperative tumors, the sensitivities of detecting MGMT promoter methylation using CSF and serum were 18/25 (72.0%) and 10/24 (41.7%), respectively, both of which were significantly higher than the corresponding values

  2. Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

    PubMed

    Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-01-01

    Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors. PMID:26530405

  3. Positive and negative tissue-specific signaling by a nematode epidermal growth factor receptor.

    PubMed Central

    Lesa, G M; Sternberg, P W

    1997-01-01

    The major determinants of receptor tissue tyrosine kinase (RTK) signaling specificity have been proposed to be Src homology 2 (SH2) binding sites, phosphotyrosine-containing oligopeptides in the cytoplasmic domain of the receptor. The Caenorhabditis elegans epidermal growth factor receptor homologue LET-23 has multiple functions during development and has eight potential SH2-binding sites in a region carboxyl terminal to its kinase domain. By analyzing transgenic nematodes for three distinct LET-23 functions, we show that six of eight potential sites function in vivo and that they are required for most, but not all, of LET-23 activity. A single site is necessary and sufficient to promote wild-type fertility. Three other sites activate the RAS pathway and are involved only in viability and vulval differentiation. A fifth site is promiscuous and can mediate all three LET-23 functions. An additional site mediates tissue-specific negative regulation. Putative SH2 binding sites are thus key effectors of both cell-specific and negative regulation in an intact organism. We suggest two distinct mechanisms for tissue-specific RTK-mediated signaling. A positive mechanism would promote RTK function through effectors present only in certain cell types. A negative mechanism would inhibit RTK function through tissue-specific negative regulators. Images PMID:9168466

  4. Tissue specific regulation of lipogenesis by thyroid hormone

    SciTech Connect

    Blennemann, B.; Freake, H. )

    1990-02-26

    Thyroid hormone stimulates long chain fatty acid synthesis in rat liver by increasing the amounts of key lipogenic enzymes. Sparse and conflicting data exist concerning its action on this pathway in other tissues. The authors recently showed that, in contrast to liver, hypothyroidism stimulates lipogenesis in brown adipose tissue and have now systematically examined the effects of thyroid state on fatty acid synthesis in other rat tissues. Lipogenesis was assessed by tritiated water incorporation. Euthyroid hepatic fatty acid synthesis (16.6um H/g/h) was reduced to 30% in hypothyroid rats and increased 3 fold in hyperthyroidism. Lipogenesis was detected in euthyroid kidney and heart and these levels were also stimulated by thyroid hormone treatment. Brown adipose tissue was unique in showing increased lipogenesis in the hypothyroid state. Hyperthyroid levels were not different from euthyroid. Effects in white adipose tissue were small and inconsistent. Brain, skin and lung were all lipogenically active, but did not respond to changes in thyroid state. Low but detectable levels of fatty acid synthesis were measured in muscle, which also were non-responsive. A wide spectrum of responses to thyroid hormone are seen in different rat tissues and thus the pathway of long chain fatty acid synthesis would appear to be an excellent model for examining the tissue specific regulation of gene expression by thyroid hormone.

  5. Tissue specific specialization of the nanoscale architecture of Arabidopsis.

    PubMed

    Liu, Jiliang; Inouye, Hideyo; Venugopalan, Nagarajan; Fischetti, Robert F; Gleber, S Charlotte; Vogt, Stefan; Cusumano, Joanne C; Kim, Jeong Im; Chapple, Clint; Makowski, Lee

    2013-11-01

    The Arabidopsis stem is composed of five tissues - the pith, xylem, phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning X-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism. PMID:24075949

  6. Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter.

    PubMed

    Fraizer, G C; Wu, Y J; Hewitt, S M; Maity, T; Ton, C C; Huff, V; Saunders, G F

    1994-03-25

    The Wilms' tumor gene, WT1, is expressed in few tissues, mainly the developing kidney, genitourinary system, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kidney and hematopoietic cell lines. We found three transcription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase II promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC-rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This finding suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identified a transcriptional enhancer located 3' of the WT1 gene > 50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested. PMID:8132626

  7. Reconstruction of Tissue-Specific Metabolic Networks Using CORDA

    PubMed Central

    Schultz, André; Qutub, Amina A.

    2016-01-01

    Human metabolism involves thousands of reactions and metabolites. To interpret this complexity, computational modeling becomes an essential experimental tool. One of the most popular techniques to study human metabolism as a whole is genome scale modeling. A key challenge to applying genome scale modeling is identifying critical metabolic reactions across diverse human tissues. Here we introduce a novel algorithm called Cost Optimization Reaction Dependency Assessment (CORDA) to build genome scale models in a tissue-specific manner. CORDA performs more efficiently computationally, shows better agreement to experimental data, and displays better model functionality and capacity when compared to previous algorithms. CORDA also returns reaction associations that can greatly assist in any manual curation to be performed following the automated reconstruction process. Using CORDA, we developed a library of 76 healthy and 20 cancer tissue-specific reconstructions. These reconstructions identified which metabolic pathways are shared across diverse human tissues. Moreover, we identified changes in reactions and pathways that are differentially included and present different capacity profiles in cancer compared to healthy tissues, including up-regulation of folate metabolism, the down-regulation of thiamine metabolism, and tight regulation of oxidative phosphorylation. PMID:26942765

  8. Tissue distribution of autoantigen specific for primary sclerosing cholangitis.

    PubMed Central

    Lo, S K; Chapman, R W; Fleming, K A

    1993-01-01

    AIM: To investigate the tissue distribution of the autoantigen specific for primary sclerosing cholangitis. METHODS: A range of normal frozen tissues including nervous system, muscle, uterus, ovary, prostate, pancreas, thyroid, salivary gland, adrenal gland, colon, gall bladder, stomach, jejunum, aorta, skin, kidney, liver, spleen and thymus was sectioned, fixed with acetone, and air-dried. Normal bone marrow and HL60, K562, and U937 cells were cytocentrifuged on to slides, air-dried, and alcohol fixed. Four sera from primary sclerosing cholangitis with high titre antibody (> 1/100) were used to screen the tissues using either two-step or APAAP immunohistochemistry. Normal sera were used as controls. RESULTS: Positive signal was detected in neutrophils in spleen with three out of four primary sclerosing cholangitis sera while one out of four primary sclerosing cholangitis sera stained spindle cells in the liver. All four sera stained mature neutrophils of the normal bone marrow. Some bone marrow neutrophil precursors (metamyelocytes and myelocytes) were also positive. All other tissues, including HL60, K562, and U937 cells, were negative. Normal sera were negative on all tissues. CONCLUSION: Antigen specific for primary sclerosing cholangitis seems to be unique to neutrophil polymorphs and is present only after myeloblast differentiation of the myeloid cell line. The antigen may be within the secondary granule of the neutrophil polymorph. Images PMID:7681855

  9. Regulating expression of cell and tissue-specific genes by modifying transcription

    SciTech Connect

    Beachy, Roger N; Dai, Shunhong

    2010-06-14

    Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Rice bZIP transcription factors RF2a, RF2b and RLP1 play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV), through their interactions with the Box II essential cis element located in the promoter (Dai et al., 2006., Dai et al., 2004., Yin et al., 1997). RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. It is equally as important to recognize that these proteins control plant development by regulating differentiation and/or function of the vascular tissues. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins will not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants. We have proposed characterize the function domains of RF2a, RF2b and RLP1 and explore the biological function of the transcription repressor RLP1.

  10. Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila.

    PubMed Central

    Benyajati, C; Ewel, A; McKeon, J; Chovav, M; Juan, E

    1992-01-01

    Chromatin footprinting in Drosophila tissue culture cells has detected the binding of a non-histone protein at +8 of the distal Adh RNA start site, on a 10-bp direct repeat motif abutting a nucleosome positioned over the inactive Adh distal promoter. Alternatively the active promoter is bound by a transcription initiation complex. We have characterized and purified a protein Adf-2 that binds specifically to this direct repeat motif 5'TCTCAGTGCA3', present at +8 and -202 of the distal RNA start site. DNase I footprinting, methylation interference, and UV-crosslinking analyses showed that both direct repeats interact in vitro with a nuclear protein of approximately 120 kilodaltons (kDa). We purified Adf-2 through multiple rounds of sequence-specific DNA affinity chromatography. Southwestern analysis showed that the purified 120 KDa polypeptide binds the Adf-2 motif efficiently as a monomer or homomultimer. In vivo titrations of Adf-2 activity with the Adf-2 motif by transient co-transfection competitions in different Drosophila cell lines suggested that Adf-2 is a cell-specific repressor. Adf-2 has been detected ubiquitously in vitro, but is functional in vivo as a sequence-specific DNA binding protein and repressor only in the cells that have the inactive distal promoter. We discuss the possibility that an activation process is required for Adf-2 protein to bind DNA and function in vivo. Images PMID:1408750

  11. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

    PubMed Central

    Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

    2014-01-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  12. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression.

    PubMed

    Ferrarese, Roberto; Harsh, Griffith R; Yadav, Ajay K; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M; Miller, Tyler E; Masilamani, Anie P; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M; Yu, Irene L Y; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W; He, Xiaolin; Prinz, Marco; Chandler, James P; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N; Carro, Maria S; Bredel, Markus

    2014-07-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  13. Controlled release strategies for modulating immune responses to promote tissue regeneration.

    PubMed

    Dumont, Courtney M; Park, Jonghyuck; Shea, Lonnie D

    2015-12-10

    Advances in the field of tissue engineering have enhanced the potential of regenerative medicine, yet the efficacy of these strategies remains incomplete, and is limited by the innate and adaptive immune responses. The immune response associated with injury or disease combined with that mounted to biomaterials, transplanted cells, proteins, and gene therapies vectors can contribute to the inability to fully restore tissue function. Blocking immune responses such as with anti-inflammatory or immunosuppressive agents are either ineffective, as the immune response contributes significantly to regeneration, or have significant side effects. This review describes targeted strategies to modulate the immune response in order to limit tissue damage following injury, promote an anti-inflammatory environment that leads to regeneration, and induce antigen (Ag)-specific tolerance that can target degenerative diseases that destroy tissues and promote engraftment of transplanted cells. Focusing on targeted immuno-modulation, we describe local delivery techniques to sites of inflammation as well as systemic approaches that preferentially target subsets of immune populations. PMID:26264833

  14. Promoter-specific expression and imprint status of marsupial IGF2.

    PubMed

    Stringer, Jessica M; Suzuki, Shunsuke; Pask, Andrew J; Shaw, Geoff; Renfree, Marilyn B

    2012-01-01

    In mice and humans, IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1-P3 in the human and mouse IGF2 locus. The expression of tammar IGF2 was predominantly from the P2 promoter, similar to humans. Expression of IGF2 was higher in pouch young than in the adult and imprinting was highly tissue and developmental-stage specific. Interestingly, while IGF2 was expressed throughout the placenta, imprinting seemed to be restricted to the vascular, trilaminar region. In addition, IGF2 was monoallelically expressed in the adult mammary gland while in the liver it switched from monoalleleic expression in the pouch young to biallelic in the adult. These data suggest a complex mode of IGF2 regulation in marsupials as seen in eutherian mammals. The conservation of the IGF2 promoters suggests they originated before the divergence of marsupials and eutherians, and have been selectively maintained for at least 160 million years. PMID:22848567

  15. Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig.

    PubMed

    Song, Yuning; Lai, Liangxue; Li, Li; Huang, Yongye; Wang, Anfeng; Tang, Xiaochun; Pang, Daxin; Li, Zhanjun; Ouyang, Hongsheng

    2016-01-01

    The Cre-loxP system is a powerful tool for genetic analysis of distinct cell lineages and tissue-specific gene knockout in animal models. VASA is specifically expressed in reproductive tissues, and is known to play important roles in spermatogenesis and germ-cell growth. In this study, Cre recombinase transgenic pigs under the control of the VASA promoter were generated by somatic cell nuclear transfer. Germ cell-specific expression of Cre recombinase in VASA-Cre transgenic pigs was shown by western blotting and immunohistochemistry. VASA-Cre transgenic pigs will be a useful tool for germ cell-specific gene knockout and a disease model for disorders of the reproductive system. PMID:27047735

  16. Calcium Channel CaVα₁ Splice Isoforms - Tissue Specificity and Drug Action.

    PubMed

    Lipscombe, Diane; Andrade, Arturo

    2015-01-01

    Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cellspecific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among CaV isoforms expressed in different tissues, and such differences may be greater than those that exist across CaV channel homologs of closely related species. Here we highlight examples of CaV isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of CaV isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of CaV channel inhibitors and agonists with improved tissue-specificity. PMID:25966698

  17. Tissue-specific cell wall hydration in sugarcane stalks.

    PubMed

    Maziero, Priscila; Jong, Jennifer; Mendes, Fernanda M; Gonçalves, Adilson R; Eder, Michaela; Driemeier, Carlos

    2013-06-19

    Plant cell walls contain water, especially under biological and wet processing conditions. The present work characterizes this water in tissues of sugarcane stalks. Environmental scanning electron microscopy shows tissue deformation upon drying. Dynamic vapor sorption determines the equilibrium and kinetics of moisture uptake. Thermoporometry by differential scanning calorimetry quantifies water in nanoscale pores. Results show that cell walls from top internodes of stalks are more deformable, slightly more sorptive to moisture, and substantially more porous. These differences of top internode are attributed to less lignified walls, which is confirmed by lower infrared spectral signal from aromatics. Furthermore, cell wall nanoscale porosity, an architectural and not directly compositional characteristic, is shown to be tissue-specific. Nanoscale porosities are ranked as follows: pith parenchyma > pith vascular bundles > rind. This ranking coincides with wall reactivity and digestibility in grasses, suggesting that nanoscale porosity is a major determinant of wall recalcitrance. PMID:23738592

  18. Tissue-specific mRNA expression profiling in grape berry tissues

    PubMed Central

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

  19. Tissue-specific alternative splicing of TCF7L2.

    PubMed

    Prokunina-Olsson, Ludmila; Welch, Cullan; Hansson, Ola; Adhikari, Neeta; Scott, Laura J; Usher, Nicolle; Tong, Maurine; Sprau, Andrew; Swift, Amy; Bonnycastle, Lori L; Erdos, Michael R; He, Zhi; Saxena, Richa; Harmon, Brennan; Kotova, Olga; Hoffman, Eric P; Altshuler, David; Groop, Leif; Boehnke, Michael; Collins, Francis S; Hall, Jennifer L

    2009-10-15

    Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r(2) = 0.84-0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164-FJ010174. PMID:19602480

  20. Galectin-1 Regulates Tissue Exit of Specific Dendritic Cell Populations*

    PubMed Central

    Thiemann, Sandra; Man, Jeanette H.; Chang, Margaret H.; Lee, Benhur; Baum, Linda G.

    2015-01-01

    During inflammation, dendritic cells emigrate from inflamed tissue across the lymphatic endothelium into the lymphatic vasculature and travel to regional lymph nodes to initiate immune responses. However, the processes that regulate dendritic cell tissue egress and migration across the lymphatic endothelium are not well defined. The mammalian lectin galectin-1 is highly expressed by vascular endothelial cells in inflamed tissue and has been shown to regulate immune cell tissue entry into inflamed tissue. Here, we show that galectin-1 is also highly expressed by human lymphatic endothelial cells, and deposition of galectin-1 in extracellular matrix selectively regulates migration of specific human dendritic cell subsets. The presence of galectin-1 inhibits migration of immunogenic dendritic cells through the extracellular matrix and across lymphatic endothelial cells, but it has no effect on migration of tolerogenic dendritic cells. The major galectin-1 counter-receptor on both dendritic cell populations is the cell surface mucin CD43; differential core 2 O-glycosylation of CD43 between immunogenic dendritic cells and tolerogenic dendritic cells appears to contribute to the differential effect of galectin-1 on migration. Binding of galectin-1 to immunogenic dendritic cells reduces phosphorylation and activity of the protein-tyrosine kinase Pyk2, an effect that may also contribute to reduced migration of this subset. In a murine lymphedema model, galectin-1−/− animals had increased numbers of migratory dendritic cells in draining lymph nodes, specifically dendritic cells with an immunogenic phenotype. These findings define a novel role for galectin-1 in inhibiting tissue emigration of immunogenic, but not tolerogenic, dendritic cells, providing an additional mechanism by which galectin-1 can dampen immune responses. PMID:26216879

  1. Tissue-specific patterns of allelically-skewed DNA methylation

    PubMed Central

    Marzi, Sarah J.; Meaburn, Emma L.; Dempster, Emma L.; Lunnon, Katie; Paya-Cano, Jose L.; Smith, Rebecca G.; Volta, Manuela; Troakes, Claire; Schalkwyk, Leonard C.; Mill, Jonathan

    2016-01-01

    ABSTRACT While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ∼220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood. PMID:26786711

  2. Tissue-specific clocks in Arabidopsis show asymmetric coupling

    PubMed Central

    Endo, Motomu; Shimizu, Hanako; Nohales, Maria A.; Araki, Takashi; Kay, Steve A.

    2014-01-01

    Many organisms rely on a circadian clock system to adapt to daily and seasonal environmental changes. The mammalian circadian clock consists of a central clock in the suprachiasmatic nucleus that is tightly coupled and synchronizes other clocks in peripheral tissues1, 2. Plants also have a circadian clock, but plant circadian clock function has long been assumed to be uncoupled3. Only a few studies have been able to show a weak, local coupling among cells4, 5, 6, 7. Here, by implementing two novel techniques, we have performed a comprehensive tissue-specific analysis of leaf tissues, and we have discovered that the vasculature and mesophyll clocks asymmetrically regulate each other in Arabidopsis. The circadian clock in the vasculature has characteristics distinct from other tissues, cycles robustly without environmental cues, and affects circadian clock regulation in other tissues. Furthermore, we found that vasculature-enriched genes that are rhythmic are preferentially expressed in the evening, whereas rhythmic mesophyll-enriched genes tend to be expressed in the morning. Our results set the stage for a deeper understanding of how the vasculature circadian clock in plants regulates key physiological responses such as flowering time. PMID:25363766

  3. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  4. Evolutionary dynamics and tissue specificity of human long noncoding RNAs in six mammals.

    PubMed

    Washietl, Stefan; Kellis, Manolis; Garber, Manuel

    2014-04-01

    Long intergenic noncoding RNAs (lincRNAs) play diverse regulatory roles in human development and disease, but little is known about their evolutionary history and constraint. Here, we characterize human lincRNA expression patterns in nine tissues across six mammalian species and multiple individuals. Of the 1898 human lincRNAs expressed in these tissues, we find orthologous transcripts for 80% in chimpanzee, 63% in rhesus, 39% in cow, 38% in mouse, and 35% in rat. Mammalian-expressed lincRNAs show remarkably strong conservation of tissue specificity, suggesting that it is selectively maintained. In contrast, abundant splice-site turnover suggests that exact splice sites are not critical. Relative to evolutionarily young lincRNAs, mammalian-expressed lincRNAs show higher primary sequence conservation in their promoters and exons, increased proximity to protein-coding genes enriched for tissue-specific functions, fewer repeat elements, and more frequent single-exon transcripts. Remarkably, we find that ∼20% of human lincRNAs are not expressed beyond chimpanzee and are undetectable even in rhesus. These hominid-specific lincRNAs are more tissue specific, enriched for testis, and faster evolving within the human lineage. PMID:24429298

  5. Species, interindividual, and tissue specificity in endocrine signaling.

    PubMed Central

    Walker, C; Ahmed, S A; Brown, T; Ho, S M; Hodges, L; Lucier, G; Russo, J; Weigel, N; Weise, T; Vandenbergh, J

    1999-01-01

    The activity of endocrine-active agents exhibits specificity at many levels. Differential responsiveness to these agents has been observed between different species and extends to interindividual differences within a species and between different tissues as well. In cases where they have been identified, the biologic and molecular mechanisms underlying this specificity are quite diverse. Determinants of species specificity include differences that exist in receptor binding, gene transcription, and cellular responses to endocrine-active compounds between species. Interindividual differences in responsiveness may be determined at the level of genetic polymorphisms in hormone-metabolizing enzymes, hormone receptors, and in those genes that are transactivated by these receptors, as well as during changing windows of susceptibility that occur as a function of age, such as prenatal and postmenopausal exposures. Extrinsic factors such as diet can also impact individual susceptibility to endocrine-active agents. Tissue-specific determinants of susceptibility are well documented, but little is known regarding the mechanisms underlying these different responses. Differences in the expression of accessory proteins for steroid hormone receptors and different patterns of receptor expression, estrogen receptor alpha and estrogen receptor beta; for example, may contribute to tissue specificity, as may differences in the pattern of expression of other genes such as hormone-metabolizing enzymes. The use of animal model systems and development of appropriate mathematical models has the potential to yield additional valuable information for elucidating the role of these determinants of specificity at low-dose exposures and for improved risk assessments for the adverse health effects of endocrine-active compounds. PMID:10421772

  6. A novel, tissue-specific, Drosophila homeobox gene.

    PubMed Central

    Barad, M; Jack, T; Chadwick, R; McGinnis, W

    1988-01-01

    The homeobox gene family of Drosophila appears to control a variety of position-specific patterning decisions during embryonic and imaginal development. Most of these patterning decisions determine groups of cells on the anterior-posterior axis of the Drosophila germ band. We have isolated a novel homeobox gene from Drosophila, designated H2.0. H2.0 has the most diverged homeobox so far characterized in metazoa, and, in contrast to all previously isolated homeobox genes, H2.0 exhibits a tissue-specific pattern of expression. The cells that accumulate transcripts for this novel gene correspond to the visceral musculature and its anlagen. Images PMID:2901348

  7. Combinatorial regulation of tissue specification by GATA and FOG factors

    PubMed Central

    Chlon, Timothy M.; Crispino, John D.

    2012-01-01

    The development of complex organisms requires the formation of diverse cell types from common stem and progenitor cells. GATA family transcriptional regulators and their dedicated co-factors, termed Friend of GATA (FOG) proteins, control cell fate and differentiation in multiple tissue types from Drosophila to man. FOGs can both facilitate and antagonize GATA factor transcriptional regulation depending on the factor, cell, and even the specific gene target. In this review, we highlight recent studies that have elucidated mechanisms by which FOGs regulate GATA factor function and discuss how these factors use these diverse modes of gene regulation to control cell lineage specification throughout metazoans. PMID:23048181

  8. Location, location, location: tissue-specific regulation of immune responses

    PubMed Central

    Hu, Wei; Pasare, Chandrashekhar

    2013-01-01

    Discovery of DCs and PRRs has contributed immensely to our understanding of induction of innate and adaptive immune responses. Activation of PRRs leads to secretion of inflammatory cytokines that regulate priming and differentiation of antigen-specific T and B lymphocytes. Pathogens enter the body via different routes, and although the same set of PRRs is likely to be activated, it is becoming clear that the route of immune challenge determines the nature of outcome of adaptive immunity. In addition to the signaling events initiated following innate-immune receptor activation, the cells of the immune system are influenced by the microenvironments in which they reside, and this has a direct impact on the resulting immune response. Specifically, immune responses could be influenced by specialized DCs, specific factors secreted by stromal cells, and also, by commensal microbiota present in certain organs. Following microbial detection, the complex interactions among DCs, stromal cells, and tissue-specific factors influence outcome of immune responses. In this review, we summarize recent findings on the phenotypic heterogeneity of innate and adaptive immune cells and how tissue-specific factors in the systemic and mucosal immune system influence the outcome of adaptive-immune responses. PMID:23825388

  9. Two tobacco AP1-like gene promoters with highly specific, tightly regulated and uniquely expressed activity during floral transition, initiation and development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biotech engineering of agronomic traits requires an array of highly specific and tightly regulated promoters in flower or other tissues. In this study, we isolated and characterized two tobacco AP1-like promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using GUS reporter and tissue-speci...

  10. Light-regulated and cell-specific methylation of the maize PEPC promoter

    PubMed Central

    Tolley, Ben J.; Woodfield, Helen; Wanchana, Samart; Bruskiewich, Richard; Hibberd, Julian M.

    2012-01-01

    The molecular mechanisms governing PEPC expression in maize remain to be fully defined. Differential methylation of a region in the PEPC promoter has been shown to correlate with transcript accumulation, however, to date, investigations into the role of DNA methylation in maize PEPC expression have relied on the use of methylation-sensitive restriction enzymes. Bisulphite sequencing was used here to provide a single-base resolution methylation map of the maize PEPC promoter. It is shown that four cytosine residues in the PEPC promoter are heavily methylated in maize root tissue. In leaves, de-methylation of these cytosines is dependent on illumination and is coincident with elevated PEPC expression. Furthermore, light-regulated de-methylation of these cytosines occurs only in mesophyll cells. No methylation was discovered in the 0.6 kb promoter required for mesophyll-specific expression indicating that cytosine methylation is not required to direct the cell-specificity of PEPC expression. This raises interesting questions regarding the function of the cell-specific cytosine de-methylation observed in the upstream region of the PEPC promoter. PMID:22143916

  11. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

    PubMed Central

    Ngô, V; Gourdji, D; Laverrière, J N

    1996-01-01

    The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo. PMID:8668139

  12. Flowering regulation by tissue specific functions of photoreceptors

    PubMed Central

    Endo, Motomu

    2008-01-01

    Flowering is one of the most important steps in a plant life cycle. Plants utilize light as an informational source to determine the timing of flowering. In Arabidopsis, phytochrome A (phyA), phyB and cryptochrome2 (cry2) are major photoreceptors that regulate flowering. These photoreceptors perceive light stimuli by leaves for the regulation of flowering. A leaf is an organ consisting of different tissues such as epidermis, mesophyll and vascular bundles. In the present study, we examined in which tissue the light signals are perceived and how those signals are integrated within a leaf to regulate flowering. For this purpose, we established transgenic Arabidopsis lines that expressed a phyB-green fluorescent protein (GFP) fusion protein or a cry2-GFP fusion protein in organ/tissue-specific manners. Consequently, phyB was shown to perceive light stimuli in mesophyll. By contrast, cry2 functioned only in vascular bundles. We further confirmed that both phyB-GFP and cry2-GFP regulated flowering by altering the expression of a key flowering gene, FT, in vascular bundles. In summary, perception sites for different spectra of light are spatially separated within a leaf and the signals are integrated through the inter-tissue communication. PMID:19704768

  13. The 14-3-3 gene expression specificity in response to stress is promoter-dependent.

    PubMed

    Aksamit, Anna; Korobczak, Alina; Skala, Jacek; Lukaszewicz, Marcin; Szopa, Jan

    2005-10-01

    Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms. PMID:16081528

  14. Regulating expressin of cell and tissue-specific genes by modifying transcription

    SciTech Connect

    Beachy, R N; Dai, Shunhong

    2009-12-15

    Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Our research supported by this program has led to the identification of rice bZIP transcription factors RF2a, RF2b and RLP1 that play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV) through their interactions with the Box II essential cis element located in the promoter. RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants and to improve biofuel feedstock.

  15. Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

    PubMed

    Vragović, Kristina; Sela, Ayala; Friedlander-Shani, Lilach; Fridman, Yulia; Hacham, Yael; Holland, Neta; Bartom, Elizabeth; Mockler, Todd C; Savaldi-Goldstein, Sigal

    2015-01-20

    The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth. PMID:25561530

  16. Regulatory Principles Governing Tissue Specificity of Developmental Enhancers

    PubMed Central

    Farley, Emma K.; Olson, Katrina M.; Levine, Michael S.

    2016-01-01

    Transcriptional enhancers are short segments of genomic DNA (50 bp to 1 kb in length) that can work over long distances (≥1 Mb) to regulate gene expression in specific cells and tissues. Genomic assays have identified on the order of 400,000 to one million putative enhancers in the human genome (e.g., ENCODE Consortium). This suggests that a typical gene is regulated by tens of enhancers, ensuring stringent regulation of gene expression in response to a variety of intrinsic and external signals. Despite the discovery of the first transcriptional enhancer more than 30 years ago, we know surprisingly little about how enhancers regulate gene expression. In particular, the relationship between primary DNA sequence and enhancer specificity remains obscure. Here we summarize recent high-throughput studies in whole embryos aimed at the systematic identification of the sequence and organizational constraints underlying enhancer function and specificity. PMID:27325706

  17. Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

    PubMed

    Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

    2013-12-01

    Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops. PMID:23974493

  18. Construction and detection of the tissue-specific pINV-HPV16 E6/7 vector

    PubMed Central

    GAO, HUI; HUANG, ZHENGFANG; SHI, CHENLONG; LI, HOUDA

    2015-01-01

    A tissue-specific promoter can control downstream gene expression in tissues or organs. The human involucrin (hINV) promoter (pINV) that contains 2474 bp of hINV upstream sequence is able to regulate tissue-specific gene expression. This tissue specificity may be important for the prevention and treatment of human papilloma virus infections. pINV was cloned by polymerase chain reaction and the human papillomavirus (HPV)16 E6/7 gene was obtained from the cancer tissue samples of patients with cervical carcinoma at the Yangzhou Maternal and China Health-Care Center of Jinagsu Province (Yangzhou, China). First, specific primers were designed according to the genomic DNA sequence of the HPV16-type standard strain that has been reported and the E6/7 gene was acquired by PCR. The carcinogenic fraction of the E6/7 gene was removed and the remaining section was cloned into T vectors, sequenced correctly and then cloned into the eukaryotic expression vector pCEP4, which was lacking the CMV promoter. The positive recombinants were identified using blue-white screening and endonuclease digestion, subsequent to sequencing and analysis, and the tissue-specific recombinant pINV-HPV16E6/7 plasmids was detected. PMID:25621060

  19. Tissue specific metal characterization of selected fish species in Pakistan.

    PubMed

    Ahmed, Mukhtiar; Ahmad, Taufiq; Liaquat, Muhammad; Abbasi, Kashif Sarfraz; Farid, Ibrahim Bayoumi Abdel; Jahangir, Muhammad

    2016-04-01

    Concentration of various metals, i.e., zinc (Zn), copper (Cu), lead (Pb), nickel (Ni), iron (Fe), manganese (Mn), chromium (Cr), and silver (Ag), was evaluated in five indigenous fish species (namely, silver carp, common carp, mahseer, thela fish, and rainbow trout), by using atomic absorption spectrophotometer. It is proved from this study that, overall, mahseer and rainbow trout had high amount of zinc, whereas thela fish and silver carp had high concentration of copper, chromium, silver, nickel, and lead, while common carp had highest amount of iron contents. Furthermore, a tissue-specific discrimination among various fish species was observed, where higher metal concentrations were noticed in fish liver, with decreasing concentration in other organs like skin, gills, and finally the least contents in fish muscle. Multivariate data analysis showed not only a variation in heavy metals among the tissues but also discrimination among the selected fish species. PMID:26951449

  20. Control of NKT cell differentiation by tissue-specific microenvironments.

    PubMed

    Yang, Yang; Ueno, Aito; Bao, Min; Wang, Zhongying; Im, Jin Seon; Porcelli, Steven; Yoon, Ji-Won

    2003-12-01

    CD1d-restricted Valpha14 NKT cells play an important role in both Th1- and Th2-type immune responses. To determine whether NKT cells develop two functionally distinct subsets that provoke different types of responses, we examined the phenotypes and cellular functions of NK1.1(+) and DX5(+) T cells. We found that both NK1.1(+) and DX5(+) T cells are CD1d-restricted Valpha14 T cells with identical Ag specificities, phenotypes, tissue locations, and functions. Similar to the NK1.1 marker, the DX5 marker (CD49b) is expressed on mature NKT cells in both NK1.1 allele-positive and allele-negative strains. However, when NK1.1(+) and DX5(+) NKT cells isolated from different tissues were compared, we found that thymic and splenic NKT cells differed not only in their cytokine profiles, but also in their phenotype and requirements for costimulatory signals. Thymic NKT cells displayed the phenotype of activated T cells and could be fully activated by TCR ligation. In contrast, splenic NKT cells displayed the phenotype of memory T cells and required a costimulatory signal for activation. Furthermore, the function and phenotype of thymic and splenic NKT cells were modulated by APCs from various tissues that expressed different levels of costimulatory molecules. Modulation of NKT cell function and differentiation may be mediated by synergic effects of costimulatory molecules on the surface of APCs. The results of the present study suggest that the costimulatory signals of tissue-specific APCs are key factors for NKT cell differentiation, and these signals cannot be replaced by anti-CD28 or anti-CD40 ligand Abs. PMID:14634102

  1. Partial characterization of specific cantharidin binding sites in mouse tissues

    SciTech Connect

    Graziano, M.J.; Pessah, I.N.; Matsuzawa, M.; Casida, J.E.

    1988-06-01

    The mode of action of cantharidin, the natural vesicant of blister beetles, is examined by radioligand binding studies with mouse tissues. (3H)Cantharidin undergoes specific and saturable binding with the liver cytosol, which is characterized as follows: Kd and Bmax values of 30 nM and 1.8 pmol/mg of protein, respectively; linearity with respect to protein concentration; pH optimum of 6.5 to 7.5; association and dissociation half-times of 20 min and 12 hr, respectively; and 50% inhibition by Mg2+ at 70 microM, Ca2+ at 224 microM, pyrophosphate at 27 microM, and nucleotide triphosphates at 52-81 microM. The binding site undergoes a loss of activity at 45 degrees or higher. The toxicological relevance of this specific (3H)cantharidin binding site of mouse liver cytosol is established in three ways. First, the potency of 15 active cantharidin analogs for inhibiting (3H)cantharidin binding is correlated with their acute toxicity to mice (r = 0.829). Second, 26 related compounds that are inactive in inhibiting (3H)cantharidin binding are also of little or no toxicity to mice. Finally, the binding of (3H) cantharidin to liver cytosol from mice poisoned with increasing amounts of unlabeled cantharidin is inhibited in a dose-dependent manner. (3H)Cantharidin also specifically binds to cytosol fractions of blood, brain, heart, kidney, lung, pancreas, skin, spleen, and stomach. The characteristics of the specific binding site in brain are very similar to those determined in liver with respect to Kd, Bmax, association/dissociation kinetics, and sensitivity to inhibitors. It therefore appears that the toxicity of cantharidin and related oxabicycloheptanes, including the herbicide endothal, is attributable to binding at a specific site in liver and possibly other tissues.

  2. Cell type-specific properties and environment shape tissue specificity of cancer genes

    PubMed Central

    Schaefer, Martin H.; Serrano, Luis

    2016-01-01

    One of the biggest mysteries in cancer research remains why mutations in certain genes cause cancer only at specific sites in the human body. The poor correlation between the expression level of a cancer gene and the tissues in which it causes malignant transformations raises the question of which factors determine the tissue-specific effects of a mutation. Here, we explore why some cancer genes are associated only with few different cancer types (i.e., are specific), while others are found mutated in a large number of different types of cancer (i.e., are general). We do so by contrasting cellular functions of specific-cancer genes with those of general ones to identify properties that determine where in the body a gene mutation is causing malignant transformations. We identified different groups of cancer genes that did not behave as expected (i.e., DNA repair genes being tissue specific, immune response genes showing a bimodal specificity function or strong association of generally expressed genes to particular cancers). Analysis of these three groups demonstrates the importance of environmental impact for understanding why certain cancer genes are only involved in the development of some cancer types but are rarely found mutated in other types of cancer. PMID:26856619

  3. Cell type-specific properties and environment shape tissue specificity of cancer genes.

    PubMed

    Schaefer, Martin H; Serrano, Luis

    2016-01-01

    One of the biggest mysteries in cancer research remains why mutations in certain genes cause cancer only at specific sites in the human body. The poor correlation between the expression level of a cancer gene and the tissues in which it causes malignant transformations raises the question of which factors determine the tissue-specific effects of a mutation. Here, we explore why some cancer genes are associated only with few different cancer types (i.e., are specific), while others are found mutated in a large number of different types of cancer (i.e., are general). We do so by contrasting cellular functions of specific-cancer genes with those of general ones to identify properties that determine where in the body a gene mutation is causing malignant transformations. We identified different groups of cancer genes that did not behave as expected (i.e., DNA repair genes being tissue specific, immune response genes showing a bimodal specificity function or strong association of generally expressed genes to particular cancers). Analysis of these three groups demonstrates the importance of environmental impact for understanding why certain cancer genes are only involved in the development of some cancer types but are rarely found mutated in other types of cancer. PMID:26856619

  4. Hyperplasia, de novo lymphangiogenesis, and lymphatic regression in mice with tissue-specific, inducible overexpression of murine VEGF-D.

    PubMed

    Lammoglia, Gabriela M; Van Zandt, Carolynn E; Galvan, Daniel X; Orozco, Jose L; Dellinger, Michael T; Rutkowski, Joseph M

    2016-08-01

    Lymphatic vessels modulate tissue fluid balance and inflammation and provide a conduit for endocrine and lipid transport. The growth of new lymphatic vessels in the adult, lymphangiogenesis, is predominantly mediated through vascular endothelial growth factor receptor-3 (VEGFR-3) signaling. We took advantage of the unique binding of murine VEGF-D specifically to VEGFR-3 and generated mice capable of inducible, tissue-specific expression of murine VEGF-D under a tightly-controlled tetracycline response element (TRE) promoter to stimulate adult tissue lymphangiogenesis. With doxycycline-activated expression, TRE-VEGF-D mouse crossed to mice with tissue-specific promoters for the lung [Clara cell secretory protein-reverse tetracycline transactivator (rtTA)] developed pulmonary lymphangiectasia. In the kidney, (kidney-specific protein-rtTA × TRE-VEGF-D) mice exhibited rapid lymphatic hyperplasia on induction of VEGF-D expression. Crossed with adipocyte-specific adiponectin-rtTA mice [Adipo-VEGF-D (VD)], chronic VEGF-D overexpression was capable of inducing de novo lymphangiogenesis in white adipose tissue and a massive expansion of brown adipose tissue lymphatics. VEGF-D expression in white adipose tissue also increased macrophage infiltration and tissue fibrosis in the tissue. Expression did not, however, measurably affect peripheral fluid transport, the blood vasculature, or basal metabolic parameters. On removal of the doxycycline stimulus, VEGF-D expression returned to normal, and the expanded adipose tissue lymphatics regressed in Adipo-VD mice. The inducible TRE-VEGF-D mouse thus provides a novel murine platform to study the adult mechanisms and therapies of an array of disease- and tissue-specific models of lymphangiogenesis. PMID:27342876

  5. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues.

    PubMed

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  6. Tissue-specific Differentiation Potency of Mesenchymal Stromal Cells from Perinatal Tissues

    PubMed Central

    Kwon, Ahlm; Kim, Yonggoo; Kim, Myungshin; Kim, Jiyeon; Choi, Hayoung; Jekarl, Dong Wook; Lee, Seungok; Kim, Jung Min; Shin, Jong-Chul; Park, In Yang

    2016-01-01

    Human perinatal tissue is an abundant source of mesenchymal stromal cells(MSCs) and lacks the ethical concerns. Perinatal MSCs can be obtained from various tissues as like amnion, chorion, and umbilical cord. Still, little is known of the distinct nature of each MSC type. In this study, we successfully isolated and cultured MSCs from amnion(AMSCs), chorion(CMSCs), and umbilical cord(UC-MSCs). Proliferation potential was different among them, that AMSCs revealed the lowest proliferation rate due to increased Annexin V and senescence-associated β-galactosidase positive cells. We demonstrated distinct characteristic gene expression according to the source of the original tissue using microarray. In particular, genes associated with apoptosis and senescence including CDKN2A were up-regulated in AMSCs. In CMSCs, genes associated with heart morphogenesis and blood circulation including HTR2B were up-regulated. Genes associated with neurological system processes including NPY were up-regulated in UC-MSCs. Quantitative RT-PCR confirmed the gene expression data. And in vitro differentiation of MSCs demonstrated that CMSCs and UC-MSCs had a more pronounced ability to differentiate into cardiomyocyte and neural cells, respectively. This study firstly demonstrated the innate tissue-specific differentiation potency of perinatal MSCs which can be helpful in choosing more adequate cell sources for better outcome in a specific disease. PMID:27045658

  7. Expression in Arabidopsis of a nucellus-specific promoter from watermelon (Citrullus lanatus).

    PubMed

    Dwivedi, Krishna K; Roche, Dominique; Carman, John G

    2010-11-01

    Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony. PMID:21802614

  8. Caries management pathways preserve dental tissues and promote oral health.

    PubMed

    Ismail, Amid I; Tellez, Marisol; Pitts, Nigel B; Ekstrand, Kim R; Ricketts, David; Longbottom, Christopher; Eggertsson, Hafsteinn; Deery, Christopher; Fisher, Julian; Young, Douglas A; Featherstone, John D B; Evans, Wendell; Zeller, Gregory G; Zero, Domenick; Martignon, Stefania; Fontana, Margherita; Zandona, Andrea

    2013-02-01

    In May 2012, cariologists, dentists, representatives of dental organizations, manufacturers, and third party payers from several countries, met in Philadelphia, Pennsylvania, to define a common mission; goals and strategic approaches for caries management in the 21th century. The workshop started with an address by Mr. Stanley Bergman, CEO of Henry Schein Inc. which focused on the imperative for change in academia, clinical practice, and public health. For decades, new scientific evidence on caries and how it should be managed have been discussed among experts in the field. However, there has been some limited change, except in some Scandinavian countries, in the models of caries management and reimbursement which have been heavily skewed toward 'drilling and filling'. There is no overall agreement on a caries' case definition or on when to surgically intervene. The participants in the workshop defined a new mission for all caries management approaches, both conventional and new. The mission of each system should be to preserve the tooth structure, and restore only when necessary. This mission marks a pivotal line for judging when to surgically intervene and when to arrest or remineralize early noncavitated lesions. Even when restorative care is necessary, the removal of hard tissues should be lesion-focused and aim to preserve, as much as possible, sound tooth structure. Continuing management of the etiological factors of caries and the use of science-based preventive regimens also will be required to prevent recurrence and re-restoration. These changes have been debated for over a decade. The Caries Management Pathways includes all systems and philosophies, conventional and new, of caries management that can be used or modified to achieve the new mission. The choice of which system to use to achieve the mission of caries management is left to the users and should be based on the science supporting each approach or philosophy, experience, utility, and ease of use

  9. Implanted scaffold-free prevascularized constructs promote tissue repair

    PubMed Central

    Czajka, Caitlin A.; Calder, Bennet W.; Yost, Michael J.; Drake, Christopher J.

    2014-01-01

    To evaluate the anastomotic potential of prevascular tissue constructs generated from scaffold-free self-assembly of human endothelial and fibroblast cells, tissue constructs were implanted into athymic mice and immune-competent rats. Analysis of xenografts placed into hind limb muscle defects showed vascular anastomotic activity by 3 days following implantation and persisting for 2 weeks. Integration of the implanted prevascular tissue constructs with the host circulatory system was evident from presence of red blood cells in the implant as early as 3 days following implantation. Additionally, analysis of 3-day xenografts in the rat model showed activation of skeletal muscle satellite cells based on Pax-7 and MyoD expression. We conclude that prevascular tissue constructs generated from scaffold-free self-assembly of human endothelial and fibroblast cells are a promising tool to provide both vascular supply and satellite cell activation toward the resolution of skeletal muscle injury. PMID:25668498

  10. Binding of a liver-specific factor to the human albumin gene promoter and enhancer

    SciTech Connect

    Frain, M.; Hardon, E.; Ciliberto, G. ); Sala-Trepat, J.M. )

    1990-03-01

    A segment of 1,022 base pairs (bp) of the 5{prime}-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: a negative element located between bp {minus}673 and {minus}486, an enhancer essential for efficient albumin transcription located between bp {minus}486 and {minus}221, and a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver {ital trans}-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-B1-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms.

  11. Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

    PubMed Central

    Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

    2015-01-01

    Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

  12. A hierarchy of ECM-mediated signalling tissue-specific gene expression regulates tissue-specific gene expression

    SciTech Connect

    Roskelley, Calvin D; Srebrow, Anabella; Bissell, Mina J

    1995-10-07

    A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution.

  13. Identification of tumor-specific Salmonella Typhimurium promoters and their regulatory logic

    PubMed Central

    Leschner, Sara; Deyneko, Igor V.; Lienenklaus, Stefan; Wolf, Kathrin; Bloecker, Helmut; Bumann, Dirk; Loessner, Holger; Weiss, Siegfried

    2012-01-01

    Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia. PMID:22140114

  14. Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    PubMed Central

    Husseiny, Mohamed I.; Kaye, Alexander; Zebadua, Emily; Kandeel, Fouad; Ferreri, Kevin

    2014-01-01

    The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy. PMID:24722187

  15. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance.

    PubMed

    Soroosh, Pejman; Doherty, Taylor A; Duan, Wei; Mehta, Amit Kumar; Choi, Heonsik; Adams, Yan Fei; Mikulski, Zbigniew; Khorram, Naseem; Rosenthal, Peter; Broide, David H; Croft, Michael

    2013-04-01

    Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3(+) iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3(+) Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma. PMID:23547101

  16. Development of vascular tissue and stress inducible hybrid-synthetic promoters through dof-1 motifs rearrangement.

    PubMed

    Ranjan, Rajiv; Dey, Nrisingha

    2012-07-01

    A Caulimovirus-based hybrid-promoter, EFCFS, was derived by fusing the distal region (-227 to -54, FUAS) of Figwort mosaic virus full-length transcript promoter (F20) with the core promoter (-151 to +12, FS3CP) domain of Figwort mosaic virus sub-genomic transcript promoter (FS3). The hybrid-promoter (EFCFS) showed enhanced activity compared to the CaMV35S, F20 and FS3 promoters; while it showed equivalent activity with that of the CAMV35S(2) promoter in both transient protoplast (Nicotiana tabacum cv. Xanthi Brad) and transgenic plants (Nicotiana tabacum; Samsun NN). Further, we have engineered the EFCFS promoter sequence by inserting additional copies of the stress-inducible 'AAAG' cis-motif (Dof-1) to generate a set of three hybrid-synthetic promoters namely; EFCFS-HS-1, EFCFS-HS-2 and EFCFS-HS-3-containing 10, 11 and 13 'AAAG' motif, respectively. Transgenic plants expressing these hybrid synthetic promoters coupled to the GUS reporter were developed and their transcriptional activities were compared with F20, FS3, 35S and 35S(2) promoters, respectively. The relative levels of uidA-mRNA accumulation in transgenic plants driven by above promoters individually were compared by qRT-PCR. Localization of GUS reporter activity in plant tissue was assayed by histochemical approach. CLSM-based study revealed that hybrid-synthetic promoters namely; EFCFS-HS-1, EFCFS-HS-2 and EFCFS-HS-3 showed enhanced activity in vascular tissue compared to the CaMV35S promoter. In the presence of abiotic stress elicitors, salicylic acid and jasmonic acid, the EFCFS-HS-1 promoters showed enhanced activity compared to the 35S promoter. Newly derived hybrid-synthetic promoter/s with enhanced activity and stress inducibility could become efficient tools for advancement of plant biotechnology. PMID:22610660

  17. Fusarium oxysporum Triggers Tissue-Specific Transcriptional Reprogramming in Arabidopsis thaliana

    PubMed Central

    Lyons, Rebecca; Stiller, Jiri; Powell, Jonathan; Rusu, Anca; Manners, John M.; Kazan, Kemal

    2015-01-01

    Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant. PMID:25849296

  18. Identification of Tissue-Specific Protein-Coding and Noncoding Transcripts across 14 Human Tissues Using RNA-seq

    PubMed Central

    Zhu, Jinhang; Chen, Geng; Zhu, Sibo; Li, Suqing; Wen, Zhuo; Bin Li; Zheng, Yuanting; Shi, Leming

    2016-01-01

    Many diseases and adverse drug reactions exhibit tissue specificity. To better understand the tissue-specific expression characteristics of transcripts in different human tissues, we deeply sequenced RNA samples from 14 different human tissues. After filtering many lowly expressed transcripts, 24,729 protein-coding transcripts and 1,653 noncoding transcripts were identified. By analyzing highly expressed tissue-specific protein-coding transcripts (TSCTs) and noncoding transcripts (TSNTs), we found that testis expressed the highest numbers of TSCTs and TSNTs. Brain, monocytes, ovary, and heart expressed more TSCTs than the rest tissues, whereas brain, placenta, heart, and monocytes expressed more TSNTs than other tissues. Co-expression network constructed based on the TSCTs and TSNTs showed that each hub TSNT was co-expressed with several TSCTs, allowing functional annotation of TSNTs. Important biological processes and KEGG pathways highly related to the specific functions or diseases of each tissue were enriched with the corresponding TSCTs. These TSCTs and TSNTs may participate in the tissue-specific physiological or pathological processes. Our study provided a unique data set and systematic analysis of expression characteristics and functions of both TSCTs and TSNTs based on 14 distinct human tissues, and could facilitate future investigation of the mechanisms behind tissue-specific diseases and adverse drug reactions. PMID:27329541

  19. Identification of Tissue-Specific Protein-Coding and Noncoding Transcripts across 14 Human Tissues Using RNA-seq.

    PubMed

    Zhu, Jinhang; Chen, Geng; Zhu, Sibo; Li, Suqing; Wen, Zhuo; Bin Li; Zheng, Yuanting; Shi, Leming

    2016-01-01

    Many diseases and adverse drug reactions exhibit tissue specificity. To better understand the tissue-specific expression characteristics of transcripts in different human tissues, we deeply sequenced RNA samples from 14 different human tissues. After filtering many lowly expressed transcripts, 24,729 protein-coding transcripts and 1,653 noncoding transcripts were identified. By analyzing highly expressed tissue-specific protein-coding transcripts (TSCTs) and noncoding transcripts (TSNTs), we found that testis expressed the highest numbers of TSCTs and TSNTs. Brain, monocytes, ovary, and heart expressed more TSCTs than the rest tissues, whereas brain, placenta, heart, and monocytes expressed more TSNTs than other tissues. Co-expression network constructed based on the TSCTs and TSNTs showed that each hub TSNT was co-expressed with several TSCTs, allowing functional annotation of TSNTs. Important biological processes and KEGG pathways highly related to the specific functions or diseases of each tissue were enriched with the corresponding TSCTs. These TSCTs and TSNTs may participate in the tissue-specific physiological or pathological processes. Our study provided a unique data set and systematic analysis of expression characteristics and functions of both TSCTs and TSNTs based on 14 distinct human tissues, and could facilitate future investigation of the mechanisms behind tissue-specific diseases and adverse drug reactions. PMID:27329541

  20. Tissue-specific targeting of cell fate regulatory genes by E2f factors.

    PubMed

    Julian, L M; Liu, Y; Pakenham, C A; Dugal-Tessier, D; Ruzhynsky, V; Bae, S; Tsai, S-Y; Leone, G; Slack, R S; Blais, A

    2016-04-01

    Cell cycle proteins are important regulators of diverse cell fate decisions, and in this capacity have pivotal roles in neurogenesis and brain development. The mechanisms by which cell cycle regulation is integrated with cell fate control in the brain and other tissues are poorly understood, and an outstanding question is whether the cell cycle machinery regulates fate decisions directly or instead as a secondary consequence of proliferative control. Identification of the genes targeted by E2 promoter binding factor (E2f) transcription factors, effectors of the pRb/E2f cell cycle pathway, will provide essential insights into these mechanisms. We identified the promoter regions bound by three neurogenic E2f factors in neural precursor cells in a genome-wide manner. Through bioinformatic analyses and integration of published genomic data sets we uncovered hundreds of transcriptionally active E2f-bound promoters corresponding to genes that control cell fate processes, including key transcriptional regulators and members of the Notch, fibroblast growth factor, Wnt and Tgf-β signaling pathways. We also demonstrate a striking enrichment of the CCCTC binding factor transcription factor (Ctcf) at E2f3-bound nervous system-related genes, suggesting a potential regulatory co-factor for E2f3 in controlling differentiation. Finally, we provide the first demonstration of extensive tissue specificity among E2f target genes in mammalian cells, whereby E2f3 promoter binding is well conserved between neural and muscle precursors at genes associated with cell cycle processes, but is tissue-specific at differentiation-associated genes. Our findings implicate the cell cycle pathway as a widespread regulator of cell fate genes, and suggest that E2f3 proteins control cell type-specific differentiation programs by regulating unique sets of target genes. This work significantly enhances our understanding of how the cell cycle machinery impacts cell fate and differentiation, and will

  1. Stress causes tissue-specific changes in the sialyltransferase activity.

    PubMed

    Dabelic, Sanja; Flögel, Mirna; Maravić, Gordana; Lauc, Gordan

    2004-01-01

    Numerous pathological conditions are associated with specific changes in glycosylation. Recent studies clearly demonstrated a link between stress and the development and course of many diseases. Biochemical mechanisms that link stress and diseases are still not fully understood, but there are some indications that changes in glycosylation are involved in this process. Influence of acute and chronic psychological stress on protein sialylation as well as the activity of sialyltransferases, enzymes that synthesize sialoglycoproteins, has been studied on Fischer rats. Liver, spleen, kidney, skeletal muscle, heart, adrenal gland, serum, cerebellum, hippocampus, medulla oblongata and cortex have been analyzed. Statistically significant tissue- and type of stress-specific changes in total sialyltransferase (ST) activity were observed. Acute stress resulted in 39% increase of ST activity in liver and spleen, while at the same time there was 43% decrease in ST activity in cerebellum. In chronic stress, ST activity increased in spleen (93%) and decreased in liver (17%), cerebellum (38%) and hippocampus (64%). Western-blot analysis using Maackia amurensis and Sambucus nigra lectins did not reveal any difference in protein sialylation. The results of serum corticosterone analysis indicate that showed increase in acute stress and decrease in chronic stress are in good accordance with the hypothesis that corticosterone has a role in the regulation of liver ST activity. PMID:15241940

  2. Sharing and Specificity of Co-expression Networks across 35 Human Tissues

    PubMed Central

    Pierson, Emma; Koller, Daphne; Battle, Alexis; Mostafavi, Sara

    2015-01-01

    To understand the regulation of tissue-specific gene expression, the GTEx Consortium generated RNA-seq expression data for more than thirty distinct human tissues. This data provides an opportunity for deriving shared and tissue specific gene regulatory networks on the basis of co-expression between genes. However, a small number of samples are available for a majority of the tissues, and therefore statistical inference of networks in this setting is highly underpowered. To address this problem, we infer tissue-specific gene co-expression networks for 35 tissues in the GTEx dataset using a novel algorithm, GNAT, that uses a hierarchy of tissues to share data between related tissues. We show that this transfer learning approach increases the accuracy with which networks are learned. Analysis of these networks reveals that tissue-specific transcription factors are hubs that preferentially connect to genes with tissue specific functions. Additionally, we observe that genes with tissue-specific functions lie at the peripheries of our networks. We identify numerous modules enriched for Gene Ontology functions, and show that modules conserved across tissues are especially likely to have functions common to all tissues, while modules that are upregulated in a particular tissue are often instrumental to tissue-specific function. Finally, we provide a web tool, available at mostafavilab.stat.ubc.ca/GNAT, which allows exploration of gene function and regulation in a tissue-specific manner. PMID:25970446

  3. An Arabidopsis tissue-specific RNAi method for studying genes essential to mitosis.

    PubMed

    Burgos-Rivera, Brunilís; Dawe, R Kelly

    2012-01-01

    A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3) and PISTILLATA (PI) promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS) RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1). Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay). A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth. PMID:23236491

  4. Comparative genomics reveals tissue-specific regulation of prolactin receptor gene expression.

    PubMed

    Schennink, Anke; Trott, Josephine F; Manjarin, Rodrigo; Lemay, Danielle G; Freking, Bradley A; Hovey, Russell C

    2015-02-01

    Prolactin (PRL), acting via the PRL receptor (PRLR), controls hundreds of biological processes across a range of species. Endocrine PRL elicits well-documented effects on target tissues such as the mammary glands and reproductive organs in addition to coordinating whole-body homeostasis during states such as lactation or adaptive responses to the environment. While changes in PRLR expression likely facilitates these tissue-specific responses to circulating PRL, the mechanisms regulating this regulation in non-rodent species has received limited attention. We performed a wide-scale analysis of PRLR 5' transcriptional regulation in pig tissues. Apart from the abundantly expressed and widely conserved exon 1, we identified alternative splicing of transcripts from an additional nine first exons of the porcine PRLR (pPRLR) gene. Notably, exon 1.5 transcripts were expressed most abundantly in the heart, while expression of exon 1.3-containing transcripts was greatest in the kidneys and small intestine. Expression of exon 1.3 mRNAs within the kidneys was most abundant in the renal cortex, and increased during gestation. A comparative analysis revealed a human homologue to exon 1.3, hE1N2, which was also principally transcribed in the kidneys and small intestines, and an exon hE1N3 was only expressed in the kidneys of humans. Promoter alignment revealed conserved motifs within the proximal promoter upstream of exon 1.3, including putative binding sites for hepatocyte nuclear factor-1 and Sp1. Together, these results highlight the diverse, conserved and tissue-specific regulation of PRLR expression in the targets for PRL, which may function to coordinate complex physiological states such as lactation and osmoregulation. PMID:25358647

  5. The Tissue-Specific Expression of a Tobacco Phytochrome B Gene.

    PubMed Central

    Adam, E.; Kozma-Bognar, L.; Kolar, C.; Schafer, E.; Nagy, F.

    1996-01-01

    We have isolated a genomic clone from Nicotiana tabacum, designated Nt-PHYB-1, encoding a type-II, "green tissue" phytochrome apoprotein. Recombinant genes, consisting of the 3319-bp promoter of the Nt-PHYB-1 gene (including the entire 5[prime] untranslated sequence but not the ATG) or its deletion derivatives and the bacterial [beta]-glucuronidase reporter gene, were constructed and transferred into tobacco. The expression patterns and levels of the endogenous Nt-PHYB-1, as well as those of the transgenes, were determined by RNase protection assays and by [beta]-glucuronidase histochemical staining. We show that (a) the PHYB-1 gene has three transcription start sites, (b) the abundance of the three PHYB-1-specific mRNAs is different, and that (c) it is not regulated by light. However, we do demonstrate that transcription of the endogenous PHYB-1 gene and that of the recombinant genes exhibit a well-defined organ and tissue specificity. This tobacco PHYB gene is relatively highly expressed in leaf, stem, and different floral organs but not in root. Deletion analysis of the Nt-PHYB-1 promoter indicates that a 382-bp region, located between -1472 and -1089, is required for high-level expression of this gene. PMID:12226242

  6. A C++ framework for creating tissue specific segmentation-pipelines

    NASA Astrophysics Data System (ADS)

    Pfeifer, Bernhard; Hanser, Friedrich; Seger, Michael; Hintermueller, Christoph; Modre-Osprian, Robert; Fischer, Gerald; Muehlthaler, Hannes; Trieb, Thomas; Tilg, Bernhard

    2005-04-01

    For a clinical application of the inverse problem of electrocardiography, a flexible and fast generation of a patient's volume conductor model is essential. The volume conductor model includes compartments like chest, lungs, ventricles, atria and the associated blood masses. It is a challenging task to create an automatic or semi-automatic segmentation procedure for each compartment. For the extraction of the lungs, as one example, a region growing algorithm can be used, to extract the blood masses of the ventricles Active Appearance Models may succeed, and to construct the atrial myocardium a multiplicity of operations are necessary. These examples illustrate that there is no common method that will succeed for all compartments like a least common denominator. Another problem is the automatization of combining different methods and the origination of a segmentation pipeline in order to extract a compartment and, accordingly, the desired model - in our case the complete volume conductor model for estimating the spread of electrical excitation in the patient's heart. On account of this, we developed a C++ framework and a special application with the goal of creating tissue-specific segmentation pipelines. The C++ framework uses different standard frameworks like DCMTK for handling medical images (http://dicom.offis.de/dcmtk.php.en), ITK (http://www.itk.org/) for some segmentation methods, and Qt (http://www.trolltech.com/) for creating user interfaces. Our Medical Segmentation Toolkit (MST) enables to combine different segmentation techniques for each compartment. In addition, the framework enables to create user-defined compartment pipelines.

  7. Multiple Promoters in the WNK1 Gene: One Controls Expression of a Kidney-Specific Kinase-Defective Isoform

    PubMed Central

    Delaloy, Celine; Lu, Jingyu; Houot, Anne-Marie; Disse-Nicodeme, Sandra; Gasc, Jean-Marie; Corvol, Pierre; Jeunemaitre, Xavier

    2003-01-01

    WNK1 is a serine-threonine kinase, the expression of which is affected in pseudohypoaldosteronism type II, a Mendelian form of arterial hypertension. We characterized human WNK1 transcripts to determine the molecular mechanisms governing WNK1 expression. We report the presence of two promoters generating two WNK1 isoforms with a complete kinase domain. Further variations are achieved by the use of two polyadenylation sites and tissue-specific splicing. We also determined the structure of a kidney-specific isoform regulated by a third promoter and starting at a novel exon. This transcript is kinase defective and has a predominant expression in the kidney compared to the other WNK1 isoforms, with, furthermore, a highly restricted expression profile in the distal convoluted tubule. We confirmed that the ubiquitous and kidney-specific promoters are functional in several cells lines and identified core promoters and regulatory elements. In particular, a strong enhancer element upstream from the kidney-specific exon seems specific to renal epithelial cells. Thus, control of human WNK1 gene expression of kinase-active or -deficient isoforms is mediated predominantly through the use of multiple transcription initiation sites and tissue-specific regulatory elements. PMID:14645531

  8. Immune modulation by MANF promotes tissue repair and regenerative success in the retina.

    PubMed

    Neves, Joana; Zhu, Jie; Sousa-Victor, Pedro; Konjikusic, Mia; Riley, Rebeccah; Chew, Shereen; Qi, Yanyan; Jasper, Heinrich; Lamba, Deepak A

    2016-07-01

    Regenerative therapies are limited by unfavorable environments in aging and diseased tissues. A promising strategy to improve success is to balance inflammatory and anti-inflammatory signals and enhance endogenous tissue repair mechanisms. Here, we identified a conserved immune modulatory mechanism that governs the interaction between damaged retinal cells and immune cells to promote tissue repair. In damaged retina of flies and mice, platelet-derived growth factor (PDGF)-like signaling induced mesencephalic astrocyte-derived neurotrophic factor (MANF) in innate immune cells. MANF promoted alternative activation of innate immune cells, enhanced neuroprotection and tissue repair, and improved the success of photoreceptor replacement therapies. Thus, immune modulation is required during tissue repair and regeneration. This approach may improve the efficacy of stem-cell-based regenerative therapies. PMID:27365452

  9. Biomimetic integrin-specific surfaces to direct osteoblastic function and tissue healing

    NASA Astrophysics Data System (ADS)

    Petrie, Timothy Andrew

    Current orthopedic implant technologies used suffer from slow rates of osseointegration, short lifetime, and lack of mechanical integrity as a result of poorly controlled cell-surface interactions. Recent biologically-inspired surface strategies (biomimetic) have focused on mimicking the biofunctionality of the extracellular matrix (ECM) by using short, adhesive oligopeptides, such as arginine-glycine-aspartic acid (RGD) present in numerous ECM components. However, these strategies have yielded mixed results in vivo and marginal bone healing responses. The central goal of this dissertation project was to engineer bioactive surfaces that specifically target integrin receptors important for osteogenic functions in order to improve bone tissue repair. In order to create integrin-specific interfaces, integrin-specific ligands reconstituting the fibronectin (FN) secondary/tertiary structure were first engineered and functionalized on material surfaces using several robust presentation schemes. We demonstrated that FN-mimetic-functionalized surfaces that directed alpha 5beta1 binding enhanced osteoblast and stromal cell integrin binding and adhesion, osteogenic signaling, and osteoblastic differentiation compared to various other RGD-based ligand-functionalized surfaces. Next, we investigated the effect of integrin-specific biointerfaces to modulate bone healing in a rat tibia implant bone model. We demonstrated, using a robust polymer brush system, that bioactive coatings on titanium implants that conferred high alpha5beta1 integrin specificity in vitro enhanced bone formation and implant integration in vivo. Moreover, we showed that integrin specificity can be engineered using different immobilization schemes, including clinically-relevant ligand dip-coating, and promote the same robust in vivo effect. Furthermore, we investigate the synergistic roles of integrin specificity and ligand clustering on cell response by engineering biointerfaces presenting trimeric and

  10. Nattokinase-promoted tissue plasminogen activator release from human cells.

    PubMed

    Yatagai, Chieko; Maruyama, Masugi; Kawahara, Tomoko; Sumi, Hiroyuki

    2008-01-01

    When heated to a temperature of 70 degrees C or higher, the strong fibrinolytic activity of nattokinase in a solution was deactivated. Similar results were observed in the case of using Suc-Ala-Ala-Pro-Phe-pNA and H-D-Val-Leu-Lys-pNA, which are synthetic substrates of nattokinase. In the current study, tests were conducted on the indirect fibrinolytic effects of the substances containing nattokinase that had been deactivated through heating at 121 degrees C for 15 min. Bacillus subtilis natto culture solutions made from three types of bacteria strain were heat-treated and deactivated, and it was found that these culture solutions had the ability to generate tissue plasminogen activators (tPA) from vascular endothelial cells and HeLa cells at certain concentration levels. For example, it was found that the addition of heat-treated culture solution of the Naruse strain (undiluted solution) raises the tPA activity of HeLa cells to about 20 times that of the control. Under the same conditions, tPA activity was raised to a level about 5 times higher for human vascular endothelial cells (HUVEC), and to a level about 24 times higher for nattokinase sold on the market. No change in cell count was observed for HeLa cells and HUVEC in the culture solution at these concentrations, and the level of activity was found to vary with concentration. PMID:19996631

  11. Adipose tissue macrophages promote myelopoiesis and monocytosis in obesity.

    PubMed

    Nagareddy, Prabhakara R; Kraakman, Michael; Masters, Seth L; Stirzaker, Roslynn A; Gorman, Darren J; Grant, Ryan W; Dragoljevic, Dragana; Hong, Eun Shil; Abdel-Latif, Ahmed; Smyth, Susan S; Choi, Sung Hee; Korner, Judith; Bornfeldt, Karin E; Fisher, Edward A; Dixit, Vishwa Deep; Tall, Alan R; Goldberg, Ira J; Murphy, Andrew J

    2014-05-01

    Obesity is associated with infiltration of macrophages into adipose tissue (AT), contributing to insulin resistance and diabetes. However, relatively little is known regarding the origin of AT macrophages (ATMs). We discovered that murine models of obesity have prominent monocytosis and neutrophilia, associated with proliferation and expansion of bone marrow (BM) myeloid progenitors. AT transplantation conferred myeloid progenitor proliferation in lean recipients, while weight loss in both mice and humans (via gastric bypass) was associated with a reversal of monocytosis and neutrophilia. Adipose S100A8/A9 induced ATM TLR4/MyD88 and NLRP3 inflammasome-dependent IL-1β production. IL-1β interacted with the IL-1 receptor on BM myeloid progenitors to stimulate the production of monocytes and neutrophils. These studies uncover a positive feedback loop between ATMs and BM myeloid progenitors and suggest that inhibition of TLR4 ligands or the NLRP3-IL-1β signaling axis could reduce AT inflammation and insulin resistance in obesity. PMID:24807222

  12. Gender-specific reproductive tissue in ratites and Tyrannosaurus rex.

    PubMed

    Schweitzer, Mary H; Wittmeyer, Jennifer L; Horner, John R

    2005-06-01

    Unambiguous indicators of gender in dinosaurs are usually lost during fossilization, along with other aspects of soft tissue anatomy. We report the presence of endosteally derived bone tissues lining the interior marrow cavities of portions of Tyrannosaurus rex (Museum of the Rockies specimen number 1125) hindlimb elements, and we hypothesize that these tissues are homologous to specialized avian tissues known as medullary bone. Because medullary bone is unique to female birds, its discovery in extinct dinosaurs solidifies the link between dinosaurs and birds, suggests similar reproductive strategies, and provides an objective means of gender differentiation in dinosaurs. PMID:15933198

  13. The bone-specific Runx2-P1 promoter displays conserved three-dimensional chromatin structure with the syntenic Supt3h promoter

    PubMed Central

    Barutcu, A. Rasim; Tai, Phillip W. L.; Wu, Hai; Gordon, Jonathan A. R.; Whitfield, Troy W.; Dobson, Jason R.; Imbalzano, Anthony N.; Lian, Jane B.; van Wijnen, André J.; Stein, Janet L.; Stein, Gary S.

    2014-01-01

    Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter. PMID:25120271

  14. Tissue Specificity of Human Angiotensin I-Converting Enzyme

    PubMed Central

    Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

    2015-01-01

    Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

  15. Scrapie-specific pathology of sheep lymphoid tissues.

    PubMed

    McGovern, Gillian; Jeffrey, Martin

    2007-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases often result in accumulation of disease-associated PrP (PrP(d)) in the lymphoreticular system (LRS), specifically in association with follicular dendritic cells (FDCs) and tingible body macrophages (TBMs) of secondary follicles. We studied the effects of sheep scrapie on lymphoid tissue in tonsils and lymph nodes by light and electron microscopy. FDCs of sheep were grouped according to morphology as immature, mature or regressing. Scrapie was associated with FDC dendrite hypertrophy and electron dense deposit or vesicles. PrP(d) was located using immunogold labelling at the plasmalemma of FDC dendrites and, infrequently, mature B cells. Abnormal electron dense deposits surrounding FDC dendrites were identified as immunoglobulins suggesting that excess immune complexes are retained and are indicative of an FDC dysfunction. Within scrapie-affected lymph nodes, macrophages outside the follicle and a proportion of germinal centre TBMs accumulated PrP(d) within endosomes and lysosomes. In addition, TBMs showed PrP(d) in association with the cell membrane, non-coated pits and vesicles, and also with discrete, large and random endoplasmic reticulum networks, which co-localised with ubiquitin. These observations suggest that PrP(d) is internalised via the caveolin-mediated pathway, and causes an abnormal disease-related alteration in endoplasmic reticulum structure. In contrast to current dogma, this study shows that sheep scrapie is associated with cytopathology of germinal centres, which we attribute to abnormal antigen complex trapping by FDCs and abnormal endocytic events in TBMs. The nature of the sub-cellular changes in FDCs and TBMs differs from those of scrapie infected neurones and glial cells suggesting that different PrP(d)/cell membrane interactions occur in different cell types. PMID:18074028

  16. Tissue-specific expression of the human brain natriuretic peptide gene in cardiac myocytes.

    PubMed

    LaPointe, M C; Wu, G; Garami, M; Yang, X P; Gardner, D G

    1996-03-01

    (70683 +/- 14744 versus 7223 +/- 3920, n=4, P < .01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricular versus atrial myocytes and essentially inactive in fibroblasts, (2) the distal BNP promoter contains both positive and negative regulatory elements, (3) a region of the proximal BNP promoter located between -127 and -40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart. PMID:8613230

  17. Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.

    PubMed

    Hill, David R; Kessler, Sean P; Rho, Hyunjin K; Cowman, Mary K; de la Motte, Carol A

    2012-08-31

    Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein. PMID:22761444

  18. Group A Streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells

    PubMed Central

    Dileepan, Thamotharampillai; Smith, Erica D.; Knowland, Daniel; Hsu, Martin; Platt, Maryann; Bittner-Eddy, Peter; Cohen, Brenda; Southern, Peter; Latimer, Elizabeth; Harley, Earl; Agalliu, Dritan; Cleary, P. Patrick

    2015-01-01

    Group A streptococcal (GAS) infection induces the production of Abs that cross-react with host neuronal proteins, and these anti-GAS mimetic Abs are associated with autoimmune diseases of the CNS. However, the mechanisms that allow these Abs to cross the blood-brain barrier (BBB) and induce neuropathology remain unresolved. We have previously shown that GAS infection in mouse models induces a robust Th17 response in nasal-associated lymphoid tissue (NALT). Here, we identified GAS-specific Th17 cells in tonsils of humans naturally exposed to GAS, prompting us to explore whether GAS-specific CD4+ T cells home to mouse brains following i.n. infection. Intranasal challenge of repeatedly GAS-inoculated mice promoted migration of GAS-specific Th17 cells from NALT into the brain, BBB breakdown, serum IgG deposition, microglial activation, and loss of excitatory synaptic proteins under conditions in which no viable bacteria were detected in CNS tissue. CD4+ T cells were predominantly located in the olfactory bulb (OB) and in other brain regions that receive direct input from the OB. Together, these findings provide insight into the immunopathology of neuropsychiatric complications that are associated with GAS infections and suggest that crosstalk between the CNS and cellular immunity may be a general mechanism by which infectious agents exacerbate symptoms associated with other CNS autoimmune disorders. PMID:26657857

  19. Tissue Discrimination by Uncorrected Autofluorescence Spectra: A Proof-of-Principle Study for Tissue-Specific Laser Surgery

    PubMed Central

    Stelzle, Florian; Knipfer, Christian; Adler, Werner; Rohde, Maximilian; Oetter, Nicolai; Nkenke, Emeka; Schmidt, Michael; Tangermann-Gerk, Katja

    2013-01-01

    Laser surgery provides a number of advantages over conventional surgery. However, it implies large risks for sensitive tissue structures due to its characteristic non-tissue-specific ablation. The present study investigates the discrimination of nine different ex vivo tissue types by using uncorrected (raw) autofluorescence spectra for the development of a remote feedback control system for tissue-selective laser surgery. Autofluorescence spectra (excitation wavelength 377 ± 50 nm) were measured from nine different ex vivo tissue types, obtained from 15 domestic pig cadavers. For data analysis, a wavelength range between 450 nm and 650 nm was investigated. Principal Component Analysis (PCA) and Quadratic Discriminant Analysis (QDA) were used to discriminate the tissue types. ROC analysis showed that PCA, followed by QDA, could differentiate all investigated tissue types with AUC results between 1.00 and 0.97. Sensitivity reached values between 93% and 100% and specificity values between 94% and 100%. This ex vivo study shows a high differentiation potential for physiological tissue types when performing autofluorescence spectroscopy followed by PCA and QDA. The uncorrected autofluorescence spectra are suitable for reliable tissue discrimination and have a high potential to meet the challenges necessary for an optical feedback system for tissue-specific laser surgery. PMID:24152930

  20. Sequence- and Structure-Based Analysis of Tissue-Specific Phosphorylation Sites

    PubMed Central

    Karabulut, Nermin Pinar; Frishman, Dmitrij

    2016-01-01

    Phosphorylation is the most widespread and well studied reversible posttranslational modification. Discovering tissue-specific preferences of phosphorylation sites is important as phosphorylation plays a role in regulating almost every cellular activity and disease state. Here we present a comprehensive analysis of global and tissue-specific sequence and structure properties of phosphorylation sites utilizing recent proteomics data. We identified tissue-specific motifs in both sequence and spatial environments of phosphorylation sites. Target site preferences of kinases across tissues indicate that, while many kinases mediate phosphorylation in all tissues, there are also kinases that exhibit more tissue-specific preferences which, notably, are not caused by tissue-specific kinase expression. We also demonstrate that many metabolic pathways are differentially regulated by phosphorylation in different tissues. PMID:27332813

  1. Construction and analyses of human large-scale tissue specific networks.

    PubMed

    Liu, Wei; Wang, Jianying; Wang, Tengjiao; Xie, Hongwei

    2014-01-01

    Construction and analyses of tissue specific networks is crucial to unveil the function and organizational structure of biological systems. As a direct method to detect protein dynamics, human proteome-wide expression data provide an valuable resource to investigate the tissue specificity of proteins and interactions. By integrating protein expression data with large-scale interaction network, we constructed 30 tissue/cell specific networks in human and analyzed their properties and functions. Rather than the tissue specificity of proteins, we mainly focused on the tissue specificity of interactions to distill tissue specific networks. Through comparing our tissue specific networks with those inferred from gene expression data, we found our networks have larger scales and higher reliability. Furthermore, we investigated the similar extent of multiple tissue specific networks, which proved that tissues with similar functions tend to contain more common interactions. Finally, we found that the tissue specific networks differed from the static network in multiple topological properties. The proteins in tissue specific networks are interacting looser and the hubs play more important roles than those in the static network. PMID:25513809

  2. On the prospect of patient-specific biomechanics without patient-specific properties of tissues.

    PubMed

    Miller, Karol; Lu, Jia

    2013-11-01

    This paper presents main theses of two keynote lectures delivered at Euromech Colloquium "Advanced experimental approaches and inverse problems in tissue biomechanics" held in Saint Etienne in June 2012. We are witnessing an advent of patient-specific biomechanics that will bring in the future personalized treatments to sufferers all over the world. It is the current task of biomechanists to devise methods for clinically-relevant patient-specific modeling. One of the obstacles standing before the biomechanics community is the difficulty in obtaining patient-specific properties of tissues to be used in biomechanical models. We postulate that focusing on reformulating computational mechanics problems in such a way that the results are weakly sensitive to the variation in mechanical properties of simulated continua is more likely to bear fruit in near future. We consider two types of problems: (i) displacement-zero traction problems whose solutions in displacements are weakly sensitive to mechanical properties of the considered continuum; and (ii) problems that are approximately statically determinate and therefore their solutions in stresses are also weakly sensitive to mechanical properties of constituents. We demonstrate that the kinematically loaded biomechanical models of the first type are applicable in the field of image-guided surgery where the current, intraoperative configuration of a soft organ is of critical importance. We show that sac-like membranes, which are prototypes of many thin-walled biological organs, are approximately statically determinate and therefore useful solutions for wall stress can be obtained without the knowledge of the wall's properties. We demonstrate the clinical applicability and effectiveness of the proposed methods using examples from modeling neurosurgery and intracranial aneurysms. PMID:23491073

  3. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter.

    PubMed

    Omidvar, V; Siti Nor Akmar, A; Marziah, M; Maheran, A A

    2008-09-01

    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms. PMID:18563415

  4. Placenta-specific Expression of the Interleukin-2 (IL-2) Receptor β Subunit from an Endogenous Retroviral Promoter*

    PubMed Central

    Cohen, Carla J.; Rebollo, Rita; Babovic, Sonja; Dai, Elizabeth L.; Robinson, Wendy P.; Mager, Dixie L.

    2011-01-01

    The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression. PMID:21865161

  5. Antiphospholipid antibodies promote tissue factor-dependent angiogenic switch and tumor progression.

    PubMed

    Wu, Yuan-Yuan; V Nguyen, Andrew; Wu, Xiao-Xuan; Loh, Mingyu; Vu, Michelle; Zou, Yiyu; Liu, Qiang; Guo, Peng; Wang, Yanhua; Montgomery, Leslie L; Orlofsky, Amos; Rand, Jacob H; Lin, Elaine Y

    2014-12-01

    Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell-derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism. PMID:25451155

  6. Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge

    PubMed Central

    Baumann, Andre; Lehmann, Rüdiger; Beckert, Annika; Vilcinskas, Andreas; Franta, Zdeněk

    2015-01-01

    The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge. PMID:26252388

  7. Tissue-specific transcriptome profiling of the citrus fruit epidermis and subepidermis using laser capture microdissection

    PubMed Central

    Matas, Antonio J.; Agustí, Javier; Tadeo, Francisco R.; Talón, Manuel; Rose, Jocelyn K. C.

    2010-01-01

    Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes, be they transcripts, proteins or metabolites, are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection. In this study, laser microdissection (LMD) was used to isolate epidermal and subepidermal cells from green, expanding Citrus clementina fruit and their transcriptomes were compared using a 20k citrus cDNA microarray and quantitative real-time PCR. The results show striking differences in gene expression profiles between the two cell types, revealing specific metabolic pathways that can be related to their respective organelle composition and cell wall specialization. Microscopy provided additional evidence of tissue specialization that could be associated with the transcript profiles with distinct differences in organelle and metabolite accumulation. Subepidermis predominant genes are primarily involved in photosynthesis- and energy-related processes, as well as cell wall biosynthesis and restructuring. By contrast, the most epidermis predominant genes are related to the biosynthesis of the cuticle, flavonoids, and defence responses. Furthermore, the epidermis transcript profile showed a high proportion of genes with no known function, supporting the original hypothesis that analysis at the tissue/cell specific levels can promote gene discovery and lead to a better understanding of the specialized contribution of each tissue to fruit physiology. PMID:20519339

  8. Proteome labelling and protein identification in specific tissues and at specific developmental stages in an animal

    PubMed Central

    Elliott, Thomas S.; Townsley, Fiona M.; Bianco, Ambra; Ernst, Russell J.; Sachdeva, Amit; Elsässer, Simon J.; Davis, Lloyd; Lang, Kathrin; Pisa, Rudolf; Greiss, Sebastian.; Lilley, Kathryn S.; Chin, Jason W.

    2014-01-01

    Identifying the proteins synthesized in defined cells at specific times in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals. PMID:24727715

  9. Lysine-specific demethylase 1 promotes tumorigenesis and predicts prognosis in gallbladder cancer.

    PubMed

    Lian, Shi Xian; Shao, Ye Bo; Liu, Hou Bao; He, Jun Yi; Lu, Wei Qi; Zhang, Yong; Jiang, Ying; Zhu, Jun

    2015-10-20

    Gallbladder Cancer (GBC), characterized by invasive growth and infiltrative dissemination, is difficult to diagnose and has poor prognosis. Emerging evidence demonstrates that Lysine-Specific Demethylase 1 (LSD1) has important roles in carcinogenesis, proliferation and metastasis. We studied the roles and molecular mechanisms of LSD1 in GBC. We examined LSD1 expression in 109 paired samples of GBC and normal gallbladder tissues. We found GBC tissues had upregulated LSD1 compared with normal gallbladder tissues (P = 0.003), and its high expression was associated with tumor-node-metastasis stage (P < 0.0001), Nevin's stage (P = 0.0093) and distant metastases (P = 0.0070). We found positive correlations between LSD1 expression and other proteins: epithelial-mesenchymal transition markers, C-myc and cyclin-related proteins. Inhibiting LSD1 expression in vitro impaired the proliferation and invasiveness of GBC cells and also downregulated c-myc expression and consequently inhibited GBC cell proliferation. LSD1 overexpression promotes GBC development and may be a predictor for a worsened prognosis. LSD1 may be a novel therapeutic target and prognostic tool for gallbladder cancer. PMID:26460616

  10. Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice

    SciTech Connect

    Vassar, R.; Rosenberg, M.; Tyner, A.; Fuchs, E. ); Ross, S. )

    1989-03-01

    A construct containing {approx}2,500 base pairs (bp) of 5{prime} upstream and {approx}700 bp of 3{prime} downstream sequence was used to drive the expression of an intronless human K14 gene in vitro and in vivo. To track the expression of the gene, a small sequence encoding the antigenic portion of neuropeptide substance P was inserted in frame 5{prime} to the TGA translation stop codon of the gene. Surprisingly, this gene was expressed promiscuously in a wide variety of cultured cells transiently transfected with the construct. In contrast, when introduced into the germ line of transgenic mice, the construct was expressed in a fashion analogous to the endogenous K14 gene--namely, in the basal layer of stratified squamous epithelia. The results suggest that some regulatory mechanism is overridden as a consequence of transient transfection but that sequences that can control proper K14 expression are present in the construct. The appropriate tissue-specific and differentiation-specific expression of K14{center dot}P in transgenic mice is an important first step in characterizing a promoter that could be employed to drive the foreign expression of drug-related genes in the epidermis of skin grafts.

  11. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    PubMed Central

    2011-01-01

    Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue. PMID:21668942

  12. Tissue-specific transcript annotation and expression profiling with complementary next-generation sequencing technologies

    PubMed Central

    Hestand, Matthew S.; Klingenhoff, Andreas; Scherf, Matthias; Ariyurek, Yavuz; Ramos, Yolande; van Workum, Wilbert; Suzuki, Makoto; Werner, Thomas; van Ommen, Gert-Jan B.; den Dunnen, Johan T.; Harbers, Matthias; 't Hoen, Peter A.C.

    2010-01-01

    Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3′-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10 000 genes expressed at levels 2 transcripts per million (0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3′-UTR usage. PMID:20615900

  13. Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

    PubMed Central

    Wilhelm, S M; Eisen, A Z; Teter, M; Clark, S D; Kronberger, A; Goldberg, G

    1986-01-01

    Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. Images PMID:3012533

  14. Identifying and functionally characterizing tissue-specific and ubiquitously expressed human lncRNAs

    PubMed Central

    Lu, Jianping; Chen, Hong; Ding, Na; Wang, Guangjuan; Xu, Juan; Li, Xia

    2016-01-01

    Recent advances in transcriptome sequencing have made it possible to distinguish ubiquitously expressed long non-coding RNAs (UE lncRNAs) from tissue-specific lncRNAs (TS lncRNAs), thereby providing clues to their cellular functions. Here, we assembled and functionally characterized a consensus lncRNA transcriptome by curating hundreds of RNA-seq datasets across normal human tissues from 16 independent studies. In total, 1,184 UE and 2,583 TS lncRNAs were identified. These different lncRNA populations had several distinct features. Specifically, UE lncRNAs were associated with genomic compaction and highly conserved exons and promoter regions. We found that UE lncRNAs are regulated at the transcriptional level (with especially strong regulation of enhancers) and are associated with epigenetic modifications and post-transcriptional regulation. Based on these observations we propose a novel way to predict the functions of UE and TS lncRNAs through analysis of their genomic location and similarities in epigenetic modifications. Our characterization of UE and TS lncRNAs may provide a foundation for lncRNA genomics and the delineation of complex disease mechanisms. PMID:26760768

  15. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

    PubMed

    Ribault, S; Neuville, P; Méchine-Neuville, A; Augé, F; Parlakian, A; Gabbiani, G; Paulin, D; Calenda, V

    2001-03-16

    Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders. PMID:11249869

  16. Identification of two candidate collecting duct cell-specific cis-acting elements in the Hoxb-7 promoter region.

    PubMed

    Plaisier, Emmanuelle; Ribes, David; Ronco, Pierre; Rossert, Jérome

    2005-02-14

    HOX genes encode highly conserved transcription factors responsible for developmental patterning and postnatal tissue homeostasis. Previous studies have shown that a 1.4-kb segment of the Hoxb-7 proximal promoter drives renal expression of reporter genes specifically in the ureteric bud and collecting ducts. In this study using stably transfected renal tubule cell lines, we have identified three short cis-acting sequences within this promoter segment that cooperate to induce high-level expression specifically in collecting duct cells. In addition to an inverted CCAAT box (-71/-67) that acts as an ubiquitous enhancer and binds the transcription factor CBF/NF-Y, two different cis-acting sequences, named CDSE-1 and CDSE-2 (for Collecting Duct Specific Element 1 and 2), allow collecting duct cell-specific promoter activation. CDSE-1 (-56/-34) is composed of two E-boxes separated by a 9-bp GC-rich sequence. Only the latter sequence enhances reporter gene expression specifically in collecting duct cells. CDSE-2 (-34/-13) contains sequence bears high homology with a segment of the Pax-2 promoter. CDSE-2 also conveys cell specificity but has no enhancer activity by itself. PMID:15716052

  17. Impact of Tissue-Specific Stem Cells on Lineage-Specific Differentiation: A Focus on the Musculoskeletal System

    PubMed Central

    Pizzute, Tyler; Lynch, Kevin; Pei, Ming

    2014-01-01

    Tissue-specific stem cells are found throughout the body and, with proper intervention and environmental cues, these stem cells exercise their capabilities for differentiation into several lineages to form cartilage, bone, muscle, and adipose tissue in vitro and in vivo. Interestingly, it has been widely demonstrated that they do not differentiate with the same efficacy during lineage-specific differentiation studies, as the tissue-specific stem cells are generally more effective when differentiating toward the tissues from which they were derived. This review focuses on four mesodermal lineages for tissue-specific stem cell differentiation: adipogenesis, chondrogenesis, myogenesis, and osteogenesis. It is intended to give insight into current multilineage differentiation and comparative research, highlight and contrast known trends regarding differentiation, and introduce supporting evidence which demonstrates particular tissue-specific stem cells’ superiority in lineage-specific differentiation, along with their resident tissue origins and natural roles. In addition, some epigenetic and transcriptomic differences between stem cells which may explain the observed trends are discussed. PMID:25113801

  18. Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded

    PubMed Central

    Hu, Lianxin; Xu, Jiajun; Yin, Meng-Xin; Zhang, Liguo; Lu, Yi; Wu, Wenqing; Xue, Zhaoyu; Ho, Margaret S; Gao, Guanjun; Zhao, Yun; Zhang, Lei

    2016-01-01

    Non-receptor tyrosine kinase activated cdc42 kinase was reported to participate in several types of cancers in mammals. It is also believed to have an anti-apoptotic function in Drosophila. Here, we report the identification of Drosophila activated cdc42 kinase as a growth promoter and a novel Hippo signaling pathway regulator. We find that activated cdc42 kinase promotes tissue growth through modulating Yorkie activity. Furthermore, we demonstrate that activated cdc42 kinase interacts with Expanded and induces tyrosine phosphorylation of Expanded on multiple sites. We propose a model that activated cdc42 kinase negatively regulates Expanded by changing its phosphorylation status to promote tissue growth. Moreover, we show that ack genetically interacts with merlin and expanded. Thus, we identify Drosophila activated cdc42 kinase as a Hippo pathway regulator. PMID:27462444

  19. A major role of insulin in promoting obesity-associated adipose tissue inflammation

    PubMed Central

    Pedersen, David J.; Guilherme, Adilson; Danai, Laura V.; Heyda, Lauren; Matevossian, Anouch; Cohen, Jessica; Nicoloro, Sarah M.; Straubhaar, Juerg; Noh, Hye Lim; Jung, DaeYoung; Kim, Jason K.; Czech, Michael P.

    2015-01-01

    Objective Adipose tissue (AT) inflammation is associated with systemic insulin resistance and hyperinsulinemia in obese rodents and humans. A longstanding concept is that hyperinsulinemia may promote systemic insulin resistance through downregulation of its receptor on target tissues. Here we tested the novel hypothesis that insulin also impairs systemic insulin sensitivity by specifically enhancing adipose inflammation. Methods Circulating insulin levels were reduced by about 50% in diet-induced and genetically obese mice by treatments with diazoxide or streptozotocin, respectively. We then examined AT crown-like structures, macrophage markers and pro-inflammatory cytokine expression in AT. AT lipogenesis and systemic insulin sensitivity was also monitored. Conversely, insulin was infused into lean mice to determine its affects on the above parameters. Results Lowering circulating insulin levels in obese mice by streptozotocin treatment decreased macrophage content in AT, enhancing insulin stimulated Akt phosphorylation and de novo lipogenesis (DNL). Moreover, responsiveness of blood glucose levels to injected insulin was improved by streptozotocin and diazoxide treatments of obese mice without changes in body weight. Remarkably, even in lean mice, infusion of insulin under constant euglycemic conditions stimulated expression of cytokines in AT. Consistent with these findings, insulin treatment of 3T3-L1 adipocytes caused a 10-fold increase in CCL2 mRNA levels within 6 h, which was blocked by the ERK inhibitor PD98059. Conclusion Taken together, these results indicate that obesity-associated hyperinsulinemia unexpectedly drives AT inflammation in obese mice, which in turn contributes to factors that suppress insulin-stimulated adipocyte DNL and systemic insulin sensitivity. PMID:26137438

  20. Epigenomic footprints across 111 reference epigenomes reveal tissue-specific epigenetic regulation of lincRNAs

    PubMed Central

    Amin, Viren; Harris, R. Alan; Onuchic, Vitor; Jackson, Andrew R.; Charnecki, Tim; Paithankar, Sameer; Lakshmi Subramanian, Sai; Riehle, Kevin; Coarfa, Cristian; Milosavljevic, Aleksandar

    2015-01-01

    Tissue-specific expression of lincRNAs suggests developmental and cell-type-specific functions, yet tissue specificity was established for only a small fraction of lincRNAs. Here, by analysing 111 reference epigenomes from the NIH Roadmap Epigenomics project, we determine tissue-specific epigenetic regulation for 3,753 (69% examined) lincRNAs, with 54% active in one of the 14 cell/tissue clusters and an additional 15% in two or three clusters. A larger fraction of lincRNA TSSs is marked in a tissue-specific manner by H3K4me1 than by H3K4me3. The tissue-specific lincRNAs are strongly linked to tissue-specific pathways and undergo distinct chromatin state transitions during cellular differentiation. Polycomb-regulated lincRNAs reside in the bivalent state in embryonic stem cells and many of them undergo H3K27me3-mediated silencing at early stages of differentiation. The exquisitely tissue-specific epigenetic regulation of lincRNAs and the assignment of a majority of them to specific tissue types will inform future studies of this newly discovered class of genes. PMID:25691256

  1. Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization

    PubMed Central

    Lin, Yi-Pin; Chen, Qiang; Ritchie, Jennifer A.; Dufour, Nicholas P.; Fischer, Joshua R.; Coburn, Jenifer; Leong, John M.

    2014-01-01

    SUMMARY Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these ‘adhesins’ bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 minutes post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization. PMID:25486989

  2. Factors Promoting Increased Rate of Tissue Regeneration: The Zebrafish Fin as a Tool for Examining Tissue Engineering Design Concepts

    PubMed Central

    Boominathan, Vijay P.

    2012-01-01

    Abstract Student interest in topics of tissue engineering is increasing exponentially as the number of universities offering programs in bioengineering are on the rise. Bioengineering encompasses all of the STEM categories: Science, Technology, Engineering, and Math. Inquiry-based learning is one of the most effective techniques for promoting student learning and has been demonstrated to have a high impact on learning outcomes. We have designed program outcomes for our bioengineering program that require tiered activities to develop problem solving skills, peer evaluation techniques, and promote team work. While it is ideal to allow students to ask unique questions and design their own experiments, this can be difficult for instructors to have reagents and supplies available for a variety of activities. Zebrafish can be easily housed, and multiple variables can be tested on a large enough group to provide statistical value, lending them well to inquiry-based learning modules. We have designed a laboratory activity that takes observation of fin regeneration to the next level: analyzing conditions that may impact regeneration. Tissue engineers seek to define the optimum conditions to grow tissue for replacement parts. The field of tissue engineering is likely to benefit from understanding natural mechanisms of regeneration and the factors that influence the rate of regeneration. We have outlined the results of varying temperature on fin regeneration and propose other inquiry modules such as the role of pH in fin regeneration. Furthermore, we have provided useful tools for developing critical thinking and peer review of research ideas, assessment guidelines, and grading rubrics for the activities associated with this exercise. PMID:23244692

  3. Factors promoting increased rate of tissue regeneration: the zebrafish fin as a tool for examining tissue engineering design concepts.

    PubMed

    Boominathan, Vijay P; Ferreira, Tracie L

    2012-12-01

    Student interest in topics of tissue engineering is increasing exponentially as the number of universities offering programs in bioengineering are on the rise. Bioengineering encompasses all of the STEM categories: Science, Technology, Engineering, and Math. Inquiry-based learning is one of the most effective techniques for promoting student learning and has been demonstrated to have a high impact on learning outcomes. We have designed program outcomes for our bioengineering program that require tiered activities to develop problem solving skills, peer evaluation techniques, and promote team work. While it is ideal to allow students to ask unique questions and design their own experiments, this can be difficult for instructors to have reagents and supplies available for a variety of activities. Zebrafish can be easily housed, and multiple variables can be tested on a large enough group to provide statistical value, lending them well to inquiry-based learning modules. We have designed a laboratory activity that takes observation of fin regeneration to the next level: analyzing conditions that may impact regeneration. Tissue engineers seek to define the optimum conditions to grow tissue for replacement parts. The field of tissue engineering is likely to benefit from understanding natural mechanisms of regeneration and the factors that influence the rate of regeneration. We have outlined the results of varying temperature on fin regeneration and propose other inquiry modules such as the role of pH in fin regeneration. Furthermore, we have provided useful tools for developing critical thinking and peer review of research ideas, assessment guidelines, and grading rubrics for the activities associated with this exercise. PMID:23244692

  4. Chronic social isolation is associated with metabolic gene expression changes specific to mammary adipose tissue

    PubMed Central

    Volden, Paul A.; Wonder, Erin L.; Skor, Maxwell N.; Carmean, Christopher M.; Patel, Feenalie N.; Ye, Honggang; Kocherginsky, Masha; McClintock, Martha K.; Brady, Matthew J.; Conzen, Suzanne D.

    2013-01-01

    Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of “triple-negative” breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e. during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2 and Acly was found in the adipocyte, rather than the epithelial fraction. Surprisingly, metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected, increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin secretion from this adipose depot. Furthermore, application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent pre-invasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer. PMID:23780289

  5. A convex optimization approach for identification of human tissue-specific interactomes

    PubMed Central

    Mohammadi, Shahin; Grama, Ananth

    2016-01-01

    Motivation: Analysis of organism-specific interactomes has yielded novel insights into cellular function and coordination, understanding of pathology, and identification of markers and drug targets. Genes, however, can exhibit varying levels of cell type specificity in their expression, and their coordinated expression manifests in tissue-specific function and pathology. Tissue-specific/tissue-selective interaction mechanisms have significant applications in drug discovery, as they are more likely to reveal drug targets. Furthermore, tissue-specific transcription factors (tsTFs) are significantly implicated in human disease, including cancers. Finally, disease genes and protein complexes have the tendency to be differentially expressed in tissues in which defects cause pathology. These observations motivate the construction of refined tissue-specific interactomes from organism-specific interactomes. Results: We present a novel technique for constructing human tissue-specific interactomes. Using a variety of validation tests (Edge Set Enrichment Analysis, Gene Ontology Enrichment, Disease-Gene Subnetwork Compactness), we show that our proposed approach significantly outperforms state-of-the-art techniques. Finally, using case studies of Alzheimer’s and Parkinson’s diseases, we show that tissue-specific interactomes derived from our study can be used to construct pathways implicated in pathology and demonstrate the use of these pathways in identifying novel targets. Availability and implementation: http://www.cs.purdue.edu/homes/mohammas/projects/ActPro.html Contact: mohammadi@purdue.edu PMID:27307623

  6. GSH2 promoter methylation in pancreatic cancer analyzed by quantitative methylation-specific polymerase chain reaction

    PubMed Central

    GAO, FEI; HUANG, HAO-JIE; GAO, JUN; LI, ZHAO-SHEN; MA, SHU-REN

    2015-01-01

    Tumor suppressor gene silencing via promoter hypermethylation is an important event in pancreatic cancer pathogenesis. Aberrant DNA hypermethylation events are highly tumor specific, and may provide a diagnostic tool for pancreatic cancer patients. The objective of the current study was to identify novel methylation-related genes that may potentially be used to establish novel therapeutic and diagnostic strategies against pancreatic cancer. The methylation status of the GS homeobox 2 (GSH2) gene was analyzed using the sodium bisulfite sequencing method. The GSH2 methylation ratio was examined in primary carcinomas and corresponding normal tissues derived from 47 patients with pancreatic cancer, using quantitative methylation-specific polymerase chain reaction. Methylation ratios were found to be associated with the patient's clinicopathological features. GSH2 gene methylation was detected in 26 (55.3%) of the 47 pancreatic cancer patients, indicating that it occurs frequently in pancreatic cancer. A significant association with methylation was observed for tumor-node-metastasis stage (P=0.031). GSH2 may be a novel methylation-sensitive tumor suppressor gene in pancreatic cancer and may be a tumor-specific biomarker of the disease. PMID:26171036

  7. Biomimetic scaffold combined with electrical stimulation and growth factor promotes tissue engineered cardiac development.

    PubMed

    Park, Hyoungshin; Larson, Benjamin L; Kolewe, Martin E; Vunjak-Novakovic, Gordana; Freed, Lisa E

    2014-02-15

    Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T. IGF-1 reduced apoptosis, promoted cell-to-cell connectivity, and lowered excitation threshold, an index of electrophysiological activity. ES promoted formation of tissue-like bundles oriented in parallel to the electrical field and a more than ten-fold increase in matrix metalloprotease-2 (MMP-2) gene expression. The combination of IGF-1 and ES increased 2D projection length, an index of overall contraction strength, and enhanced expression of the gap junction protein connexin-43 and sarcomere development. This culture environment, designed to combine cardiac-like scaffold architecture and biomechanics with molecular and biophysical signals, enabled functional assembly of engineered heart muscle from dissociated cells and could serve as a template for future studies on the hierarchy of various signaling domains relative to cardiac tissue development. PMID:24240126

  8. Tissue-Specific Effects of Vitamin E Supplementation

    PubMed Central

    Jansen, Eugene; Viezeliene, Dale; Beekhof, Piet; Gremmer, Eric; Ivanov, Leonid

    2016-01-01

    A multivitamin and mineral supplementation study of 6 weeks was conducted with male and female mice. The control group received a standard dose of vitamins and minerals of 1× the Recommended Daily Intake (RDI), whereas a second group received 3× RDI. A third group received a high dose of vitamin E (25× RDI), close to the upper limit of toxicity (UL), but still recommended and considered to be harmless and beneficial. The high dose of vitamin E caused a number of beneficial, but also adverse effects. Different biomarkers of tissue toxicity, oxidative stress related processes and inflammation were determined. These biomarkers did not change in plasma and erythrocytes to a large extent. In the liver of male mice, some beneficial effects were observed by a lower concentration of several biomarkers of inflammation. However, in the kidney of male mice, a number of biomarkers increased substantially with the higher dose of vitamin E, indicating tissue toxicity and an increased level of inflammation. Since this dose of vitamin E, which is lower than the UL, cause some adverse effects, even after a short exposure period, further studies are required to reconsider the UL for vitamin E. PMID:27447613

  9. Tissue-Specific Effects of Vitamin E Supplementation.

    PubMed

    Jansen, Eugene; Viezeliene, Dale; Beekhof, Piet; Gremmer, Eric; Ivanov, Leonid

    2016-01-01

    A multivitamin and mineral supplementation study of 6 weeks was conducted with male and female mice. The control group received a standard dose of vitamins and minerals of 1× the Recommended Daily Intake (RDI), whereas a second group received 3× RDI. A third group received a high dose of vitamin E (25× RDI), close to the upper limit of toxicity (UL), but still recommended and considered to be harmless and beneficial. The high dose of vitamin E caused a number of beneficial, but also adverse effects. Different biomarkers of tissue toxicity, oxidative stress related processes and inflammation were determined. These biomarkers did not change in plasma and erythrocytes to a large extent. In the liver of male mice, some beneficial effects were observed by a lower concentration of several biomarkers of inflammation. However, in the kidney of male mice, a number of biomarkers increased substantially with the higher dose of vitamin E, indicating tissue toxicity and an increased level of inflammation. Since this dose of vitamin E, which is lower than the UL, cause some adverse effects, even after a short exposure period, further studies are required to reconsider the UL for vitamin E. PMID:27447613

  10. Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes

    PubMed Central

    Adir, Idan; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-01-01

    Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal’s lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene’s promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

  11. Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes.

    PubMed

    Sagi, Dror; Rak, Roni; Gingold, Hila; Adir, Idan; Maayan, Gadi; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-08-01

    Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

  12. Inflammasome Activation Can Mediate Tissue-Specific Pathogenesis or Protection in Staphylococcus aureus Infection.

    PubMed

    Melehani, Jason H; Duncan, Joseph A

    2016-01-01

    Staphylococcus aureus is a Gram-positive coccus that interacts with human hosts on a spectrum from quiet commensal to deadly pathogen. S. aureus is capable of infecting nearly every tissue in the body resulting in cellulitis, pneumonia, osteomyelitis, endocarditis, brain abscesses, bacteremia, and more. S. aureus has a wide range of factors that promote infection, and each site of infection triggers a different response in the human host. In particular, the different patterns of inflammasome activation mediate tissue-specific pathogenesis or protection in S. aureus infection. Although still a nascent field, understanding the unique host-pathogen interactions in each infection and the role of inflammasomes in mediating pathogenesis may lead to novel strategies for treating S. aureus infections. Reviews addressing S. aureus virulence and pathogenesis (Thammavongsa et al. 2015), as well as epidemiology and pathophysiology (Tong et al. 2015), have recently been published. This review will focus on S. aureus factors that activate inflammasomes and their impact on innate immune signaling and bacterial survival. PMID:27460814

  13. Intermittent fasting results in tissue-specific changes in bioenergetics and redox state.

    PubMed

    Chausse, Bruno; Vieira-Lara, Marcel A; Sanchez, Angélica B; Medeiros, Marisa H G; Kowaltowski, Alicia J

    2015-01-01

    Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart. PMID:25749501

  14. Promoting Social Communication in a Child with Specific Language Impairment

    ERIC Educational Resources Information Center

    O'Handley, Roderick D.; Radley, Keith C.; Lum, John D. K.

    2016-01-01

    Social difficulties represent a major area of concern in children with specific language impairment (SLI). Social skills interventions targeting communication or language skills of children with SLI have been generally ineffective. The current study tested the efficacy of a social skills intervention consisting of multiple behavioral interventions…

  15. Do Specific Relevance Instructions Promote Transfer Appropriate Processing?

    ERIC Educational Resources Information Center

    McCrudden, Matthew T.

    2011-01-01

    This study examined whether specific relevance instructions affect transfer appropriate processing. Undergraduates (n = 52) were randomly assigned to one of three pre-reading question conditions that asked them what-questions, why-questions, or to read for understanding (i.e., control condition). There were no differences in reading time across…

  16. Cloning, expression, and regulation of tissue-specific genes in Drosophila

    SciTech Connect

    Korochkin, L.I.

    1995-08-01

    The family of esterase genes was studied in various Drosophilia species. These genes are classified as tissue-specific and housekeeping ones. The expression of tissue-specific esterases in the male reproductive system of Drosophilia species from the virilis and melanogaster groups was thoroughly examined. Modifier genes controlling activity level, time of synthesis, and distribution in cells of the tissue-specific esterase isozyme from the ejaculatory bulb were revealed. The structural gene coding of this enzyme was isolated, cloned, and sequenced. This gene was shown to be similar in different Drosophilia species; the transcriptional level of tissue specificity of this gene was determined. The possibility of transformating the tissue-specific gene into a housekeeping one was demonstrated. In different Drosophilia species, this gene can be expressed in different parts of the reproductive system. In transgenic males carrying the gene of another species, the foreign gene is expressed as in the donor. 68 refs., 11 figs.

  17. Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions.

    PubMed

    Jost, J P; Saluz, H P; McEwan, I; Feavers, I M; Hughes, M; Reiber, S; Liang, H M; Vaccaro, M

    1990-01-30

    The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor. PMID:1968660

  18. Whole-genome bisulfite sequencing maps from multiple human tissues reveal novel CpG islands associated with tissue-specific regulation

    PubMed Central

    Mendizabal, Isabel; Yi, Soojin V.

    2016-01-01

    CpG islands (CGIs) are one of the most widely studied regulatory features of the human genome, with critical roles in development and disease. Despite such significance and the original epigenetic definition, currently used CGI sets are typically predicted from DNA sequence characteristics. Although CGIs are deeply implicated in practical analyses of DNA methylation, recent studies have shown that such computational annotations suffer from inaccuracies. Here we used whole-genome bisulfite sequencing from 10 diverse human tissues to identify a comprehensive, experimentally obtained, single-base resolution CGI catalog. In addition to the unparalleled annotation precision, our method is free from potential bias due to arbitrary sequence features or probe affinity differences. In addition to clarifying substantial false positives in the widely used University of California Santa Cruz (UCSC) annotations, our study identifies numerous novel epigenetic loci. In particular, we reveal significant impact of transposable elements on the epigenetic regulatory landscape of the human genome and demonstrate ubiquitous presence of transcription initiation at CGIs, including alternative promoters in gene bodies and non-coding RNAs in intergenic regions. Moreover, coordinated DNA methylation and chromatin modifications mark tissue-specific enhancers at novel CGIs. Enrichment of specific transcription factor binding from ChIP-seq supports mechanistic roles of CGIs on the regulation of tissue-specific transcription. The new CGI catalog provides a comprehensive and integrated list of genomic hotspots of epigenetic regulation. PMID:26512062

  19. Whole-genome bisulfite sequencing maps from multiple human tissues reveal novel CpG islands associated with tissue-specific regulation.

    PubMed

    Mendizabal, Isabel; Yi, Soojin V

    2016-01-01

    CpG islands (CGIs) are one of the most widely studied regulatory features of the human genome, with critical roles in development and disease. Despite such significance and the original epigenetic definition, currently used CGI sets are typically predicted from DNA sequence characteristics. Although CGIs are deeply implicated in practical analyses of DNA methylation, recent studies have shown that such computational annotations suffer from inaccuracies. Here we used whole-genome bisulfite sequencing from 10 diverse human tissues to identify a comprehensive, experimentally obtained, single-base resolution CGI catalog. In addition to the unparalleled annotation precision, our method is free from potential bias due to arbitrary sequence features or probe affinity differences. In addition to clarifying substantial false positives in the widely used University of California Santa Cruz (UCSC) annotations, our study identifies numerous novel epigenetic loci. In particular, we reveal significant impact of transposable elements on the epigenetic regulatory landscape of the human genome and demonstrate ubiquitous presence of transcription initiation at CGIs, including alternative promoters in gene bodies and non-coding RNAs in intergenic regions. Moreover, coordinated DNA methylation and chromatin modifications mark tissue-specific enhancers at novel CGIs. Enrichment of specific transcription factor binding from ChIP-seq supports mechanistic roles of CGIs on the regulation of tissue-specific transcription. The new CGI catalog provides a comprehensive and integrated list of genomic hotspots of epigenetic regulation. PMID:26512062

  20. Transgenic reporter mice with promoter region of murine LRAT specifically marks lens and meiosis spermatocytes.

    PubMed

    Prukova, D; Ileninova, Z; Antosova, B; Kasparek, P; Gregor, M; Sedlacek, R

    2015-01-01

    Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs. PMID:25317684

  1. Distal cis-regulatory elements are required for tissue-specific expression of enamelin (Enam)

    PubMed Central

    Hu, Yuanyuan; Papagerakis, Petros; Ye, Ling; Feng, Jerry Q.; Simmer, James P.; Hu, Jan C-C.

    2009-01-01

    Enamel formation is orchestrated by the sequential expression of genes encoding enamel matrix proteins; however, the mechanisms sustaining the spatio–temporal order of gene transcription during amelogenesis are poorly understood. The aim of this study was to characterize the cis-regulatory sequences necessary for normal expression of enamelin (Enam). Several enamelin transcription regulatory regions, showing high sequence homology among species, were identified. DNA constructs containing 5.2 or 3.9 kb regions upstream of the enamelin translation initiation site were linked to a LacZ reporter and used to generate transgenic mice. Only the 5.2-Enam–LacZ construct was sufficient to recapitulate the endogenous pattern of enamelin tooth-specific expression. The 3.9-Enam–LacZ transgenic lines showed no expression in dental cells, but ectopic β-galactosidase activity was detected in osteoblasts. Potential transcription factor-binding sites were identified that may be important in controlling enamelin basal promoter activity and in conferring enamelin tissue-specific expression. Our study provides new insights into regulatory mechanisms governing enamelin expression. PMID:18353004

  2. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.

    PubMed

    Ablain, Julien; Durand, Ellen M; Yang, Song; Zhou, Yi; Zon, Leonard I

    2015-03-23

    CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish. PMID:25752963

  3. Core Promoter Plasticity Between Maize Tissues and Genotypes Contrasts with Predominance of Sharp Transcription Initiation Sites.

    PubMed

    Mejía-Guerra, María Katherine; Li, Wei; Galeano, Narmer F; Vidal, Mabel; Gray, John; Doseff, Andrea I; Grotewold, Erich

    2015-12-01

    Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop. PMID:26628745

  4. Genome-wide de Novo Prediction of Proximal and Distal Tissue-Specific Enhancers

    SciTech Connect

    Loots, G G; Ovcharenko, I V

    2005-11-03

    Determining how transcriptional regulatory networks are encoded in the human genome is essential for understanding how cellular processes are directed. Here, we present a novel approach for systematically predicting tissue specific regulatory elements (REs) that blends genome-wide expression profiling, vertebrate genome comparisons, and pattern analysis of transcription factor binding sites. This analysis yields 4,670 candidate REs in the human genome with distinct tissue specificities, the majority of which reside far away from transcription start sites. We identify key transcription factors (TFs) for 34 distinct tissues and demonstrate that tissue-specific gene expression relies on multiple regulatory pathways employing similar, but different cohorts of interacting TFs. The methods and results we describe provide a global view of tissue specific gene regulation in humans, and propose a strategy for deciphering the transcriptional regulatory code in eukaryotes.

  5. Neuregulin 1 promotes excitatory synapse development specifically in GABAergic interneurons

    PubMed Central

    Ting, Annie K.; Chen, Yongjun; Wen, Lei; Yin, Dong-Min; Shen, Chengyong; Tao, Yanmei; Liu, Xihui; Xiong, Wen-Cheng; Mei, Lin

    2011-01-01

    Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Taken together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism. PMID:21209185

  6. Microbiota depletion promotes browning of white adipose tissue and reduces obesity.

    PubMed

    Suárez-Zamorano, Nicolas; Fabbiano, Salvatore; Chevalier, Claire; Stojanović, Ozren; Colin, Didier J; Stevanović, Ana; Veyrat-Durebex, Christelle; Tarallo, Valentina; Rigo, Dorothée; Germain, Stéphane; Ilievska, Miroslava; Montet, Xavier; Seimbille, Yann; Hapfelmeier, Siegfried; Trajkovski, Mirko

    2015-12-01

    Brown adipose tissue (BAT) promotes a lean and healthy phenotype and improves insulin sensitivity. In response to cold or exercise, brown fat cells also emerge in the white adipose tissue (WAT; also known as beige cells), a process known as browning. Here we show that the development of functional beige fat in the inguinal subcutaneous adipose tissue (ingSAT) and perigonadal visceral adipose tissue (pgVAT) is promoted by the depletion of microbiota either by means of antibiotic treatment or in germ-free mice. This leads to improved glucose tolerance and insulin sensitivity and decreased white fat and adipocyte size in lean mice, obese leptin-deficient (ob/ob) mice and high-fat diet (HFD)-fed mice. Such metabolic improvements are mediated by eosinophil infiltration, enhanced type 2 cytokine signaling and M2 macrophage polarization in the subcutaneous white fat depots of microbiota-depleted animals. The metabolic phenotype and the browning of the subcutaneous fat are impaired by the suppression of type 2 cytokine signaling, and they are reversed by recolonization of the antibiotic-treated or germ-free mice with microbes. These results provide insight into the microbiota-fat signaling axis and beige-fat development in health and metabolic disease. PMID:26569380

  7. Inhibition of Notch signaling by Dll4-Fc promotes reperfusion of acutely ischemic tissues

    SciTech Connect

    Liu, Ren; Trindade, Alexandre; Sun, Zhanfeng; Kumar, Ram; Weaver, Fred A.; Krasnoperov, Valery; Naga, Kranthi; Duarte, Antonio; Gill, Parkash S.

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Low dose Dll4-Fc increases vascular proliferation and overall perfusion. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in hindlimb ischemia model. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in skin flap model. Black-Right-Pointing-Pointer Dll4 heterozygous deletion promotes vascular injury recovery. Black-Right-Pointing-Pointer Dll4 overexpression delays vascular injury recovery. -- Abstract: Notch pathway regulates vessel development and maturation. Dll4, a high-affinity ligand for Notch, is expressed predominantly in the arterial endothelium and is induced by hypoxia among other factors. Inhibition of Dll4 has paradoxical effects of reducing the maturation and perfusion in newly forming vessels while increasing the density of vessels. We hypothesized that partial and/or intermittent inhibition of Dll4 may lead to increased vascular response and still allow vascular maturation to occur. Thus tissue perfusion can be restored rapidly, allowing quicker recovery from ischemia or tissue injury. Our studies in two different models (hindlimb ischemia and skin flap) show that inhibition of Dll4 at low dose allows faster recovery from vascular and tissue injury. This opens a new possibility for Dll4 blockade's therapeutic application in promoting recovery from vascular injury and restoring blood supply to ischemic tissues.

  8. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

    PubMed

    Yang, Zhiyong; Edenberg, Howard J; Davis, Ronald L

    2005-01-01

    To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes. PMID:16204451

  9. A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue.

    PubMed

    Shore, Andrew; Emes, Richard D; Wessely, Frank; Kemp, Paul; Cillo, Clemente; D'Armiento, Maria; Hoggard, Nigel; Lomax, Michael A

    2012-01-01

    The thermoregulatory function of brown adipose tissue (BAT) is due to the tissue-specific expression of uncoupling protein 1 (UCP1) which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1 transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulfite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1 expression between white and BAT. Tissue-specific expression of UCP1 has been proposed to be regulated by a conserved 5' distal enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in five eutherians as well as marsupials, monotremes, amphibians, and fish, is present in pigs despite UCP1 having become a pseudogene, and that absence of the enhancer element does not relate to BAT-specific UCP1 expression. We identify an additional putative 5' regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1 expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1 5' untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1. PMID:23293654

  10. Stable CpG Hypomethylation of Adipogenic Promoters in Freshly Isolated, Cultured, and Differentiated Mesenchymal Stem Cells from Adipose Tissue

    PubMed Central

    Noer, Agate; Sørensen, Anita L.; Boquest, Andrew C.

    2006-01-01

    Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells. PMID:16760426

  11. Dissection of the erythroid-specific transcriptional promoter used by the gene encoding aminolevulinic acid dehydratase (ALAD)

    SciTech Connect

    Bishop, T.R.; Schaffer, T.; Pien, B.

    1994-09-01

    The gene encoding delta-aminolevulinate dehydratase (ALAD), the second enzyme of the heme biosynthetic pathway, exists as a single gene in most mammalian genomes and we have sequenced over 12 kb from overlapping lambda clones containing the murine ALAD gene. The gene has a dual promoter driving expression of two different first exons; exon1A is expressed in all tissues and exon1B only in erythroid cells, where heme production is induced to exceptionally high levels for hemoglobin synthesis. Erythroid-specific expression of the ALAD gene is presumably accomplished by using the exon1B promoter which we hypothesize is responsive to erythroid-specific transcriptional activators. In order to test this, we have used gel mobility shift assays and DNase footprint analyses to dissect and identify the critical upstream regulatory elements. Nuclear extracts, prepared from murine erythroleukemia cells (MELC), human chronic myelogenous leukemia cell line (K562) and human fibroblast cell line (HeLa), were used as sources of proteins to analyze DNA binding sites in the ALAD erythroid-specific promoter from -307 to +1. In this region, there are three potential GATA1 sites, two CACCC boxes, a CCAAT box and a GGTGG box. NF-E2 sites were explored by using in vitro translation products of cloned p18 and p45, the two heterologous components of NF-E2, and successfully gel-shifted a 29 bp double-stranded oligo found at 2.6 kb in front of the ALAD gene. Thus, the ALAD gene utilizes both a housekeeping and a tissue-specific promoter.

  12. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs.

    PubMed

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-07-18

    In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV

  13. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut

    PubMed Central

    Miller, Hugh R P; Pemberton, Alan D

    2002-01-01

    Serine proteinases with trypsin-like (tryptase) and chymotrypsin-like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa-specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa-specific chymase, mouse mast cell proteinase-1 (mMCP-1), is regulated by transforming growth factor-β1 (TGF-β1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase-activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin-I to angiotensin-II. Mucosa-specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in

  14. Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

    PubMed Central

    Iatrou, K; Meidinger, R G

    1990-01-01

    A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

  15. Identification of the control region for tissue-specific imprinting of the stimulatory G protein α-subunit

    PubMed Central

    Liu, Jie; Chen, Min; Deng, Chuxia; Bourc'his, Déborah; Nealon, Julie G.; Erlichman, Beth; Bestor, Timothy H.; Weinstein, Lee S.

    2005-01-01

    Gnas is a complex gene with multiple imprinted promoters. The upstream Nesp and Nespas/Gnasxl promoters are paternally and maternally methylated, respectively. The downstream promoter for the stimulatory G protein α-subunit (Gsα) is unmethylated, although in some tissues (e.g., renal proximal tubules), Gsα is poorly expressed from the paternal allele. Just upstream of the Gsα promoter is a primary imprint mark (1A region) where maternal-specific methylation is established during oogenesis. Pseudohypoparathyroidism type 1B, a disorder of renal parathyroid hormone resistance, is associated with loss of 1A methylation. Analysis of embryos of Dnmt3L–/– mothers (which cannot methylate maternal imprint marks) showed that Nesp, Nespas/Gnasxl, and 1A imprinting depend on one or more maternal primary imprint marks. We generated mice with deletion of the 1A differentially methylated region. These mice had normal Nesp-Nespas/Gnasxl imprinting, indicating that the Gnas locus contains two independent imprinting domains (Nespas-Nespas/Gnasxl and 1A-Gsα) controlled by distinct maternal primary imprint marks. Paternal, but not maternal, 1A deletion resulted in Gsα overexpression in proximal tubules and evidence for increased parathyroid hormone sensitivity but had no effect on Gsα expression in other tissues where Gsα is normally not imprinted. The 1A region is a maternal imprint mark that contains one or more methylation-sensitive cis-acting elements that suppress Gsα expression from the paternal allele in a tissue-specific manner. PMID:15811946

  16. The stearoyl-acyl-carrier-protein desaturase promoter (Des) from oil palm confers fruit-specific GUS expression in transgenic tomato.

    PubMed

    Saed Taha, Rima; Ismail, Ismanizan; Zainal, Zamri; Abdullah, Siti Nor Akmar

    2012-09-01

    The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits. PMID:22658816

  17. Spatio-Temporal Imaging of Promoter Activity in Intact Plant Tissues.

    PubMed

    Xiong, Tou Cheu; Sanchez, Frédéric; Briat, Jean-François; Gaymard, Frédéric; Dubos, Christian

    2016-01-01

    Localization and quantification of expression levels of genes help to determine their function. Localization of gene expression is often achieved through the study of their promoter activity. Three main reporter genes β-glucuronidase (GUS), green fluorescent protein (GFP), and luciferase (LUC) have been intensively used to characterize promoter activities, each having its own specificities and advantages. Among them, the LUC reporter gene is best suitable for the analysis of the promoter activity of genes in intact living plants. Here, we describe a LUC-based method that allows to precisely localize and quantify promoter activity at the whole plant level, and to study the mechanisms that are involved in long-distance regulation of gene expression in Arabidopsis thaliana. Imaging LUC signals with a low-light CCD camera allows monitoring promoter activity in time and space in the transgenic plant harboring the promoter fused with the LUC gene. In addition, it allows quantifying change of promoter activities in plant during several hours. PMID:27557763

  18. Tissue-Specific Signals Control Reversible Program of Localization and Functional Polarization of Macrophages

    PubMed Central

    Okabe, Yasutaka; Medzhitov, Ruslan

    2014-01-01

    SUMMARY Tissue-resident macrophages are highly heterogeneous in terms of their functions and phenotypes as a consequence of adaptation to different tissue environments. Local tissue-derived signals are thought to control functional polarization of resident macrophages; however, the identity of these signals remains largely unknown. It is also unknown whether functional heterogeneity is a result of irreversible lineage-specific differentiation or a consequence of continuous but reversible induction of diverse functional programs. Here, we identified retinoic acid as a signal that induces tissue-specific localization and functional polarization of peritoneal macrophages through the reversible induction of transcription factor GATA6. We further found that GATA6 in macrophages regulates gut IgA production through peritoneal B-1 cells. These results provide insight into the regulation of tissue-resident macrophage functional specialization by tissue-derived signals. PMID:24792964

  19. Cloning of the human activated leukocyte cell adhesion molecule promoter and identification of its tissue-independent transcriptional activation by Sp1.

    PubMed

    Tan, Fang; Mbunkui, Flaubert; Ofori-Acquah, Solomon F

    2012-12-01

    Activated leukocyte cell adhesion molecule (ALCAM) belongs to the immunoglobulin cell adhesion molecule super family. ALCAM is implicated in tumor progression, inflammation, and the differentiation of hematopoietic stem cells. Hitherto, the identity of regulatory DNA elements and cognate transcription factors responsible for ALCAM gene expression remained unknown. In this report, the human ALCAM promoter was cloned and its transcriptional mechanisms elucidated. The promoter is TATA-less and contains multiple GC-boxes. A proximal 650-bp promoter fragment conferred tissue-independent activation, whereas two contiguous regions upstream of this region negatively influenced promoter activity in a tissue-specific manner. The positive regulatory promoter region was mapped to a core 50 base pair sequence containing a conical Sp1 element. Mutation analysis revealed that this element alone or in tandem with elements immediately upstream was required for maximal promoter activity. Chromatin analysis revealed that Sp1 binds exclusively to the canonical binding sequence in vivo, but not to DNA sequence immediately upstream. Finally, we showed that over-expression of Sp1 significantly increased the basal promoter activity. Thus, Sp1 activated the ALCAM promoter in most cells. These findings have important ramifications for unraveling the roles of ALCAM in inflammation and tumorigenesis. PMID:22941204

  20. Tissue-specific imprinting of the mouse insulin-like growth factor II receptor gene correlates with differential allele-specific DNA methylation.

    PubMed

    Hu, J F; Oruganti, H; Vu, T H; Hoffman, A R

    1998-02-01

    Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r. PMID:9482664

  1. Promoting external inosculation of prevascularised tissue constructs by pre-cultivation in an angiogenic extracellular matrix.

    PubMed

    Laschke, M W; Mussawy, H; Schuler, S; Eglin, D; Alini, M; Menger, M D

    2010-01-01

    The engineering of preformed microvessels offers the promising opportunity to rapidly vascularise implanted tissue constructs by the process of inosculation. Herein, we analyzed whether this process may further be accelerated by cultivation of prevascularised tissue constructs in Matrigel before implantation. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were implanted into the flank of FVB/N-TgN (Tie2/GFP) 287 Sato mice to allow the ingrowth of a granulation tissue with green fluorescent protein (GFP)-positive blood vessels. After harvesting, these prevascularised constructs were then transferred into dorsal skinfold chambers of FVB/N recipient mice to study the process of inosculation. The constructs were implanted directly after embedding in Matrigel or after 3 days of cultivation in the extracellular matrix. Matrigel-free constructs served as control. Cultivation in Matrigel resulted in the outgrowth of CD31/GFP-positive vascular sprouts. Vascularisation of these constructs was markedly improved when compared to the other two groups, as indicated by a significantly elevated functional microvessel density between days 6 to 14 after implantation into the dorsal skinfold chamber. This was associated with an increased number of GFP-positive blood vessels growing into the surrounding host tissue. Thus, the blood supply to prevascularised tissue constructs can be accelerated by their pre-cultivation in an angiogenic extracellular matrix, promoting external inosculation of the preformed microvascular networks with the host microvasculature. PMID:21154242

  2. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    SciTech Connect

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P.; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  3. Understanding multicellular function and disease with human tissue-specific networks

    PubMed Central

    Greene, Casey S.; Krishnan, Arjun; Wong, Aaron K.; Ricciotti, Emanuela; Zelaya, Rene A.; Himmelstein, Daniel S.; Zhang, Ran; Hartmann, Boris M.; Zaslavsky, Elena; Sealfon, Stuart C.; Chasman, Daniel I.; FitzGerald, Garret A.; Dolinski, Kara; Grosser, Tilo; Troyanskaya, Olga G.

    2016-01-01

    Tissue and cell-type identity lie at the core of human physiology and disease. Understanding the genetic underpinnings of complex tissues and individual cell lineages is crucial for developing improved diagnostics and therapeutics. We present genome-wide functional interaction networks for 144 human tissues and cell types developed using a data-driven Bayesian methodology that integrates thousands of diverse experiments spanning tissue and disease states. Tissue-specific networks predict lineage-specific responses to perturbation, reveal genes’ changing functional roles across tissues, and illuminate disease-disease relationships. We introduce NetWAS, which combines genes with nominally significant GWAS p-values and tissue-specific networks to identify disease-gene associations more accurately than GWAS alone. Our webserver, GIANT, provides an interface to human tissue networks through multi-gene queries, network visualization, analysis tools including NetWAS, and downloadable networks. GIANT enables systematic exploration of the landscape of interacting genes that shape specialized cellular functions across more than one hundred human tissues and cell types. PMID:25915600

  4. Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter

    PubMed Central

    Rama, Ana R.; Hernandez, Rosa; Perazzoli, Gloria; Burgos, Miguel; Melguizo, Consolación; Vélez, Celia; Prados, Jose

    2015-01-01

    Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA) to direct E gene expression (pCEA-E) towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells. PMID:26053394

  5. Tissue-specific Regulation of Porcine Prolactin Receptor Expression by Estrogen, Progesterone and Prolactin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. ...

  6. Tissue-specific disallowance of housekeeping genes: The other face of cell differentiation

    PubMed Central

    Thorrez, Lieven; Laudadio, Ilaria; Van Deun, Katrijn; Quintens, Roel; Hendrickx, Nico; Granvik, Mikaela; Lemaire, Katleen; Schraenen, Anica; Van Lommel, Leentje; Lehnert, Stefan; Aguayo-Mazzucato, Cristina; Cheng-Xue, Rui; Gilon, Patrick; Van Mechelen, Iven; Bonner-Weir, Susan; Lemaigre, Frédéric; Schuit, Frans

    2011-01-01

    We report on a hitherto poorly characterized class of genes that are expressed in all tissues, except in one. Often, these genes have been classified as housekeeping genes, based on their nearly ubiquitous expression. However, the specific repression in one tissue defines a special class of “disallowed genes.” In this paper, we used the intersection-union test to screen for such genes in a multi-tissue panel of genome-wide mRNA expression data. We propose that disallowed genes need to be repressed in the specific target tissue to ensure correct tissue function. We provide mechanistic data of repression with two metabolic examples, exercise-induced inappropriate insulin release and interference with ketogenesis in liver. Developmentally, this repression is established during tissue maturation in the early postnatal period involving epigenetic changes in histone methylation. In addition, tissue-specific expression of microRNAs can further diminish these repressed mRNAs. Together, we provide a systematic analysis of tissue-specific repression of housekeeping genes, a phenomenon that has not been studied so far on a genome-wide basis and, when perturbed, can lead to human disease. PMID:21088282

  7. SPECTRA: An Integrated Knowledge Base for Comparing Tissue and Tumor-Specific PPI Networks in Human.

    PubMed

    Micale, Giovanni; Ferro, Alfredo; Pulvirenti, Alfredo; Giugno, Rosalba

    2015-01-01

    Protein-protein interaction (PPI) networks available in public repositories usually represent relationships between proteins within the cell. They ignore the specific set of tissues or tumors where the interactions take place. Indeed, proteins can form tissue-selective complexes, while they remain inactive in other tissues. For these reasons, a great attention has been recently paid to tissue-specific PPI networks, in which nodes are proteins of the global PPI network whose corresponding genes are preferentially expressed in specific tissues. In this paper, we present SPECTRA, a knowledge base to build and compare tissue or tumor-specific PPI networks. SPECTRA integrates gene expression and protein interaction data from the most authoritative online repositories. We also provide tools for visualizing and comparing such networks, in order to identify the expression and interaction changes of proteins across tissues, or between the normal and pathological states of the same tissue. SPECTRA is available as a web server at http://alpha.dmi.unict.it/spectra. PMID:26005672

  8. Transgenic characterization of two testis-specific promoters in the silkworm, Bombyx mori.

    PubMed

    Xu, J; Bi, H; Chen, R; Aslam, A F M; Li, Z; Ling, L; Zeng, B; Huang, Y; Tan, A

    2015-04-01

    Sex-specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female-specific modification system whereas little success was reported on male-specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene-based, female-specific lethality system has been established based on sex-specific alternative splicing factors and a female-specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male-specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis-specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta-tubulin 4 gene (Bmβ4) were introduced using piggybac-based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis-specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis-specific gene expression. Identification of these testis-specific promoters not only contributes to a better understanding of testis-specific gene function in insects, but also has potential applications in sterile insect techniques for pest management. PMID:25387604

  9. Microbiota depletion promotes browning of white adipose tissue and reduces obesity

    PubMed Central

    Chevalier, Claire; Stojanović, Ozren; Colin, Didier J.; Stevanović, Ana; Veyrat-Durebex, Christelle; Tarallo, Valentina; Rigo, Dorothée; Germain, Stéphane; Ilievska, Miroslava; Montet, Xavier; Seimbille, Yann; Hapfelmeier, Siegfried; Trajkovski, Mirko

    2015-01-01

    Brown adipose tissue (BAT) promotes a lean and healthy phenotype and improves insulin sensitivity1. In response to cold or exercise brown fat cells also emerge in the white adipose tissue (named beige cells), a process known as browning2,3,4. Here, we show that the development of functional beige fat is promoted by microbiota depletion either by antibiotic treatment or in germ-free mice within the inguinal subcutaneous and perigonadal visceral adipose tissues (ingSAT and pgVAT, respectively). This leads to improved glucose tolerance, insulin sensitivity and decreased white fat and adipocyte size in lean mice and obese leptin-deficient (ob/ob) and high fat diet (HFD)-fed mice. These metabolic improvements are mediated by eosinophil infiltration and enhanced type 2 cytokine signaling and M2 macrophage polarization in the subcutaneous white fat depots of microbiota-depleted animals. The metabolic phenotype and the browning of the subcutaneous fat are impaired by suppression of the type 2 signaling and are reversed by recolonization of the antibiotic-treated, or the germ-free mice with microbes. These results provide insight into microbiota-fat signaling axis and beige fat development in health and metabolic disease. PMID:26569380

  10. Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.

    PubMed

    Cianfarani, Francesca; Toietta, Gabriele; Di Rocco, Giuliana; Cesareo, Eleonora; Zambruno, Giovanna; Odorisio, Teresa

    2013-01-01

    Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers. PMID:23627689

  11. Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs

    PubMed Central

    Pihlajamaa, Päivi; Sahu, Biswajyoti; Lyly, Lauri; Aittomäki, Viljami; Hautaniemi, Sampsa; Jänne, Olli A

    2014-01-01

    Androgen receptor (AR) binds male sex steroids and mediates physiological androgen actions in target tissues. ChIP-seq analyses of AR-binding events in murine prostate, kidney and epididymis show that in vivo AR cistromes and their respective androgen-dependent transcription programs are highly tissue specific mediating distinct biological pathways. This high order of tissue specificity is achieved by the use of exclusive collaborating factors in the three androgen-responsive tissues. We find two novel collaborating factors for AR signaling in vivo—Hnf4α (hepatocyte nuclear factor 4α) in mouse kidney and AP-2α (activating enhancer binding protein 2α) in mouse epididymis—that define tissue-specific AR recruitment. In mouse prostate, FoxA1 serves for the same purpose. FoxA1, Hnf4α and AP-2α motifs are over-represented within unique AR-binding loci, and the cistromes of these factors show substantial overlap with AR-binding events distinct to each tissue type. These licensing or pioneering factors are constitutively bound to chromatin and guide AR to specific genomic loci upon hormone exposure. Collectively, liganded receptor and its DNA-response elements are required but not sufficient for establishment of tissue-specific transcription programs. PMID:24451200

  12. Effects of Adeno-Associated Virus Serotype and Tissue-Specific Expression on Circulating Biomarkers of Propionic Acidemia

    PubMed Central

    Guenzel, Adam J.; Hillestad, Matthew L.; Matern, Dietrich

    2014-01-01

    Abstract Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca−/−(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

  13. Effects of adeno-associated virus serotype and tissue-specific expression on circulating biomarkers of propionic acidemia.

    PubMed

    Guenzel, Adam J; Hillestad, Matthew L; Matern, Dietrich; Barry, Michael A

    2014-09-01

    Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca(-/-)(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

  14. Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury: mechanisms of brain tissue repair.

    PubMed

    Zhang, Zhen-Qiang; Song, Jun-Ying; Jia, Ya-Quan; Zhang, Yun-Ke

    2016-03-01

    Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. In this study, rats were intragastrically given Buyanghuanwu decoction, 15 mL/kg, for 3 days. A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion. In rats administered Buyanghuanwu decoction, infarct volume was reduced, serum vascular endothelial growth factor and integrin αvβ3 levels were increased, and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals. These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor (administered through the lateral ventricle for 7 consecutive days). These data suggest that Buyanghuanwu decoction promotes angiogenesis, improves cerebral circulation, and enhances brain tissue repair after cerebral ischemia/reperfusion injury. PMID:27127482

  15. Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury: mechanisms of brain tissue repair

    PubMed Central

    Zhang, Zhen-qiang; Song, Jun-ying; Jia, Ya-quan; Zhang, Yun-ke

    2016-01-01

    Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. In this study, rats were intragastrically given Buyanghuanwu decoction, 15 mL/kg, for 3 days. A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion. In rats administered Buyanghuanwu decoction, infarct volume was reduced, serum vascular endothelial growth factor and integrin αvβ3 levels were increased, and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals. These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor (administered through the lateral ventricle for 7 consecutive days). These data suggest that Buyanghuanwu decoction promotes angiogenesis, improves cerebral circulation, and enhances brain tissue repair after cerebral ischemia/reperfusion injury. PMID:27127482

  16. Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells.

    PubMed

    Xu, Jian; Pope, Scott D; Jazirehi, Ali R; Attema, Joanne L; Papathanasiou, Peter; Watts, Jason A; Zaret, Kenneth S; Weissman, Irving L; Smale, Stephen T

    2007-07-24

    Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state. PMID:17640912

  17. Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters.

    PubMed

    Sallenave, J M; Xing, Z; Simpson, A J; Graham, F L; Gauldie, J

    1998-03-01

    This report describes the design and construction of three recombinant adenoviruses of serotype 5 (Ad5) expressing elafin (EL), also called elastase-specific inhibitor. Three promoters were chosen to drive the synthesis of elafin: the small (380 bp) human cytomegalovirus promoter (HCMV), the Ad2 major late promoter (MLP) and the mouse cytomegalovirus (MCMV) promoter. Human alveolar epithelial cells (A549), as well as rat and human primary pulmonary fibroblasts were infected with Ad5-HCMV-EL, Ad5-MLP-EL, Ad5-MCMV-EL and with the control Ad5-dl70/3. The MCMV promoter was the most efficient promoter in all cells studied. MLP was the least efficient promoter Intermediate between MCMV and MLP was HCMV which was able to induce significant amounts of elafin, particularly in human A549 cells. When compared in vivo in rat lungs, results were similar; MCMV was the only promoter which induced significant amounts of elafin as assessed by Northern blot analysis and ELISA, even with a low dose of virus (3 x 10(8) p.f.u.). Our data indicate that the MCMV promoter is the promoter of choice for the strong induction of adenovirus-mediated transgenes in the lung and suggest its suitability both in rodent experimental models and in humans for investigative and therapeutic purposes. PMID:9614555

  18. Tissue-specific hypomethylation of the human c-K-ras gene.

    PubMed Central

    Metter, J; Cho, C

    1989-01-01

    Methylation of the c-K-ras gene was examined in a wide variety of human tissues using the methylation sensitive restriction endonuclease HpaII. All tissues showed hypomethylation in the region of exon zero. Specific hypomethylation of a particular HpaII site in the second intron was observed in gastrointestinal and tracheobronchial epithelial cell DNAs. Specific hypomethylation was also observed in a cluster of HpaII sites within the first intron in sperm, endometrium and placenta DNAs. These regions were predominantly methylated in a wide variety of other tissues, including fetal gut. The possible implications are discussed. Images PMID:2476726

  19. The receptor tyrosine kinase Pvr promotes tissue closure by coordinating corpse removal and epidermal zippering.

    PubMed

    Garlena, Rebecca A; Lennox, Ashley L; Baker, Lewis R; Parsons, Trish E; Weinberg, Seth M; Stronach, Beth E

    2015-10-01

    A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. We find that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants, such as in the JNK pathway, and we demonstrate that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation, consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and the protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue-specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in hemocytes for embryonic survival. Taken together, we argue that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos whereby dorsal midline closure is incomplete. Based on these observations, we suggest that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus might underlie some congenital birth defects. PMID:26293306

  20. Correlating Molecular Character of NIR Imaging Agents with Tissue-Specific Uptake

    PubMed Central

    Owens, Eric A.; Hyun, Hoon; Tawney, Joseph G.; Choi, Hak Soo; Henary, Maged

    2015-01-01

    Near-infrared (NIR) fluorescent contrast agents are emerging in optical imaging as sensitive, cost-effective, and nonharmful alternatives to current agents that emit harmful ionizing radiation. Developing spectrally distinct NIR fluorophores to visualize sensitive vital tissues to selectively avoid them during surgical resection of diseased tissue is of great significance. Herein, we report the synthetic variation of pentamethine cyanine fluorophores with modifications of physicochemical properties toward prompting tissue-specific uptake into sensitive tissues (i.e., endocrine glands). Tissue-specific targeting and biodistribution studies revealed localization of contrast agents in the adrenal and pituitary glands, pancreas, and lymph nodes with dependence on molecular characteristics. Incorporation of hydrophobic heterocyclic rings, alkyl groups, and halogens allowed a fine-tuning capability to the hydrophobic character and dipole moment for observing perturbation in biological activity in response to minor structural alterations. These NIR contrast agents have potential for clinical translation for intraoperative imaging in the delineation of delicate glands. PMID:25923454

  1. Computational dissection of tissue contamination for identification of colon cancer-specific expression profiles.

    PubMed

    Türeci, Ozlem; Ding, Jiayi; Hilton, Holly; Bian, Hongjin; Ohkawa, Hitomi; Braxenthaler, Michael; Seitz, Gerhard; Raddrizzani, Laura; Friess, Helmut; Buchler, Markus; Sahin, Ugur; Hammer, Juergen

    2003-03-01

    Microarray profiles of bulk tumor tissues reflect gene expression corresponding to malignant cells as well as to many different types of contaminating normal cells. In this report, we assess the feasibility of querying baseline multitissue transcriptome databases to dissect disease-specific genes. Using colon cancer as a model tumor, we show that the application of Boolean operators (AND, OR, BUTNOT) for database searches leads to genes with expression patterns of interest. The BUTNOT operator for example allows the assignment of "expression signatures" to normal tissue specimens. These expression signatures were then used to computationally identify contaminating cells within conventionally dissected tissue specimens. The combination of several logic operators together with an expression database based on multiple human tissue specimens can resolve the problem of tissue contamination, revealing novel cancer-specific gene expression. Several markers, previously not known to be colon cancer associated, are provided. PMID:12631577

  2. Molecular Signatures of Tissue-Specific Microvascular Endothelial Cell Heterogeneity in Organ Maintenance and Regeneration

    PubMed Central

    Nolan, Daniel J.; Ginsberg, Michael; Israely, Edo; Palikuqi, Brisa; Poulos, Michael G.; James, Daylon; Ding, Bi-Sen; Schachterle, William; Liu, Ying; Rosenwaks, Zev; Butler, Jason M.; Xiang, Jenny; Rafii, Arash; Shido, Koji; Rabbany, Sina Y.; Elemento, Olivier; Rafii, Shahin

    2013-01-01

    SUMMARY Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration. PMID:23871589

  3. Human cancers overexpress genes that are specific to a variety of normal human tissues

    PubMed Central

    Lotem, Joseph; Netanely, Dvir; Domany, Eytan; Sachs, Leo

    2005-01-01

    We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential. PMID:16339305

  4. Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue

    PubMed Central

    Aranjuez, George; Burtscher, Ashley; Sawant, Ketki; Majumder, Pralay; McDonald, Jocelyn A.

    2016-01-01

    Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6−10 cells, traversing a network of large germ line–derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues. PMID:27122602

  5. Oxidized plasma albumin promotes platelet-endothelial crosstalk and endothelial tissue factor expression

    PubMed Central

    Pasterk, Lisa; Lemesch, Sandra; Leber, Bettina; Trieb, Markus; Curcic, Sanja; Stadlbauer, Vanessa; Schuligoi, Rufina; Schicho, Rudolf; Heinemann, Akos; Marsche, Gunther

    2016-01-01

    Plasma advanced oxidation protein products (AOPPs), a class of pro-inflammatory pathogenic mediators, accumulate in subjects with chronic kidney disease. Whether AOPPs contribute to coagulation abnormalities, which are frequently seen in uremic patients, is unknown. Here we report that AOPPs activate platelets via a CD36-mediated signaling pathway. Activation of signaling pathways by AOPP-platelet interaction resulted in the expression of several platelet activation markers and rapidly induced the expression of CD40 ligand, triggering platelet adhesion to endothelial cells and promoting endothelial tissue factor expression. AOPPs and serum tissue factor levels were considerably increased in end stage renal disease patients on hemodialysis and a significant correlation of AOPPs and serum tissue factor was found. Interestingly, serum levels of AOPPs and tissue factor were substantially lower in stable kidney transplant patients when compared with hemodialysis patients. Given that CD36 is known to transduce the effects of oxidized lipids into platelet hyperactivity, our findings reveal previously unknown pro-thrombotic activities of oxidized plasma albumin via a CD36 dependent pathway. PMID:26905525

  6. Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue.

    PubMed

    Aranjuez, George; Burtscher, Ashley; Sawant, Ketki; Majumder, Pralay; McDonald, Jocelyn A

    2016-06-15

    Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germ line-derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues. PMID:27122602

  7. Resolvin D1 prevents smoking-induced emphysema and promotes lung tissue regeneration

    PubMed Central

    Kim, Kang-Hyun; Park, Tai Sun; Kim, You-Sun; Lee, Jae Seung; Oh, Yeon-Mok; Lee, Sang-Do; Lee, Sei Won

    2016-01-01

    Purpose Emphysema is an irreversible disease that is characterized by destruction of lung tissue as a result of inflammation caused by smoking. Resolvin D1 (RvD1), derived from docosahexaenoic acid, is a novel lipid that resolves inflammation. The present study tested whether RvD1 prevents smoking-induced emphysema and promotes lung tissue regeneration. Materials and methods C57BL/6 mice, 8 weeks of age, were randomly divided into four groups: control, RvD1 only, smoking only, and smoking with RvD1 administration. Four different protocols were used to induce emphysema and administer RvD1: mice were exposed to smoking for 4 weeks with poly(I:C) or to smoking only for 24 weeks, and RvD1 was injected within the smoking exposure period to prevent regeneration or after completion of smoking exposure to assess regeneration. The mean linear intercept and inflammation scores were measured in the lung tissue, and inflammatory cells and cytokines were measured in the bronchoalveolar lavage fluid. Results Measurements of mean linear intercept showed that RvD1 significantly attenuated smoking-induced lung destruction in all emphysema models. RvD1 also reduced smoking-induced inflammatory cell infiltration, which causes the structural derangements observed in emphysema. In the 4-week prevention model, RvD1 reduced the smoking-induced increase in eosinophils and interleukin-6 in the bronchoalveolar lavage fluid. In the 24-week prevention model, RvD1 also reduced the increased neutrophils and total cell counts induced by smoking. Conclusion RvD1 attenuated smoking-induced emphysema in vivo by reducing inflammation and promoting tissue regeneration. This result suggests that RvD1 may be useful in the prevention and treatment of emphysema. PMID:27313451

  8. 25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

    SciTech Connect

    Ichikawa, Tomohiro; Sugiura, Hisatoshi; Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki; Ichinose, Masakazu

    2013-05-01

    Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblasts (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P<0.001) and collagen I (P<0.001) expression in HFL-1. 25-HC also significantly enhanced the release and activation of matrix metallaoproteinase (MMP)-2 (P<0.001) and MMP-9 (P<0.001) without any significant effect on the production of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. 25-HC stimulated transforming growth factor (TGF)-β{sub 1} production (P<0.01) and a neutralizing anti-TGF-β antibody restored these 25-HC-augmented pro-fibrotic responses. 25-HC significantly promoted the translocation of nuclear factor (NF)-κB p65 into the nuclei (P<0.01), but not phospholylated-c-jun, a complex of activator protein-1. Pharmacological inhibition of NF-κB restored the 25-HC-augmented pro-fibrotic responses and TGF-β{sub 1} release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway.

  9. Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues.

    PubMed

    Libantová, Jana; Kämäräinen, Terttu; Moravcíková, Jana; Matusíková, Ildikó; Salaj, Jan

    2009-05-01

    The round-leaf sundew (Drosera rotundifolia L.) is a carnivorous plant expressing a wide range of chitinolytic enzymes playing role in many different processes. In this study the intact plants were analyzed for the presence of chitinase transcripts and chitinolytic activities in different organs. In situ hybridization with chitnase fragment as a probe has revealed the presence of chitinases in the mesophyll cells of leaves and vascular elements of stems of healthy, non-stressed plants. More pronounced expression was observed in cortex and stele cells of roots as well as in ovules and anthers of reproductive organs. Similarly, higher chitinase enzyme activity was typical for flowers and roots suggesting a more specific role of chitinases in these tissues. In addition to endochitinases of different substrate specificities, chitobiosidases contributed to overall chitinolytic activity of tissue extracts. The activity of chitobiosidases was again typical for flowers and roots, while their role in plant physiology remains to be elucidated. PMID:18437530

  10. A macrophage-specific synthetic promoter for therapeutic application of adiponectin

    PubMed Central

    Kang, W S; Kwon, J S; Kim, H B; Jeong, H-y; Kang, H J; Jeong, M H; Cho, J G; Park, J C; Kim, Y S; Ahn, Y

    2014-01-01

    Foam cell formation from macrophage is a major cause of atherosclerosis. An efficient macrophage-specific promoter is required for the targeting to macrophages. In this study, we develop a macrophage-specific synthetic promoter for the therapeutic application of adiponectin (APN), an antiatherogenic gene. Synthetic promoter-146 (SP146), registered on the NCBI website (http://www.ncbi.nlm.nih.gov/nuccore/DQ107383), was tested for promoter activities in two non-macrophage cell lines (293 T, HeLa) and a macrophage cell line (RAW264.7, bone marrow-derived macrophages). To enforce macrophage specificity, partial elements of p47phox including the PU.1 site with various lengths (-C1, -C2 and -C3) were inserted next to the synthetic promoters. SP146-C1 showed the highest specificity and efficacy in RAW264.7 cells and was selected for development of an APN-carrying macrophage-specific promoter. Green fluorescent protein (GFP)- or APN-expressing lentivirus under SP146-C1 (Lenti-SP-GFP or Lenti-SP-APN, respectively) showed the highest expression efficacy in RAW264.7 cells compared with the non-macrophage cell lines. APN overexpression in RAW264.7 cells successfully inhibited intracellular lipid accumulation, and atherosclerotic lesions and lipid accumulation were significantly reduced by Lenti-SP-APN in ApoE−/− atherosclerosis mice. In conclusion, the synthetic promoter SP146-C1, combined with a p47phox promoter element, was successfully developed to target macrophage, and macrophage-specific introduction of APN under SP146-C1 was shown to ameliorate the atherosclerotic pathology. PMID:24500526

  11. A macrophage-specific synthetic promoter for therapeutic application of adiponectin.

    PubMed

    Kang, W S; Kwon, J S; Kim, H B; Jeong, H-Y; Kang, H J; Jeong, M H; Cho, J G; Park, J C; Kim, Y S; Ahn, Y

    2014-04-01

    Foam cell formation from macrophage is a major cause of atherosclerosis. An efficient macrophage-specific promoter is required for the targeting to macrophages. In this study, we develop a macrophage-specific synthetic promoter for the therapeutic application of adiponectin (APN), an antiatherogenic gene. Synthetic promoter-146 (SP146), registered on the NCBI website (http://www.ncbi.nlm.nih.gov/nuccore/DQ107383), was tested for promoter activities in two non-macrophage cell lines (293 T, HeLa) and a macrophage cell line (RAW264.7, bone marrow-derived macrophages). To enforce macrophage specificity, partial elements of p47(phox) including the PU.1 site with various lengths (-C1, -C2 and -C3) were inserted next to the synthetic promoters. SP146-C1 showed the highest specificity and efficacy in RAW264.7 cells and was selected for development of an APN-carrying macrophage-specific promoter. Green fluorescent protein (GFP)- or APN-expressing lentivirus under SP146-C1 (Lenti-SP-GFP or Lenti-SP-APN, respectively) showed the highest expression efficacy in RAW264.7 cells compared with the non-macrophage cell lines. APN overexpression in RAW264.7 cells successfully inhibited intracellular lipid accumulation, and atherosclerotic lesions and lipid accumulation were significantly reduced by Lenti-SP-APN in ApoE-/- atherosclerosis mice. In conclusion, the synthetic promoter SP146-C1, combined with a p47(phox) promoter element, was successfully developed to target macrophage, and macrophage-specific introduction of APN under SP146-C1 was shown to ameliorate the atherosclerotic pathology. PMID:24500526

  12. Tissue-Specific Effects of Loss of Estrogen during Menopause and Aging

    PubMed Central

    Wend, Korinna; Wend, Peter; Krum, Susan A.

    2012-01-01

    The roles of estrogens have been best studied in the breast, breast cancers, and in the female reproductive tract. However, estrogens have important functions in almost every tissue in the body. Recent clinical trials such as the Women’s Health Initiative have highlighted both the importance of estrogens and how little we know about the molecular mechanism of estrogens in these other tissues. In this review, we illustrate the diverse functions of estrogens in the bone, adipose tissue, skin, hair, brain, skeletal muscle and cardiovascular system, and how the loss of estrogens during aging affects these tissues. Early transcriptional targets of estrogen are reviewed in each tissue. We also describe the tissue-specific effects of selective estrogen receptor modulators (SERMs) used for the treatment of breast cancers and postmenopausal symptoms. PMID:22654856

  13. Comparative Transcriptome Analysis Reveals Substantial Tissue Specificity in Human Aortic Valve

    PubMed Central

    Wang, Jun; Wang, Ying; Gu, Weidong; Ni, Buqing; Sun, Haoliang; Yu, Tong; Gu, Wanjun; Chen, Liang; Shao, Yongfeng

    2016-01-01

    RNA sequencing (RNA-seq) has revolutionary roles in transcriptome identification and quantification of different types of tissues and cells in many organisms. Although numerous RNA-seq data derived from many types of human tissues and cell lines, little is known on the transcriptome repertoire of human aortic valve. In this study, we sequenced the total RNA prepared from two calcified human aortic valves and reported the whole transcriptome of human aortic valve. Integrating RNA-seq data of 13 human tissues from Human Body Map 2 Project, we constructed a transcriptome repertoire of human tissues, including 19,505 protein-coding genes and 4,948 long intergenic noncoding RNAs (lincRNAs). Among them, 263 lincRNAs were identified as novel noncoding transcripts in our data. By comparing transcriptome data among different human tissues, we observed substantial tissue specificity of RNA transcripts, both protein-coding genes and lincRNAs, in human aortic valve. Further analysis revealed that aortic valve-specific lincRNAs were more likely to be recently derived from repetitive elements in the primate lineage, but were less likely to be conserved at the nucleotide level. Expression profiling analysis showed significant lower expression levels of aortic valve-specific protein-coding genes and lincRNA genes, when compared with genes that were universally expressed in various tissues. Isoform-level expression analysis also showed that a majority of mRNA genes had a major isoform expressed in the human aortic valve. To our knowledge, this is the first comparative transcriptome analysis between human aortic valve and other human tissues. Our results are helpful to understand the transcriptome diversity of human tissues and the underlying mechanisms that drive tissue specificity of protein-coding genes and lincRNAs in human aortic valve. PMID:27493474

  14. Comparative Transcriptome Analysis Reveals Substantial Tissue Specificity in Human Aortic Valve.

    PubMed

    Wang, Jun; Wang, Ying; Gu, Weidong; Ni, Buqing; Sun, Haoliang; Yu, Tong; Gu, Wanjun; Chen, Liang; Shao, Yongfeng

    2016-01-01

    RNA sequencing (RNA-seq) has revolutionary roles in transcriptome identification and quantification of different types of tissues and cells in many organisms. Although numerous RNA-seq data derived from many types of human tissues and cell lines, little is known on the transcriptome repertoire of human aortic valve. In this study, we sequenced the total RNA prepared from two calcified human aortic valves and reported the whole transcriptome of human aortic valve. Integrating RNA-seq data of 13 human tissues from Human Body Map 2 Project, we constructed a transcriptome repertoire of human tissues, including 19,505 protein-coding genes and 4,948 long intergenic noncoding RNAs (lincRNAs). Among them, 263 lincRNAs were identified as novel noncoding transcripts in our data. By comparing transcriptome data among different human tissues, we observed substantial tissue specificity of RNA transcripts, both protein-coding genes and lincRNAs, in human aortic valve. Further analysis revealed that aortic valve-specific lincRNAs were more likely to be recently derived from repetitive elements in the primate lineage, but were less likely to be conserved at the nucleotide level. Expression profiling analysis showed significant lower expression levels of aortic valve-specific protein-coding genes and lincRNA genes, when compared with genes that were universally expressed in various tissues. Isoform-level expression analysis also showed that a majority of mRNA genes had a major isoform expressed in the human aortic valve. To our knowledge, this is the first comparative transcriptome analysis between human aortic valve and other human tissues. Our results are helpful to understand the transcriptome diversity of human tissues and the underlying mechanisms that drive tissue specificity of protein-coding genes and lincRNAs in human aortic valve. PMID:27493474

  15. HdhQ111 Mice Exhibit Tissue Specific Metabolite Profiles that Include Striatal Lipid Accumulation

    PubMed Central

    Carroll, Jeffrey B.; Deik, Amy; Fossale, Elisa; Weston, Rory M.; Guide, Jolene R.; Arjomand, Jamshid; Kwak, Seung; Clish, Clary B.; MacDonald, Marcy E.

    2015-01-01

    The HTT CAG expansion mutation causes Huntington’s Disease and is associated with a wide range of cellular consequences, including altered metabolism. The mutant allele is expressed widely, in all tissues, but the striatum and cortex are especially vulnerable to its effects. To more fully understand this tissue-specificity, early in the disease process, we asked whether the metabolic impact of the mutant CAG expanded allele in heterozygous B6.HdhQ111/+ mice would be common across tissues, or whether tissues would have tissue-specific responses and whether such changes may be affected by diet. Specifically, we cross-sectionally examined steady state metabolite concentrations from a range of tissues (plasma, brown adipose tissue, cerebellum, striatum, liver, white adipose tissue), using an established liquid chromatography-mass spectrometry pipeline, from cohorts of 8 month old mutant and wild-type littermate mice that were fed one of two different high-fat diets. The differential response to diet highlighted a proportion of metabolites in all tissues, ranging from 3% (7/219) in the striatum to 12% (25/212) in white adipose tissue. By contrast, the mutant CAG-expanded allele primarily affected brain metabolites, with 14% (30/219) of metabolites significantly altered, compared to wild-type, in striatum and 11% (25/224) in the cerebellum. In general, diet and the CAG-expanded allele both elicited metabolite changes that were predominantly tissue-specific and non-overlapping, with evidence for mutation-by-diet interaction in peripheral tissues most affected by diet. Machine-learning approaches highlighted the accumulation of diverse lipid species as the most genotype-predictive metabolite changes in the striatum. Validation experiments in cell culture demonstrated that lipid accumulation was also a defining feature of mutant HdhQ111 striatal progenitor cells. Thus, metabolite-level responses to the CAG expansion mutation in vivo were tissue specific and most evident

  16. Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis.

    PubMed

    Thoma, S; Hecht, U; Kippers, A; Botella, J; De Vries, S; Somerville, C

    1994-05-01

    Nonspecific lipid transfer proteins (LTPs) from plants are characterized by their ability to stimulate phospholipid transfer between membranes in vitro. However, because these proteins are generally located outside of the plasma membrane, it is unlikely that they have a similar role in vivo. As a step toward identifying the function of these proteins, one of several LTP genes from Arabidoposis has been cloned and the expression pattern of the gene has been examined by analysis of the tissue specificity of beta-glucuronidase (GUS) activity in transgenic plants containing LTP promoter-GUS fusions and by in situ mRNA localization. The LTP1 promoter was active early in development in protoderm cells of embryos, vascular tissues, lignified tips of cotyledons, shoot meristem, and stipules. In adult plants, the gene was expressed in epidermal cells of young leaves and the stem. In flowers, expression was observed in the epidermis of all developing influorescence and flower organ primordia, the epidermis of the siliques and the outer ovule wall, the stigma, petal tips, and floral nectaries of mature flowers, and the petal/sepal abscission zone of mature siliques. The presence of GUS activity in guard cells, lateral roots, pollen grains, leaf vascular tissue, and internal cells of stipules and nectaries was not confirmed by in situ hybridizations, supporting previous observations that suggest that the reporter gene is subject to artifactual expression. These results are consistent with a role for the LTP1 gene product in some aspect of secretion or deposition of lipophilic substances in the cell walls of expanding epidermal cells and certain secretory tissues. The LTP1 promoter region contained sequences homologous to putative regulatory elements of genes in the phenylpropanoid biosynthetic pathway, suggesting that the expression of the LTP1 gene may be regulated by the same or similar mechanisms as genes in the phenylpropanoid pathway. PMID:8029357

  17. Differential interaction of nuclear factors with the leukocyte-specific pp52 promoter in B and T cells.

    PubMed

    Omori, S A; Smale, S; O'Shea-Greenfield, A; Wall, R

    1997-08-15

    The leukocyte-specific, cytoskeleton-binding pp52 (LSP-1, WP-34) protein is widely expressed in multiple leukocyte lineages, including B and T lymphocytes, granulocytes, and macrophages. We previously detected a tissue-specific promoter preceding the exon encoding the N terminus of the pp52 leukocyte protein. Here we describe the functional characterization of this promoter and identification of the factors in B and T cells that regulate its activity. The pp52 promoter contains an initiator specifying the unique 5' terminus of pp52 mRNA, tandem pairs of Ets and SP1 motifs, and a lone C/EBP motif. All these motifs are essential and collectively control transcriptional activity. DNA binding studies and Ab supershift assays revealed that different combinations of factors interact with these motifs in B cells vs T cells. The Ets motifs are preferentially bound by PU-1 in B cell extracts from all stages of development, whereas a different Ets family member reacts with these motifs in T cell extracts. The C/EBP motif is bound by Ig/EBP-1 in pre-B cell and T cell extracts, but is replaced by nuclear factor-IL-6beta or a nuclear factor-IL-6beta-Ig/EBP-1 heterodimer in plasmacytoma cell extracts. Despite its reported role as a negative regulator of transcription, Ig/EBP-1 appears to exert a stimulatory effect on this promoter. These findings reveal the features controlling the pp52 promoter in B and T cells and provide the foundation for determining the regulation of this promoter in other leukocyte lineages. PMID:9257843

  18. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues

    SciTech Connect

    Croop, J.M.; Arceci, R.J. ); Raymond, M.; Gros, P.; Devault, A. . Dept. of Chemistry); Haber, D. ); Housman, D.E. )

    1989-03-01

    The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family. The authors have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse. Expression levels of each mdr gene are dramatically different in various mouse tissues. Specific mdr RNA transcripts of approximately 4.5, 5 and 6 kilobases have been detected. Each of the mdr genes has a specific RNA transcript pattern. These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.

  19. Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression.

    PubMed Central

    Yanagisawa, S; Sheen, J

    1998-01-01

    Dof is a novel family of plant proteins that share a unique and highly conserved DNA binding domain with one C2-C2 zinc finger motif. Although multiple Dof proteins associated with diverse gene promoters have recently been identified in a variety of plants, their physiological functions and regulation remain elusive. In maize, Dof1 (MNB1a) is constitutively expressed in leaves, stems, and roots, whereas the closely related Dof2 is expressed mainly in stems and roots. Here, by using a maize leaf protoplast transient assay, we show that Dof1 is a transcriptional activator, whereas Dof2 can act as a transcriptional repressor. Thus, differential expression of Dof1 and Dof2 may permit leaf-specific gene expression. Interestingly, in vivo analyses showed that although DNA binding activity of Dof1 is regulated by light-dependent development, its transactivation activity and nuclear localization are not. Moreover, in vivo transcription and in vitro electrophoretic mobility shift assays revealed that Dof1 can interact specifically with the maize C4 phosphoenolpyruvate carboxylase gene promoter and enhance its promoter activity, which displays a light-regulated expression pattern matching Dof1 activity. We propose that the evolutionarily conserved Dof proteins can function as transcriptional activators or repressors of tissue-specific and light-regulated gene expression in plants. PMID:9477573

  20. Prostate cancer specific integrin αvβ3 modulates bone metastatic growth and tissue remodeling

    PubMed Central

    McCabe, NP; De, S; Vasanji, A; Brainard, J; Byzova, TV

    2009-01-01

    The management of pain and morbidity owing to the spreading and growth of cancer within bone remains to be a paramount problem in clinical care. Cancer cells actively transform bone, however, the molecular requirements and mechanisms of this process remain unclear. This study shows that functional modulation of the αvβ3 integrin receptor in prostate cancer cells is required for progression within bone and determines tumor-induced bone tissue transformation. Using histology and quantitative microCT analysis, we show that αvβ3 integrin is required not only for tumor growth within the bone but for tumor-induced bone gain, a response resembling bone lesions in prostate cancer patients. Expression of normal, fully functional αvβ3 enabled tumor growth in bone (incidence: 4/4), whereas αvβ3 (—), inactive or constitutively active mutants of αvβ3 did not (incidence: 0/4, 0/6 and 1/7, respectively) within a 35-day-period. This response appeared to be bone-specific in comparison to the subcutis where tumor incidence was greater than 60% for all groups. Interestingly, bone residing prostate cancer cells expressing normal or dis-regulated αvβ3 (either inactive of constitutively active), but not those lacking β3 promoted bone gain or afforded protection from bone loss in the presence or absence of histologically detectable tumor 35 days following implantation. As bone is replete with ligands for β3 integrin, we next demonstrated that αvβ3 integrin activation on tumor cells is essential for the recognition of key bone-specific matrix proteins. As a result, prostate cancer cells expressing fully functional but not dis-regulated αvβ3 integrin are able to control their own adherence and migration to bone matrix, functions that facilitate tumor growth and control bone lesion development. PMID:17369840

  1. Insulation of the ubiquitous Rxrb promoter from the cartilage-specific adjacent gene, Col11a2.

    PubMed

    Murai, Junko; Ikegami, Daisuke; Okamoto, Mina; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2008-10-10

    The retinoid X receptor beta gene (Rxrb) is located just upstream of the alpha2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF. PMID:18682388

  2. Isolation and sequence analysis of napin seed specific promoter from Iranian Rapeseed (Brassica napus L.).

    PubMed

    Sohrabi, Maryam; Zebarjadi, Alireza; Najaphy, Abdollah; Kahrizi, Danial

    2015-06-01

    Rapeseed (Brassica napus L.) has become an important crop during the last 30years. In addition to a high lipid level, the seeds also have a significant protein content, which constitutes 20-25% of the dry seed weight. The synthesis of storage proteins is primarily controlled at transcriptional level and seed-specific expression has been shown to be conferred upon the promoter regions of many storage protein genes. Napin is one of the main storage proteins in rapeseed(')s embryo that is produced in seed developing stage. Its promoter region located at 5' upstream of the napin gene has already been isolated (GenBank number, EU416279.1). In current research, seed-specific promoter (napin) of Iranian B. napus L. was isolated from the genomic DNA and cloned into pBI121 plant binary vector to use in future researches. For this purpose, the napin promoter was amplified by PCR method using specific primers, cloned in pSK(+) vector and sequenced. Sequencing analysis showed that the cloned promoter contained all of conserved motifs such as TATA box (TATAAA), RY repeats (CATGCA), dist-B (TCAAACACC) and prox-B elements (GCCACTTGTC), G-box (CACGTG) and CAAT Motifs, which constituted the seed-specific promoter activity and according to this analysis, the seed-specific promoter activity of cloned sequence was predicted. Based on sequence distances of nucleotide sequences, our sequence had the highest similarity (99.8%) whit B. napus sequence (with EU416279.1 accession number). Finally the promoter obtained might be interesting not only as a useful tool for biotechnological application but also for fundamental research. PMID:25797503

  3. Different promoter affinities account for specificity in MYC-dependent gene regulation

    PubMed Central

    Lorenzin, Francesca; Benary, Uwe; Baluapuri, Apoorva; Walz, Susanne; Jung, Lisa Anna; von Eyss, Björn; Kisker, Caroline; Wolf, Jana; Eilers, Martin; Wolf, Elmar

    2016-01-01

    Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells. DOI: http://dx.doi.org/10.7554/eLife.15161.001 PMID:27460974

  4. A Minimal Set of Tissue-Specific Hypomethylated CpGs Constitute Epigenetic Signatures of Developmental Programming

    PubMed Central

    Pagadala, Vijayakanth; Kittur, Jaya; Staffa, Nickolas G.; Peddada, Shyamal D.; Isganaitis, Elvira; Patti, Mary Elizabeth; Birnbaumer, Lutz

    2013-01-01

    Background Cell specific states of the chromatin are programmed during mammalian development. Dynamic DNA methylation across the developing embryo guides a program of repression, switching off genes in most cell types. Thus, the majority of the tissue specific differentially methylated sites (TS-DMS) must be un-methylated CpGs. Methodology and Principal Findings Comparison of expanded Methyl Sensitive Cut Counting data (eMSCC) among four tissues (liver, testes, brain and kidney) from three C57BL/6J mice, identified 138,052 differentially methylated sites of which 23,270 contain CpGs un-methylated in only one tissue (TS-DMS). Most of these CpGs were located in intergenic regions, outside of promoters, CpG islands or their shores, and up to 20% of them overlapped reported active enhancers. Indeed, tissue-specific enhancers were up to 30 fold enriched in TS-DMS. Testis showed the highest number of TS-DMS, but paradoxically their associated genes do not appear to be specific to the germ cell functions, but rather are involved in organism development. In the other tissues the differentially methylated genes are associated with tissue-specific physiological or anatomical functions. The identified sets of TS-DMS quantify epigenetic distances between tissues, generated during development. We applied this concept to measure the extent of reprogramming in the liver of mice exposed to in utero or early postnatal nutritional stress. Different protocols of food restriction reprogrammed the liver methylome in different but reproducible ways. Conclusion and Significance Thus, each identified set of differentially methylated sites constituted an epigenetic signature that traced the developmental programing or the early nutritional reprogramming of each exposed mouse. We propose that our approach has the potential to outline a number of disease-associated epigenetic states. The composition of differentially methylated CpGs may vary with each situation, behaving as a composite

  5. Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

    PubMed Central

    Brendel, Christian; Hänseler, Walther; Wohlgensinger, Vital; Bianchi, Matteo; Tokmak, Serap; Chen-Wichmann, Linping; Kuzmenko, Elena; Cesarovic, Nikola; Nicholls, Flora; Reichenbach, Janine; Seger, Reinhard; Grez, Manuel

    2013-01-01

    Abstract Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47phox and gp91phox in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47phox, gp91phox, and green fluorescent protein (GFP) within self-inactivating (SIN) gamma- and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47phox patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter–mediated p47phox expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance. PMID:23489116

  6. Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

    PubMed Central

    Zhou, Quan; Wu, Xiongyan; Chen, Xuehua; Li, Jianfang; Zhu, Zhenggang; Liu, Bingya; Su, Liping

    2016-01-01

    Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. PMID:26771235

  7. A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis

    PubMed Central

    Chen, Jun; Xu, Na; Huang, Huanwei; Cai, Tao; Xi, Rongwen

    2016-01-01

    Homeostatic renewal of many adult tissues requires balanced self-renewal and differentiation of local stem cells, but the underlying mechanisms are poorly understood. Here we identified a novel feedback mechanism in controlling intestinal regeneration and tumorigenesis in Drosophila. Sox21a, a group B Sox protein, is preferentially expressed in the committed progenitor named enteroblast (EB) to promote enterocyte differentiation. In Sox21a mutants, EBs do not divide, but cannot differentiate properly and have increased expression of mitogens, which then act as paracrine signals to promote intestinal stem cell (ISC) proliferation. This leads to a feedback amplification loop for rapid production of differentiation-defective EBs and tumorigenesis. Notably, in normal intestine following damage, Sox21a is temporally downregulated in EBs to allow the activation of the ISC-EB amplification loop for epithelial repair. We propose that executing a feedback amplification loop between stem cells and their progeny could be a common mechanism underlying tissue regeneration and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.14330.001 PMID:27187149

  8. Hair growth promoting potential of phospholipids purified from porcine lung tissues.

    PubMed

    Choi, Seong-Hyun; Moon, Jeong-Su; Jeon, Byung-Suk; Jeon, Yeon-Jeong; Yoon, Byung-Il; Lim, Chang-Jin

    2015-03-01

    BP201, porcine lung tissue-derived phospholipids, consists of phosphatidylcholine as a major phospholipid species. BP201 promoted hair growth after application onto the shaved backs of BALB/c and C3H mice. Its effect was enhanced when applied together with minoxidil (MNX) in C3H mice. When the tissue specimens prepared from the shaved skins of BP201-treated and control mice were microscopically examined, the total numbers of hair follicles in both anagen and telogen phases of BP201-treated mice were significantly higher than those of control mice. The numbers of hair follicles in the anagen phase of BP201-treated mice were also higher than those of control mice. In combination with MNX, BP201 further increased the total number of hair follicles, but did not alter the percentage of hair follicles in the anagenic phase. BP201 also increased the proliferation of human hair follicle dermal papilla cells. Collectively, BP201 possesses hair growth promoting potential, which would suggest its use singly or in combination for hair growth products. PMID:25767686

  9. Hair Growth Promoting Potential of Phospholipids Purified from Porcine Lung Tissues

    PubMed Central

    Choi, Seong-Hyun; Moon, Jeong-Su; Jeon, Byung-Suk; Jeon, Yeon-Jeong; Yoon, Byung-Il; Lim, Chang-Jin

    2015-01-01

    BP201, porcine lung tissue-derived phospholipids, consists of phosphatidylcholine as a major phospholipid species. BP201 promoted hair growth after application onto the shaved backs of BALB/c and C3H mice. Its effect was enhanced when applied together with minoxidil (MNX) in C3H mice. When the tissue specimens prepared from the shaved skins of BP201-treated and control mice were microscopically examined, the total numbers of hair follicles in both anagen and telogen phases of BP201-treated mice were significantly higher than those of control mice. The numbers of hair follicles in the anagen phase of BP201-treated mice were also higher than those of control mice. In combination with MNX, BP201 further increased the total number of hair follicles, but did not alter the percentage of hair follicles in the anagenic phase. BP201 also increased the proliferation of human hair follicle dermal papilla cells. Collectively, BP201 possesses hair growth promoting potential, which would suggest its use singly or in combination for hair growth products. PMID:25767686

  10. Tissue-Specific Immune Gene Expression in the Migratory Locust, Locusta Migratoria.

    PubMed

    Pulpitel, Tamara; Pernice, Mathieu; Simpson, Stephen J; Ponton, Fleur

    2015-01-01

    The ability of hosts to respond to infection involves several complex immune recognition pathways. Broadly conserved pathogen-associated molecular patterns (PAMPs) allow individuals to target a range of invading microbes. Recently, studies on insect innate immunity have found evidence that a single pathogen can activate different immune pathways across species. In this study, expression changes in immune genes encoding peptidoglycan-recognition protein SA (PGRP-SA), gram-negative binding protein 1 (GNBP1) and prophenoloxidase (ProPO) were investigated in Locusta migratoria, following an immune challenge using injected lipopolysaccharide (LPS) solution from Escherichia coli. Since immune activation might also be tissue-specific, gene expression levels were followed across a range of tissue types. For PGRP-SA, expression increased in response to LPS within all seven of the tissue-types assayed and differed significantly between tissues. Expression of GNBP1 similarly varied across tissue types, yet showed no clear expression difference between LPS-injected and uninfected locusts. Increases in ProPO expression in response to LPS, however, could only be detected in the gut sections. This study has revealed tissue-specific immune response to add a new level of complexity to insect immune studies. In addition to variation in recognition pathways identified in previous works, tissue-specificity should be carefully considered in similar works. PMID:26463191

  11. Tissue-Specific Venom Composition and Differential Gene Expression in Sea Anemones.

    PubMed

    Macrander, Jason; Broe, Michael; Daly, Marymegan

    2016-01-01

    Cnidarians represent one of the few groups of venomous animals that lack a centralized venom transmission system. Instead, they are equipped with stinging capsules collectively known as nematocysts. Nematocysts vary in abundance and type across different tissues; however, the venom composition in most species remains unknown. Depending on the tissue type, the venom composition in sea anemones may be vital for predation, defense, or digestion. Using a tissue-specific RNA-seq approach, we characterize the venom assemblage in the tentacles, mesenterial filaments, and column for three species of sea anemone (Anemonia sulcata, Heteractis crispa, and Megalactis griffithsi). These taxa vary with regard to inferred venom potency, symbiont abundance, and nematocyst diversity. We show that there is significant variation in abundance of toxin-like genes across tissues and species. Although the cumulative toxin abundance for the column was consistently the lowest, contributions to the overall toxin assemblage varied considerably among tissues for different toxin types. Our gene ontology (GO) analyses also show sharp contrasts between conserved GO groups emerging from whole transcriptome analysis and tissue-specific expression among GO groups in our differential expression analysis. This study provides a framework for future characterization of tissue-specific venom and other functionally important genes in this lineage of simple bodied animals. PMID:27389690

  12. Comprehensive Tissue-Specific Transcriptome Analysis Reveals Distinct Regulatory Programs during Early Tomato Fruit Development.

    PubMed

    Pattison, Richard J; Csukasi, Fabiana; Zheng, Yi; Fei, Zhangjun; van der Knaap, Esther; Catalá, Carmen

    2015-08-01

    Fruit formation and early development involve a range of physiological and morphological transformations of the various constituent tissues of the ovary. These developmental changes vary considerably according to tissue type, but molecular analyses at an organ-wide level inevitably obscure many tissue-specific phenomena. We used laser-capture microdissection coupled to high-throughput RNA sequencing to analyze the transcriptome of ovaries and fruit tissues of the wild tomato species Solanum pimpinellifolium. This laser-capture microdissection-high-throughput RNA sequencing approach allowed quantitative global profiling of gene expression at previously unobtainable levels of spatial resolution, revealing numerous contrasting transcriptome profiles and uncovering rare and cell type-specific transcripts. Coexpressed gene clusters linked specific tissues and stages to major transcriptional changes underlying the ovary-to-fruit transition and provided evidence of regulatory modules related to cell division, photosynthesis, and auxin transport in internal fruit tissues, together with parallel specialization of the pericarp transcriptome in stress responses and secondary metabolism. Analysis of transcription factor expression and regulatory motifs indicated putative gene regulatory modules that may regulate the development of different tissues and hormonal processes. Major alterations in the expression of hormone metabolic and signaling components illustrate the complex hormonal control underpinning fruit formation, with intricate spatiotemporal variations suggesting separate regulatory programs. PMID:26099271

  13. Tissue-Specific Venom Composition and Differential Gene Expression in Sea Anemones

    PubMed Central

    Macrander, Jason; Broe, Michael; Daly, Marymegan

    2016-01-01

    Cnidarians represent one of the few groups of venomous animals that lack a centralized venom transmission system. Instead, they are equipped with stinging capsules collectively known as nematocysts. Nematocysts vary in abundance and type across different tissues; however, the venom composition in most species remains unknown. Depending on the tissue type, the venom composition in sea anemones may be vital for predation, defense, or digestion. Using a tissue-specific RNA-seq approach, we characterize the venom assemblage in the tentacles, mesenterial filaments, and column for three species of sea anemone (Anemonia sulcata, Heteractis crispa, and Megalactis griffithsi). These taxa vary with regard to inferred venom potency, symbiont abundance, and nematocyst diversity. We show that there is significant variation in abundance of toxin-like genes across tissues and species. Although the cumulative toxin abundance for the column was consistently the lowest, contributions to the overall toxin assemblage varied considerably among tissues for different toxin types. Our gene ontology (GO) analyses also show sharp contrasts between conserved GO groups emerging from whole transcriptome analysis and tissue-specific expression among GO groups in our differential expression analysis. This study provides a framework for future characterization of tissue-specific venom and other functionally important genes in this lineage of simple bodied animals. PMID:27389690

  14. Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue. [Mice

    SciTech Connect

    Ogawa, Y.; Imanaka, K.; Ashida, C.; Takashima, H.; Imajo, Y.; Kimura, S.

    1983-04-01

    Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue was studied on the transplanted MM46 tumor of female C3H/He mice after radiotherapy. MM46 tumor cells were inoculated into the right hind paws of mice. On the 5th day, irradiation with the dose irradiated tumor tissue (2000 rad on the fifth day), were injected into the left hind paws of the tumor-bearing mice. Effectiveness of this active specific immunotherapy against tumor was evaluated by the regression of tumor and survival rate of mice. Tumor was markedly regressed and survival rate was significantly increased by the active specific immunitherapy.

  15. Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.

    PubMed

    Khan, Tahsin N; Mooster, Jana L; Kilgore, Augustus M; Osborn, Jossef F; Nolz, Jeffrey C

    2016-05-30

    Tissue-resident memory (Trm) CD8(+) T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against reinfection. Whether local recognition of antigen in nonlymphoid tissues during infection can impact the formation of Trm populations remains unresolved. Using skin infections with vaccinia virus (VacV)-expressing model antigens, we found that local antigen recognition had a profound impact on Trm formation. Activated CD8(+) T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent, manner. However, after viral clearance, there was a subsequent ∼50-fold increase in Trm formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in nonlymphoid tissue caused CD8(+) T cells to rapidly express CD69 and be retained at the site of infection. Finally, Trm CD8(+) T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the nonlymphoid tissue microenvironment plays a critical role in the formation of functional Trm CD8(+) T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders. PMID:27217536

  16. Biokinetics of radiolabeled monoclonal antibodies in heterotransplanted nude rats: Evaluation of corrected specific tissue uptake

    SciTech Connect

    Ingvar, C.; Norrgren, K.; Strand, S.E.; Brodin, T.; Joensson, P.E.S.; Sjoegren, H.O. )

    1989-07-01

    A tumor model is presented to study the biokinetics and localization of radiolabeled monoclonal antibodies (MAb) in the nude rat (Rowett RNu/RNu) heterotransplanted with human melanoma metastases. The nude rat is larger, less sensitive, and lives longer than the nude mouse. It is, therefore, well suited for in vivo studies of tumor localization with radiolabeled monoclonal antibodies. The tumor-to-host weight ratio was closer to the human situation for the nude rat than for the mouse, and quantitative imaging could be performed with a parallel hole collimator. We followed the antibody biokinetics for as long as 8 days, with repeated blood sampling and imaging. Specific uptake of MAb was higher in tumor tissue than in all other tissues except blood. Initial high uptake was also recorded in the bone marrow. The lymph glands showed a slow uptake of specific and control antibody. A simple in vitro correction procedure is described to calculate the corrected specific tissue uptake (STUcorr) that takes the blood activity into account. Thus it was shown that 80% of the tissue uptake in the dissected liver at 30 hr was due to labeled antibodies circulating in the blood. The specific tissue uptake ratio of antibodies 96.5 and OKT3 (nonspecific control) was unity for all other organs except for tumor tissue, where the ratio was greater than two and even higher when correction for blood content of labeled antibody was made.

  17. Putative storage root specific promoters from cassava and yam: cloning and evaluation in transgenic carrots as a model system.

    PubMed

    Arango, Jacobo; Salazar, Bertha; Welsch, Ralf; Sarmiento, Felipe; Beyer, Peter; Al-Babili, Salim

    2010-06-01

    A prerequisite for biotechnological improvements of storage roots is the availability of tissue-specific promoters enabling high expression of transgenes. In this work, we cloned two genomic fragments, pMe1 and pDJ3S, controlling the expression of a gene with unknown function from cassava (Manihot esculenta) and of the storage protein dioscorin 3 small subunit gene from yam (Dioscorea japonica), respectively. Using beta-glucuronidase as a reporter, the activities of pMe1 and pDJ3S were evaluated in independent transgenic carrot lines and compared to the constitutive CaMV35S and the previously described cassava p15 promoters. Activities of pMe1 and pDJ3S in storage roots were assessed using quantitative GUS assays that showed pDJ3S as the most active one. To determine organ specificities, uidA transcript levels in leaves, stems and roots were measured by real-time RT-PCR analyses showing highest storage root specificity for pDJ3S. Root cross sections revealed that pMe1 was highly active in secondary xylem. In contrast, pDJ3S was active in all root tissues except for the central xylem. The expression patterns caused by the cassava p15 promoter in carrot storage roots were consistent with its previously described activities for the original storage organ. Our data demonstrate that the pDJ3S and, to a lesser extent, the pMe1 regulatory sequences represent feasible candidates to drive high and preferential expression of genes in carrot storage roots. PMID:20369359

  18. Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis.

    PubMed

    Ayllón, Nieves; Villar, Margarita; Galindo, Ruth C; Kocan, Katherine M; Šíma, Radek; López, Juan A; Vázquez, Jesús; Alberdi, Pilar; Cabezas-Cruz, Alejandro; Kopáček, Petr; de la Fuente, José

    2015-03-01

    Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A

  19. Simultaneous immunochemical detection of four banned antibiotic growth promoters in raw and cooked poultry tissue.

    PubMed

    McNamee, S E; Cunningham, R; Elliott, C T

    2013-01-01

    Spiramycin, tylosin, bacitracin and virginiamycin are among a group of antibiotic growth promoters that have been banned in the European Union since the 1999 Council. This was due to concerns over the development of resistant bacteria emerging between humans and animals with the threat of antibiotics no longer being able to be used effectively to treat human infections. A sensitive and fast immunochemical method is presented for the determination of these four antibiotic growth promoters simultaneously in poultry tissue. The method employs methanol extraction followed by sample clean-up by solid-phase extraction (SPE) with determination by enzyme-linked immunoabsorbant assay (ELISA). The limit of detection (LOD) was less than 1 ng g(-1) and the detection capability (CCβ) was 3 ng g(-1) or less for all four antibiotic growth promoters. Validation was completed with both raw and cooked chicken, therefore either matrix could be used for the monitoring of these banned drugs. In a feeding trial no residues of either bacitracin or virginiamycin were found in medicated birds even without a withdrawal period. In the case of tylosin and spiramycin much higher residues level were detected immunochemically than was the case by mass spectrometry. PMID:23789918

  20. Efficient, Glucose Responsive, and Islet-Specific Transgene Expression by a Modified Rat Insulin Promoter

    PubMed Central

    Chai, Renjie; Chen, Shuyuan; Ding, Jiahuan; Grayburn, Paul A

    2009-01-01

    This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various rat insulin promoter lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells, and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is 5-fold more active in INS-1 cells than the full length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent alpha cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified rat insulin promoter, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas. PMID:19727136

  1. Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1.

    PubMed Central

    Parekh, R B; Tse, A G; Dwek, R A; Williams, A F; Rademacher, T W

    1987-01-01

    To examine the extent to which protein structure and tissue-type influence glycosylation, we have determined the oligosaccharide structures at each of the three glycosylation sites (Asn-23, 74 and 98) of the cell surface glycoprotein Thy-1 isolated from rat brain and thymus. The results show that there is tissue-specificity of glycosylation and that superimposed on this is a significant degree of site-specificity. On the basis of the site distribution of oligosaccharides, we find that no Thy-1 molecules are in common between the two tissues despite the amino acid sequences being identical. We suggest, therefore, that by controlling N-glycosylation a tissue creates an unique set of glycoforms (same polypeptide but with oligosaccharides that differ either in sequence or disposition). The structures at each of the three sites were also determined for the thymocyte Thy-1 that binds to lentil lectin (Thy-1 L+) and for that which does not (Thy-1 L-). Segregation of intact thymus Thy-1 into two distinct sets of glycoforms by lentil lectin was found to be due to the structures at site 74. Analysis of oligosaccharide structures at the 'passenger' sites (23 and 98) suggests that either Thy-1 L+ and Thy-1 L- molecules are made in different cell-types or that the biosynthesis of oligosaccharides at one site is influenced by the glycosylation at other sites. PMID:2886334

  2. The Adipose Tissue Microenvironment Regulates Depot-Specific Adipogenesis in Obesity.

    PubMed

    Jeffery, Elise; Wing, Allison; Holtrup, Brandon; Sebo, Zachary; Kaplan, Jennifer L; Saavedra-Peña, Rocio; Church, Christopher D; Colman, Laura; Berry, Ryan; Rodeheffer, Matthew S

    2016-07-12

    The sexually dimorphic distribution of adipose tissue influences the development of obesity-associated pathologies. The accumulation of visceral white adipose tissue (VWAT) that occurs in males is detrimental to metabolic health, while accumulation of subcutaneous adipose tissue (SWAT) seen in females may be protective. Here, we show that adipocyte hyperplasia contributes directly to the differential fat distribution between the sexes. In male mice, high-fat diet (HFD) induces adipogenesis specifically in VWAT, while in females HFD induces adipogenesis in both VWAT and SWAT in a sex hormone-dependent manner. We also show that the activation of adipocyte precursors (APs), which drives adipocyte hyperplasia in obesity, is regulated by the adipose depot microenvironment and not by cell-intrinsic mechanisms. These findings indicate that APs are plastic cells, which respond to both local and systemic signals that influence their differentiation potential independent of depot origin. Therefore, depot-specific AP niches coordinate adipose tissue growth and distribution. PMID:27320063

  3. Change in the Specific Heat Capacity of Parenchymal Tissues of Apples due to Dehydration

    NASA Astrophysics Data System (ADS)

    Mikhailik, V. A.; Dmitrenko, N. V.; Snezhkin, Yu. F.

    2014-01-01

    We present the results of measurements of the heat capacity of parenchymal tissues of apples by the differential scanning calorimetry method. An analytical dependence of the specific heat capacity of these tissues on their temperature (10-90°C) and moisture (6.8-90%) is proposed. We have considered the boundary conditions under which it is possible to calculate the heat capacity of moist parenchymal tissues of apples containing simultaneously free and bound water by an additive model. Reliable values of the heat capacity of tissues containing only bound water can be obtained only experimentally. In parenchymal tissues of apples with a low moisture content (0.6-0.43%) in the positive temperature range, a stepwise change in the heat capacity has been revealed.

  4. Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

    PubMed

    Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

    2016-05-01

    Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. PMID:26957309

  5. The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

    PubMed

    Liu, Xiaoqing; Yang, Wenzhu; Li, Ye; Li, Suzhen; Zhou, Xiaojin; Zhao, Qianqian; Fan, Yunliu; Lin, Min; Chen, Rumei

    2016-07-01

    Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters. PMID:27279278

  6. Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities.

    PubMed Central

    Julien, B; Calendar, R

    1996-01-01

    Transcription from the late promoters of bacteriophage P2 and its satellite phage P4 is activated by a unique class of small, zinc-binding proteins. Using plasmid expression systems, we compared activators from two P2-like (helper) phages with those encoded by two satellite phages. The helper phage activators have more activity on the P4 phage sid promoter. In contrast, the satellite phage activators function better on the four late P2 promoters and on the P4 late leftward promoter. We purified one activator encoded by a P2-like phage and an activator from a satellite phage and determined their binding sites within the P2 and P4 late promoters. Differences in activity levels correlate with binding specificities; promoters that function best with the satellite phage activators have only one activator binding site centered at -55, while the P4 sid promoter, which has more activity with helper phage activators, has a second binding site centered at -18. Surprisingly, DNase I footprinting revealed only very minor differences in promoter binding by the two activators reported here and the P4 activator reported previously. Thus, the differences in transcriptional activity are probably due to interactions between the activators and RNA polymerase, rather than interactions between the activators and DNA. PMID:8824611

  7. Expression Profile and Tissue-Specific Distribution of the Receptor-Interacting Protein 3 in BALB/c Mice.

    PubMed

    Wang, Qingnan; Yu, Meng; Zhang, Kaizhao; Liu, Jianxin; Tao, Pan; Ge, Shikun; Ning, Zhangyong

    2016-08-01

    RIP3, a member of receptor-interacting protein family, is serine/threonine kinase that contributes to necrosis and promotes systematic inflammation. However, detailed information of the expression pattern and tissue distribution in BALB/c mice, a commonly used laboratory animal model, is still unavailable. Here, we provided the basic data of expression profile and histologic distribution of RIP3 in tissues of BALB/c mice. Rip3 mRNA expression levels and tissue distribution were detected by real-time quantitative PCR and immunohistochemical detection, respectively. Rip3 mRNA expression showed the highest level in the spleen and duodenum, while with the lowest level in brain. Immunohistochemical detection revealed this protein located in different type cells in different tissues. What's more, the obvious positive staining in nuclear was detected in liver cells and neurons in cerebral cortex of the brain, while cells in other organs, including heart, spleen, lung, kidney, stomach, duodenum and trachea, showed strong positive mainly in cytoplasm. The results will help us to further understand the site-specific functions of RIP3 in necrosis and inflammatory responses. PMID:26969469

  8. Adipose tissue-specific inactivation of the retinoblastoma protein protects against diabesity because of increased energy expenditure

    PubMed Central

    Dali-Youcef, Nassim; Mataki, Chikage; Coste, Agnès; Messaddeq, Nadia; Giroud, Sylvain; Blanc, Stéphane; Koehl, Christian; Champy, Marie-France; Chambon, Pierre; Fajas, Lluis; Metzger, Daniel; Schoonjans, Kristina; Auwerx, Johan

    2007-01-01

    The role of the tumor suppressor retinoblastoma protein (pRb) has been firmly established in the control of cell cycle, apoptosis, and differentiation. Recently, it was demonstrated that lack of pRb promotes a switch from white to brown adipocyte differentiation in vitro. We used the Cre-Lox system to specifically inactivate pRb in adult adipose tissue. Under a high-fat diet, pRb-deficient (pRbad−/−) mice failed to gain weight because of increased energy expenditure. This protection against weight gain was caused by the activation of mitochondrial activity in white and brown fat as evidenced by histologic, electron microscopic, and gene expression studies. Moreover, pRb−/− mouse embryonic fibroblasts displayed higher proliferation and apoptosis rates than pRb+/+ mouse embryonic fibroblasts, which could contribute to the altered white adipose tissue morphology. Taken together, our data support a direct role of pRb in adipocyte cell fate determination in vivo and suggest that pRb could serve as a potential therapeutic target to trigger mitochondrial activation in white adipose tissue and brown adipose tissue, favoring an increase in energy expenditure and subsequent weight loss. PMID:17556545

  9. А new Gal/GalNAc-specific lectin from the mussel Mytilus trossulus: Structure, tissue specificity, antimicrobial and antifungal activity.

    PubMed

    Chikalovets, Irina V; Kovalchuk, Svetlana N; Litovchenko, Alina P; Molchanova, Valentina I; Pivkin, Mikhail V; Chernikov, Oleg V

    2016-03-01

    In the present study, a new Gal/GalNAc specific lectin from the mussel Mytilus trossulus (designated as MTL) was identified, and its expression levels, both in tissues and toward pathogen stimulation, were then characterized. The MTL primary structure was determined via cDNA sequencing. Deduced sequence of 150 amino acid residues showed 89% similarity to lectins from the mussels Crenomytilus grayanus and Mytilus galloprovincialis that were the first members of a new family of zoolectins. The results indicated that the MTL might be involved in immune response toward pathogen infection, and it might perform different recognition specificity toward bacteria or fungi. PMID:26802895

  10. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  11. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    PubMed

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

  12. Urinary bladder matrix promotes site appropriate tissue formation following right ventricle outflow tract repair

    PubMed Central

    Remlinger, Nathaniel T; Gilbert, Thomas W; Yoshida, Masahiro; Guest, Brogan N; Hashizume, Ryotaro; Weaver, Michelle L; Wagner, William R; Brown, Bryan N; Tobita, Kimimasa; Wearden, Peter D

    2013-01-01

    The current prevalence and severity of heart defects requiring functional replacement of cardiac tissue pose a serious clinical challenge. Biologic scaffolds are an attractive tissue engineering approach to cardiac repair because they avoid sensitization associated with homograft materials and theoretically possess the potential for growth in similar patterns as surrounding native tissue. Both urinary bladder matrix (UBM) and cardiac ECM (C-ECM) have been previously investigated as scaffolds for cardiac repair with modest success, but have not been compared directly. In other tissue locations, bone marrow derived cells have been shown to play a role in the remodeling process, but this has not been investigated for UBM in the cardiac location, and has never been studied for C-ECM. The objectives of the present study were to compare the effectiveness of an organ-specific C-ECM patch with a commonly used ECM scaffold for myocardial tissue repair of the right ventricle outflow tract (RVOT), and to examine the role of bone marrow derived cells in the remodeling response. A chimeric rat model in which all bone marrow cells express green fluorescent protein (GFP) was generated and used to show the ability of ECM scaffolds derived from the heart and bladder to support cardiac function and cellular growth in the RVOT. The results from this study suggest that urinary bladder matrix may provide a more appropriate substrate for myocardial repair than cardiac derived matrices, as shown by differences in the remodeling responses following implantation, as well as the presence of site appropriate cells and the formation of immature, myocardial tissue. PMID:23974174

  13. A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression

    PubMed Central

    Warburton, Alix; Breen, Gerome; Bubb, Vivien J.; Quinn, John P.

    2016-01-01

    Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk. PMID:26429811

  14. A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression.

    PubMed

    Warburton, Alix; Breen, Gerome; Bubb, Vivien J; Quinn, John P

    2016-07-01

    Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk. PMID:26429811

  15. The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

    PubMed Central

    Ip, H S; Wilson, D B; Heikinheimo, M; Tang, Z; Ting, C N; Simon, M C; Leiden, J M; Parmacek, M S

    1994-01-01

    The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development. Images PMID:7935467

  16. Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

    SciTech Connect

    Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick; Smith, Richard D.; Kehr, Julia

    2005-07-14

    Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

  17. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.

    PubMed

    Kuda, Ondrej; Rombaldova, Martina; Janovska, Petra; Flachs, Pavel; Kopecky, Jan

    2016-01-15

    Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA. PMID:26707880

  18. Human Umbilical Tissue-Derived Cells Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins

    PubMed Central

    Koh, Sehwon; Kim, Namsoo; Yin, Henry H.; Harris, Ian R.; Dejneka, Nadine S.

    2015-01-01

    Cell therapy demonstrates great potential for the treatment of neurological disorders. Human umbilical tissue-derived cells (hUTCs) were previously shown to have protective and regenerative effects in animal models of stroke and retinal degeneration, but the underlying therapeutic mechanisms are unknown. Because synaptic dysfunction, synapse loss, degeneration of neuronal processes, and neuronal death are hallmarks of neurological diseases and retinal degenerations, we tested whether hUTCs contribute to tissue repair and regeneration by stimulating synapse formation, neurite outgrowth, and neuronal survival. To do so, we used a purified rat retinal ganglion cell culture system and found that hUTCs secrete factors that strongly promote excitatory synaptic connectivity and enhance neuronal survival. Additionally, we demonstrated that hUTCs support neurite outgrowth under normal culture conditions and in the presence of the growth-inhibitory proteins chondroitin sulfate proteoglycan, myelin basic protein, or Nogo-A (reticulon 4). Furthermore, through biochemical fractionation and pharmacology, we identified the major hUTC-secreted synaptogenic factors as the thrombospondin family proteins (TSPs), TSP1, TSP2, and TSP4. Silencing TSP expression in hUTCs, using small RNA interference, eliminated both the synaptogenic function of these cells and their ability to promote neurite outgrowth. However, the majority of the prosurvival functions of hUTC-conditioned media was spared after TSP knockdown, indicating that hUTCs secrete additional neurotrophic factors. Together, our findings demonstrate that hUTCs affect multiple aspects of neuronal health and connectivity through secreted factors, and each of these paracrine effects may individually contribute to the therapeutic function of these cells. SIGNIFICANCE STATEMENT Human umbilical tissue-derived cells (hUTC) are currently under clinical investigation for the treatment of geographic atrophy secondary to age-related macular

  19. Research Resource: Tissue- and Pathway-Specific Metabolomic Profiles of the Steroid Receptor Coactivator (SRC) Family

    PubMed Central

    York, Brian; Sagen, Jørn V.; Tsimelzon, Anna; Louet, Jean-Francios; Chopra, Atul R.; Reineke, Erin L.; Zhou, Suoling; Stevens, Robert D.; Wenner, Brett R.; Ilkayeva, Olga; Bain, James R.; Xu, Jianming; Hilsenbeck, Susan G.; Newgard, Christopher B.

    2013-01-01

    The rapidly growing family of transcriptional coregulators includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have emerged as important regulators of metabolic homeostasis, including the p160 steroid receptor coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis, fat absorption and storage in the liver, and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of the SRC family members, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockouts (KOs) of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 KO mice and their wild-type littermates under fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC. Specifically, SRC-1 ablation revealed the most significant impact on hepatic metabolism, whereas SRC-2 appeared to impact cardiac metabolism. Conversely, ablation of SRC-3 primarily affected brain and skeletal muscle metabolism. Surprisingly, we identified very few metabolites that changed universally across the three SRC KO models. The findings of this Research Resource demonstrate that coactivator function has very limited metabolic redundancy even within the homologous SRC family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators. PMID:23315938

  20. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells

    SciTech Connect

    Pendse, G.J.; Bailey, J.E. . Dept. of Chemical Engineering)

    1994-12-01

    Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

  1. Identification and characterization of a prostate-specific androgen-independent protein-binding site in the probasin promoter.

    PubMed Central

    Yeung, Lillian H Y; Read, Jason T; Sorenson, Pernille; Nelson, Colleen C; Jia, William; Rennie, Paul S

    2003-01-01

    In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the prostate-specific probasin (PB) promoter. Using DNaseI in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides -286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Four of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10 nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of the presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoretic mobility-shift assay. Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield slightly different protection patterns for some of the protein complexes formed with non-prostate-derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. Site-directed mutagenesis of the protected nucleotides within FPII resulted in a significant reduction in expression from the PB promoter. Identification of proteins binding to the FPII region revealed the participation of nuclear factor I (NF-I) or a closely related protein, although other unknown proteins are also involved. Defining the DNA and protein components that dictate prostate-specific expression of the PB promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen-independent prostate cancer. PMID:12540291

  2. Tissue-Specific Metabolic Profile Study of Moringa oleifera L. Using Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Mahmud, Iqbal; Chowdhury, Kamal

    2014-01-01

    Moringa oleifera, an important multipurpose crop, is rich in various phytochemicals: flavonoids, antioxidants, vitamins, minerals and carotenes. The purpose of this study was to profile the groups of metabolites in leaf and stem tissues of M. oleifera. Various sugars, amino acids, and organic acid derivatives were found in all of the M. oleifera tissues with different profiles/peak intensities depending on the tissue. 1D proton nuclear magnetic resonance (NMR) was applied for collecting metabolite spectra. Approximately 30 metabolites with 2 unknown peaks were identified with Chenomx and verified with MMCD databases using carbon data. Among these metabolites, 22 metabolites were identified as common in both leaf and stem tissues. Of the remaining 8 metabolites, 4-aminobutyrate, adenosine, guanosine, tyrosine, and p-cresol were found only in leaf tissues; however, glutamate, glutamine, and tryptophan were found only in stem tissues. Biochemical pathway analysis revealed that 28 identified metabolites were interconnected with 36 different pathways as well as related to different fatty acids and secondary metabolites synthesis biochemical pathways. It is well known that different tissues of M. oleifera have nutritional, medicinal, and therapeutic values; therefore, our main objective is to provide a publicly available M. oliefera tissue specific metabolite database. PMID:26366209

  3. In Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

    PubMed Central

    Horstmann, Sebastian; Annoura, Takeshi; del Portillo, Hernando A.; Khan, Shahid M.; Heussler, Volker T.

    2015-01-01

    Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems. PMID:25874388

  4. The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner.

    PubMed

    Fu, G K; Markovitz, D M

    1998-02-17

    LON proteases, which are ATP-dependent and exhibit ATPase activity, are found in bacteria, yeast, and humans. In Escherichia coli, LON is known to regulate gene expression by targeting specific regulatory proteins for degradation. The yeast and human LON proteins are encoded in the nucleus but localize to the mitochondrial matrix. In yeast, LON has been shown to be essential for the maintenance of the integrity of the mitochondrial genome. E. coli Lon has long been known to bind DNA, but we have only recently demonstrated that it binds preferentially to a specific TG-rich double-stranded sequence. We now show that human LON recognizes a very similar site in both the light and heavy chain promoters of the mitochondrial genome, in a region which is involved in regulating both DNA replication and transcription. Unlike E. coli Lon, however, human LON specifically binds to the TG-rich element only when it is presented in the context of a single DNA strand. These findings suggest that the human LON protease might regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters. PMID:9485316

  5. Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz).

    PubMed

    Koehorst-van Putten, Herma J J; Wolters, Anne-Marie A; Pereira-Bertram, Isolde M; van den Berg, Hans H J; van der Krol, Alexander R; Visser, Richard G F

    2012-12-01

    In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava. PMID:23132522

  6. De novo assembly and analysis of tissue-specific transcriptomes revealed the tissue-specific genes and profile of immunity from Strongylocentrotus intermedius.

    PubMed

    Chen, Yadong; Chang, Yaqing; Wang, Xiuli; Qiu, Xuemei; Liu, Yang

    2015-10-01

    Strongylocentrotus intermedius is an important marine species in north China and Japan. Recent years, diseases are threating the sea urchin aquaculture industry seriously. To provide a genetic resource for S. intermedius as well as overview the immune-related genes of S. intermedius, we performed transcriptome sequencing of three cDNA libraries representing three tissues, coelomocytes, gut and peristomial membrane respectively. In total 138,421 contigs were assembled from all sequencing data. 96,764 contigs were annotated according to bioinformatics databases, including NT, nr, Swiss-Prot, KEGG, COG. 49,336 Contigs were annotated as CDS. In this study, we obtained 24,778 gene families from S. intermedius transcriptome. The gene expression analysis revealed that more genes were expressed in gut, more high expression level genes in coelomocytes when compared with other tissues. Specific expressed contigs in coelomocytes, gut, and peristomial membrane were 546, 1136, and 1012 respectively. Pathway analysis suggested 25, 17 and 36 potential specifically pathways may specific progressed in peristomial membrane, gut and coelomocytes respectively. Similarities and differences between S. intermedius and other echinoderms were analyzed. S. intermedius was more homology to Strongylocentrotus purpuratus than others sea urchin. Of 24,778 genes, 1074 genes are immune-related, immune genes were expressed with a higher level in coelomocytes than other tissues. Complement system may be the most important immune system in sea urchin. We also identified 2438 SSRs and 16,236 SNPs for S. intermedius. These results provide a transcriptome resource and foundation to study molecular mechanisms of sea urchin immune system. PMID:26253994

  7. Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

    PubMed

    Venables, Julian P; Vignal, Emmanuel; Baghdiguian, Stephen; Fort, Philippe; Tazi, Jamal

    2012-01-01

    Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific

  8. Tissue Specificity and Sex-Specific Regulatory Variation Permit the Evolution of Sex-Biased Gene Expression.

    PubMed

    Dean, Rebecca; Mank, Judith E

    2016-09-01

    Genetic correlations between males and females are often thought to constrain the evolution of sexual dimorphism. However, sexually dimorphic traits and the underlying sexually dimorphic gene expression patterns are often rapidly evolving. We explore this apparent paradox by measuring the genetic correlation in gene expression between males and females (Cmf) across broad evolutionary timescales, using two RNA-sequencing data sets spanning multiple populations and multiple species. We find that unbiased genes have higher Cmf than sex-biased genes, consistent with intersexual genetic correlations constraining the evolution of sexual dimorphism. However, we found that highly sex-biased genes (both male and female biased) also had higher tissue specificity, and unbiased genes had greater expression breadth, suggesting that pleiotropy may constrain the breakdown of intersexual genetic correlations. Finally, we show that genes with high Cmf showed some degree of sex-specific changes in gene expression in males and females. Together, our results suggest that genetic correlations between males and females may be less important in constraining the evolution of sex-biased gene expression than pleiotropy. Sex-specific regulatory variation and tissue specificity may resolve the paradox of widespread sex bias within a largely shared genome. PMID:27501094

  9. Mechanism of the tissue-specific action of the selective androgen receptor modulator S-101479.

    PubMed

    Furuya, Kazuyuki; Yamamoto, Noriko; Ohyabu, Yuki; Morikyu, Teruyuki; Ishige, Hirohide; Albers, Michael; Endo, Yasuhisa

    2013-01-01

    Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce anabolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimulation of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction assays revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, β-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tissues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues. PMID:23449329

  10. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    PubMed

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  11. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

    PubMed Central

    Huang, Chao; Sachse, Frank B.; Hitchcock, Robert W.; Kaza, Aditya K.

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  12. The impact of laser ablation on optical soft tissue differentiation for tissue specific laser surgery-an experimental ex vivo study

    PubMed Central

    2012-01-01

    Background Optical diffuse reflectance can remotely differentiate various bio tissues. To implement this technique in an optical feedback system to guide laser surgery in a tissue-specific way, the alteration of optical tissue properties by laser ablation has to be taken into account. It was the aim of this study to evaluate the general feasibility of optical soft tissue differentiation by diffuse reflectance spectroscopy under the influence of laser ablation, comparing the tissue differentiation results before and after laser intervention. Methods A total of 70 ex vivo tissue samples (5 tissue types) were taken from 14 bisected pig heads. Diffuse reflectance spectra were recorded before and after Er:YAG-laser ablation. The spectra were analyzed and differentiated using principal component analysis (PCA), followed by linear discriminant analysis (LDA). To assess the potential of tissue differentiation, area under the curve (AUC), sensitivity and specificity was computed for each pair of tissue types before and after laser ablation, and compared to each other. Results Optical tissue differentiation showed good results before laser exposure (total classification error 13.51%). However, the tissue pair nerve and fat yielded lower AUC results of only 0.75. After laser ablation slightly reduced differentiation results were found with a total classification error of 16.83%. The tissue pair nerve and fat showed enhanced differentiation (AUC: 0.85). Laser ablation reduced the sensitivity in 50% and specificity in 80% of the cases of tissue pair comparison. The sensitivity of nerve–fat differentiation was enhanced by 35%. Conclusions The observed results show the general feasibility of tissue differentiation by diffuse reflectance spectroscopy even under conditions of tissue alteration by laser ablation. The contrast enhancement for the differentiation between nerve and fat tissue after ablation is assumed to be due to laser removal of the surrounding lipid-rich nerve

  13. eQTL mapping identifies insertion- and deletion-specific eQTLs in multiple tissues.

    PubMed

    Huang, Jinyan; Chen, Jun; Esparza, Jorge; Ding, Jun; Elder, James T; Abecasis, Goncalo R; Lee, Young-Ae; Mark Lathrop, G; Moffatt, Miriam F; Cookson, William O C; Liang, Liming

    2015-01-01

    Genome-wide gene expression quantitative trait loci (eQTL) mapping have been focused on single-nucleotide polymorphisms and have helped interpret findings from diseases mapping studies. The functional effect of structure variants, especially short insertions and deletions (indel) has not been well investigated. Here we impute 1,380,133 indels based on the latest 1,000 Genomes Project panel into three eQTL data sets from multiple tissues. Imputation of indels increased 9.9% power and identifies indel-specific eQTLs for 325 genes. We find introns and vicinities of UTRs are more enriched of indel eQTLs and 3.6 (single-tissue)-9.2%(multi-tissue) of previous identified eSNPs were taggers of eindels. Functional analyses identifies epigenetics marks, gene ontology categories and disease GWAS loci affected by SNPs and indels eQTLs showing tissue-consistent or tissue-specific effects. This study provides new insights into the underlying genetic architecture of gene expression across tissues and new resource to interpret function of diseases and traits associated structure variants. PMID:25951796

  14. eQTL mapping identify insertion and deletion specific eQTLs in multiple tissues

    PubMed Central

    Huang, Jinyan; Chen, Jun; Esparza, Jorge; Ding, Jun; Elder, James; Abecasis, Goncalo R; Lee, Young-Ae; Lathrop, G. Mark; Moffatt, Miriam F; Cookson, William O C; Liang, Liming

    2016-01-01

    GenomeC wide gene expression quantitative trait loci (eQTL) mapping have been focused on single nucleotide polymorphisms and have helped interpret findings from diseases mapping studies. The functional effect of structure variants, especially short insertions and deletions (indel) has not been well investigated. Here we imputed 1,380,133 indels based on the latest 1000 Genomes Project panel into 3 eQTL datasets from multiple tissues. Imputation of indels increased 9.9% power and identified indel specific eQTLs for 325 genes. We found introns and vicinities of UTRs were more enriched of indel eQTLs and 3.6 (singleC tissue)C 9.2%(multiC tissue) of previous identified eSNPs were taggers of eindels. Functional analyses identified epigenetics marks, gene ontology categories and disease GWAS loci affected by SNPs and indels eQTLs showing tissueC consistent or tissueC specific effects. This study provides new insights into the underlying genetic architecture of gene expression across tissues and new resource to interpret function of diseases and traits associated structure variants. PMID:25951796

  15. Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit.

    PubMed

    Opitz, Nina; Marcon, Caroline; Paschold, Anja; Malik, Waqas Ahmed; Lithio, Andrew; Brandt, Ronny; Piepho, Hans-Peter; Nettleton, Dan; Hochholdinger, Frank

    2016-02-01

    Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the cortex of the mature root zone and in the elongation zone. The most prominent functional categories among differentially expressed genes in all tissues were 'transcriptional regulation' and 'hormone metabolism', indicating global reprogramming of cellular metabolism as an adaptation to water deficit. Additionally, the most significant transcriptomic changes in the root tip were associated with cell wall reorganization, leading to continued root growth despite water deficit conditions. This study provides insight into tissue-specific water deficit responses and will be a resource for future genetic analyses and breeding strategies to develop more drought-tolerant maize cultivars. PMID:26463995

  16. Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit

    PubMed Central

    Opitz, Nina; Marcon, Caroline; Paschold, Anja; Malik, Waqas Ahmed; Lithio, Andrew; Brandt, Ronny; Piepho, Hans-Peter; Nettleton, Dan; Hochholdinger, Frank

    2016-01-01

    Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the cortex of the mature root zone and in the elongation zone. The most prominent functional categories among differentially expressed genes in all tissues were ‘transcriptional regulation’ and ‘hormone metabolism’, indicating global reprogramming of cellular metabolism as an adaptation to water deficit. Additionally, the most significant transcriptomic changes in the root tip were associated with cell wall reorganization, leading to continued root growth despite water deficit conditions. This study provides insight into tissue-specific water deficit responses and will be a resource for future genetic analyses and breeding strategies to develop more drought-tolerant maize cultivars. PMID:26463995

  17. Visualizing Oxazine 4 nerve-specific fluorescence ex vivo in frozen tissue sections

    NASA Astrophysics Data System (ADS)

    Barth, Connor W.; Gibbs, Summer L.

    2016-03-01

    Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

  18. Characterization of the Promoter Regions of Two Sheep Keratin-Associated Protein Genes for Hair Cortex-Specific Expression

    PubMed Central

    Zhao, Zhichao; Liu, Guangbin; Li, Xinyun; Huang, Ji; Xiao, Yujing; Du, Xiaoyong; Yu, Mei

    2016-01-01

    The keratin-associated proteins (KAPs) are the structural proteins of hair fibers and are thought to play an important role in determining the physical properties of hair fibers. These proteins are activated in a striking sequential and spatial pattern in the keratinocytes of hair fibers. Thus, it is important to elucidate the mechanism that underlies the specific transcriptional activity of these genes. In this study, sheep KRTAP 3–3 and KRTAP11-1 genes were found to be highly expressed in wool follicles in a tissue-specific manner. Subsequently, the promoter regions of the two genes that contained the 5′ flanking/5′ untranslated regions and the coding regions were cloned. Using an in vivo transgenic approach, we found that the promoter regions from the two genes exhibited transcriptional activity in hair fibers. A much stronger and more uniformly expressed green fluorescent signal was observed in the KRTAP11-1-ZsGreen1 transgenic mice. In situ hybridization revealed the symmetrical expression of sheep KRTAP11-1 in the entire wool cortex. Consistently, immunohistochemical analysis demonstrated that the pattern of ZsGreen1 expression in the hair cortex of transgenic mice matches that of the endogenous KRTAP11-1 gene, indicating that the cloned promoter region contains elements that are sufficient to govern the wool cortex-specific transcription of KRTAP11-1. Furthermore, regulatory regions in the 5′ upstream sequence of the sheep KRTAP11-1 gene that may regulate the observed hair keratinocyte specificity were identified using in vivo reporter assays. PMID:27100288

  19. Tissue-specific expression of Le(Y) antigen in high endothelial venules of human lymphoid tissues.

    PubMed

    Tanegashima, A; Ushiyama, I; Nishi, K; Yamamoto, H; Fukunaga, T

    1999-12-01

    In this study, we demonstrated that the anti-Le(Y) antibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The Le(Y) expression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the Le(Y) expression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of Le(Y)-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of Le(Y) was observed in vessels of Payer's patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-Le(Y) antibody increased after enzyme digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs. PMID:11133021

  20. Tissue-specific Patterning of the Host Innate Immune Response by Staphylococcus aureus α-toxin

    PubMed Central

    Becker, Russell E. N.; Berube, Bryan J.; Sampedro, Georgia R.; DeDent, Andrea C.; Wardenburg, Juliane Bubeck

    2014-01-01

    Immunomodulatory cytotoxins are prominent virulence factors produced by Staphylococcus aureus, a leading cause of bacterial sepsis, skin infection, and pneumonia. S. aureus α-toxin is a pore-forming toxin that utilizes a widely-expressed receptor, ADAM10, to injure the host epithelium, endothelium, and immune cells. As each host tissue is characterized by a unique composition of resident cells and recruited immune cells, the outcome of α-toxin-mediated injury may depend on the infected tissue environment. Utilizing myeloid lineage-specific Adam10 knockout mice, we show that α-toxin exerts tissue-specific effects on innate immunity to staphylococcal infection. Loss of ADAM10 expression exacerbates skin infection, yet affords protection against lethal pneumonia. These diverse outcomes are not related to altered immune cell recruitment, but rather correlate with a defect in toxin-induced IL-1β production. Extension of these studies through analysis of ADAM10 double knockout mice affecting both the myeloid lineage and either the skin or lung epithelium highlight the prominence of toxin-induced injury to the epithelium in governing the outcome of infection. Together, these studies provide evidence of tissue specificity of pore-forming cytotoxin action in modulation of host immunity, and illustrate that the outcome of infection is a collective manifestation of all effects of the toxin within the tissue microenvironment. PMID:24820433

  1. Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

    PubMed Central

    Stoflet, E S; Schmidt, L J; Elder, P K; Korf, G M; Foster, D N; Strauch, A R; Getz, M J

    1992-01-01

    Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways. Images PMID:1421567

  2. Curcumin promotes browning of white adipose tissue in a norepinephrine-dependent way.

    PubMed

    Wang, Shan; Wang, Xiuchao; Ye, Zichen; Xu, Chengming; Zhang, Ming; Ruan, Banjun; Wei, Ming; Jiang, Yinghao; Zhang, Ying; Wang, Li; Lei, Xiaoying; Lu, Zifan

    2015-10-16

    Brown adipose tissue converts energy from food into heat via the mitochondrial uncoupling protein UCP1, defending against cold. In some conditions, inducible 'brown-like' adipocytes, also known as beige adipocytes, can develop within white adipose tissue (WAT). These beige adipocytes have characteristics similar to classical brown adipocytes and thus can burn lipids to produce heat. In the current study, we demonstrated that curcumin (50 or 100 mg/kg/day) decreased bodyweight and fat mass without affecting food intake in mice. We further demonstrated that curcumin improves cold tolerance in mice. This effect was possibly mediated by the emergence of beige adipocytes and the increase of thermogenic gene expression and mitochondrial biogenesis in inguinal WAT. In addition, curcumin promotes β3AR gene expression in inguinal WAT and elevates the levels of plasma norepinephrine, a hormone that can induce WAT browning. Taken together, our data suggest that curcumin can potentially prevent obesity by inducing browning of inguinal WAT via the norepinephrine-β3AR pathway. PMID:26362189

  3. Tissue Restricted Splice Junctions Originate Not Only from Tissue-Specific Gene Loci, but Gene Loci with a Broad Pattern of Expression

    PubMed Central

    Hestand, Matthew S.; Zeng, Zheng; Coleman, Stephen J.; Liu, Jinze; MacLeod, James N.

    2015-01-01

    Cellular mechanisms that achieve protein diversity in eukaryotes are multifaceted, including transcriptional components such as RNA splicing. Through alternative splicing, a single protein-coding gene can generate multiple mRNA transcripts and protein isoforms, some of which are tissue-specific. We have conducted qualitative and quantitative analyses of the Bodymap 2.0 messenger RNA-sequencing data from 16 human tissue samples and identified 209,363 splice junctions. Of these, 22,231 (10.6%) were not previously annotated and 21,650 (10.3%) were expressed in a tissue-restricted pattern. Tissue-restricted alternative splicing was found to be widespread, with approximately 65% of expressed multi-exon genes containing at least one tissue-specific splice junction. Interestingly, we observed many tissue-specific splice junctions not only in genes expressed in one or a few tissues, but also from gene loci with a broad pattern of expression. PMID:26713731

  4. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    PubMed

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation. PMID:27111282

  5. Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in Response to Environmental Water Samples

    PubMed Central

    Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki S.; Halpern, Marnie E.

    2014-01-01

    Background: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization. Results: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves. Citation: Gorelick DA, Iwanowicz LR, Hung AL, Blazer VS, Halpern ME. 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to

  6. Adaptive growth factor delivery from a polyelectrolyte coating promotes synergistic bone tissue repair and reconstruction

    PubMed Central

    Shah, Nisarg J.; Hyder, Md. Nasim; Quadir, Mohiuddin A.; Dorval Courchesne, Noémie-Manuelle; Seeherman, Howard J.; Nevins, Myron; Spector, Myron; Hammond, Paula T.

    2014-01-01

    Traumatic wounds and congenital defects that require large-scale bone tissue repair have few successful clinical therapies, particularly for craniomaxillofacial defects. Although bioactive materials have demonstrated alternative approaches to tissue repair, an optimized materials system for reproducible, safe, and targeted repair remains elusive. We hypothesized that controlled, rapid bone formation in large, critical-size defects could be induced by simultaneously delivering multiple biological growth factors to the site of the wound. Here, we report an approach for bone repair using a polyelectrolye multilayer coating carrying as little as 200 ng of bone morphogenetic protein-2 and platelet-derived growth factor-BB that were eluted over readily adapted time scales to induce rapid bone repair. Based on electrostatic interactions between the polymer multilayers and growth factors alone, we sustained mitogenic and osteogenic signals with these growth factors in an easily tunable and controlled manner to direct endogenous cell function. To prove the role of this adaptive release system, we applied the polyelectrolyte coating on a well-studied biodegradable poly(lactic-co-glycolic acid) support membrane. The released growth factors directed cellular processes to induce bone repair in a critical-size rat calvaria model. The released growth factors promoted local bone formation that bridged a critical-size defect in the calvaria as early as 2 wk after implantation. Mature, mechanically competent bone regenerated the native calvaria form. Such an approach could be clinically useful and has significant benefits as a synthetic, off-the-shelf, cell-free option for bone tissue repair and restoration. PMID:25136093

  7. Sex- and Tissue-specific Functions of Drosophila Doublesex Transcription Factor Target Genes

    PubMed Central

    Clough, Emily; Jimenez, Erin; Kim, Yoo-Ah; Whitworth, Cale; Neville, Megan C.; Hempel, Leonie; Pavlou, Hania J.; Chen, Zhen-Xia; Sturgill, David; Dale, Ryan; Smith, Harold E.; Przytycka, Teresa M.; Goodwin, Stephen F.; Van Doren, Mark; Oliver, Brian

    2014-01-01

    Primary sex determination “switches” evolve rapidly, but Doublesex (DSX) related transcription factors (DMRTs) act downstream of these switches to control sexual development in most animal species. Drosophila dsx encodes female- and male-specific isoforms (DSXF and DSXM), but little is known about how dsx controls sexual development, whether DSXF and DSXM bind different targets, or how DSX proteins direct different outcomes in diverse tissues. We undertook genome-wide analyses to identify DSX targets using in vivo occupancy, binding site prediction, and evolutionary conservation. We find that DSXF and DSXM bind thousands of the same targets in multiple tissues in both sexes, yet these targets have sex- and tissue-specific functions. Interestingly, DSX targets show considerable overlap with targets identified for mouse DMRT1. DSX targets include transcription factors and signaling pathway components providing for direct and indirect regulation of sex-biased expression. PMID:25535918

  8. Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice.

    PubMed Central

    Short, M K; Clouthier, D E; Schaefer, I M; Hammer, R E; Magnuson, M A; Beale, E G

    1992-01-01

    The cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene is expressed in multiple tissues and is regulated in a complex tissue-specific manner. To map the cis-acting DNA elements that direct this tissue-specific expression, we made transgenic mice containing truncated PEPCK-human growth hormone (hGH) fusion genes. The transgenes contained PEPCK promoter fragments with 5' endpoints at -2088, -888, -600, -402, and -207 bp, while the 3' endpoint was at +69 bp. Immunohistochemical analysis showed that the -2088 transgene was expressed in the correct cell types (hepatocytes, proximal tubular epithelium of the kidney, villar epithelium of the small intestine, epithelium of the colon, smooth muscle of the vagina and lungs, ductal epithelium of the sublingual gland, and white and brown adipocytes). Solution hybridization of hGH mRNA expressed from the transgenes indicated that white and brown fat-specific elements are located distally (-2088 to -888 bp) and that liver-, gut-, and kidney-specific elements are located proximally (-600 to +69 bp). However, elements outside of the region tested are necessary for the correct developmental pattern and level of PEPCK expression in kidney. Both the -2088 and -402 transgenes responded in a tissue-specific manner to dietary stimuli, and the -2088 transgene responded to glucocorticoid stimuli. Thus, different tissues utilize distinct cell-specific cis-acting elements to direct and regulate the PEPCK gene. Images PMID:1545785

  9. GLITTER: a web-based application for gene link inspection through tissue-specific coexpression.

    PubMed

    Liu, Xiangtao; Yu, Pengfei; Cheng, Chao; Potash, James B; Han, Shizhong

    2016-01-01

    Accumulating evidence supports the polygenic nature of most complex diseases, suggesting the involvement of many susceptibility genes with small effect sizes. Although hundreds of genes may underlie the genetic architecture of complex diseases, those involved in a given disease are probably not randomly distributed, but likely to be functionally related. Protein-protein interaction networks have been used to evaluate the functional relatedness of susceptibility genes. However, these networks do not account for tissue specificity, are limited to protein-coding genes, and are typically biased by incomplete biological knowledge. Here, we present Gene Link Inspector Through Tissue-specific coExpRession (GLITTER), a web-based application for assessing the functional relatedness of susceptibility genes, either coding or noncoding, according to tissue-specific gene expression profiles. GLITTER can also shed light on the specific tissues in which susceptibility genes might exert their functions. We further demonstrate examples of how GLITTER can evaluate the functional relatedness of susceptibility genes underlying schizophrenia and breast cancer, and provide clues about etiology. PMID:27623690

  10. Regulation of tissue-specific expression of SPATULA, a bHLH gene involved in carpel development, seedling germination, and lateral organ growth in Arabidopsis.

    PubMed

    Groszmann, Michael; Bylstra, Yasmin; Lampugnani, Edwin R; Smyth, David R

    2010-03-01

    SPATULA is a bHLH transcription factor that promotes growth of tissues arising from the carpel margins, including the septum and transmitting tract. It is also involved in repressing germination of newly harvested seeds, and in inhibiting cotyledon, leaf, and petal expansion. Using a reporter gene construct, its expression profile was fully defined. Consistent with its known functions, SPT was expressed in developing carpel margin tissues, and in the hypocotyls and cotyledons of germinating seedlings, and in developing leaves and petals. It was also strongly expressed in tissues where no functions have been identified to date, including the dehiscence zone of fruits, developing anthers, embryos, and in the epidermal initials and new stele of root tips. The promoter region of SPT was dissected by truncation and deletion, and two main regions occupied by tissue-specific enhancers were identified. These were correlated with eight regions conserved between promoter regions of Arabidopsis, Brassica oleracea, and Brassica rapa. When transformed into Arabidopsis, the B. oleracea promoter drove expression in reproductive tissues mostly comparable to the equivalent Arabidopsis promoter. There is genetic evidence that SPT function in the gynoecium is associated with the perception of auxin. However, site-directed mutagenesis of three putative auxin-response elements had no detectable effect on SPT expression patterns. Even so, disruption of a putative E-box variant adjacent to one of these resulted in a loss of valve dehiscence zone expression. This expression was also specifically lost in mutants of another bHLH gene INDEHISCENT, indicating that IND may directly regulate SPT expression through this variant E-box. PMID:20176890

  11. Regulation of tissue-specific expression of SPATULA, a bHLH gene involved in carpel development, seedling germination, and lateral organ growth in Arabidopsis

    PubMed Central

    Groszmann, Michael; Bylstra, Yasmin; Lampugnani, Edwin R.; Smyth, David R.

    2010-01-01

    SPATULA is a bHLH transcription factor that promotes growth of tissues arising from the carpel margins, including the septum and transmitting tract. It is also involved in repressing germination of newly harvested seeds, and in inhibiting cotyledon, leaf, and petal expansion. Using a reporter gene construct, its expression profile was fully defined. Consistent with its known functions, SPT was expressed in developing carpel margin tissues, and in the hypocotyls and cotyledons of germinating seedlings, and in developing leaves and petals. It was also strongly expressed in tissues where no functions have been identified to date, including the dehiscence zone of fruits, developing anthers, embryos, and in the epidermal initials and new stele of root tips. The promoter region of SPT was dissected by truncation and deletion, and two main regions occupied by tissue-specific enhancers were identified. These were correlated with eight regions conserved between promoter regions of Arabidopsis, Brassica oleracea, and Brassica rapa. When transformed into Arabidopsis, the B. oleracea promoter drove expression in reproductive tissues mostly comparable to the equivalent Arabidopsis promoter. There is genetic evidence that SPT function in the gynoecium is associated with the perception of auxin. However, site-directed mutagenesis of three putative auxin-response elements had no detectable effect on SPT expression patterns. Even so, disruption of a putative E-box variant adjacent to one of these resulted in a loss of valve dehiscence zone expression. This expression was also specifically lost in mutants of another bHLH gene INDEHISCENT, indicating that IND may directly regulate SPT expression through this variant E-box. PMID:20176890

  12. Both the constitutive Cauliflower Mosaic Virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of multiple enhancers and promoters within a single vector often provokes complicated mutual interaction and crosstalk, thereby, altering promoter specificity, which causes serious problems for precisely engineering gene function and agronomic traits in transgenic plants. Enhancer elem...

  13. The Suppressor of Hairy-Wing Protein Regulates the Tissue-Specific Expression of the Drosophila Gypsy Retrotransposon

    PubMed Central

    Smith, P. A.; Corces, V. G.

    1995-01-01

    The gypsy retrotransposon of Drosophila melanogaster causes mutations that show temporal and tissue-specific phenotypes. These mutant phenotypes can be reversed by mutations in su(Hw), a gene that also regulates the transcription of the gypsy element. Gypsy encodes a full-length 7.0-kb RNA that is expressed in the salivary gland precursors and fat body of the embryo, imaginal discs and fat body of larvae, and fat body and ovaries of adult females. The su(Hw)-binding region inserted upstream of the promoter of a lacZ reporter gene can induce β-galactosidase expression in a subset of the embryonic and larval tissues where gypsy is normally transcribed. This expression is dependent on the presence of a functional su(Hw) product, suggesting that this protein is a positive activator of gypsy transcription. Flies transformed with a construct in which the 5' LTR and leader sequences of gypsy are fused to lacZ show β-galactosidase expression in all tissues where gypsy is normally expressed, indicating that sequences other than the su(Hw)-binding site are required for proper spatial and temporal expression of gypsy. Mutations in the zinc fingers of su(Hw) affect its ability to bind DNA and to induce transcription of the lacZ reporter gene. Two other structural domains of su(Hw) also play an important role in transcriptional regulation of gypsy. Deletion of the amino-terminal acidic domain results in the loss of lacZ expression in larval fat body and adult ovaries, whereas mutations in the leucine zipper region result in an increase of lacZ expression in larval fat body and a decrease in adult ovaries. These effects might be the result of interactions of su(Hw) with activator and repressor proteins through the acidic and leucine zipper domains to produce the final pattern of tissue-specific expression of gypsy. PMID:7705625

  14. The Petal-Specific InMYB1 Promoter Functions by Recognizing Petaloid Cells.

    PubMed

    Azuma, Mirai; Mitsuda, Nobutaka; Goto, Koji; Oshima, Yoshimi; Ohme-Takagi, Masaru; Otagaki, Shungo; Matsumoto, Shogo; Shiratake, Katsuhiro

    2016-03-01

    The InMYB1 gene in Japanese morning glory (Ipomoea nil) is a member of the MYB transcription factor family. The promoter of InMYB1 has been reported to induce petal-specific gene expression in Arabidopsis and Eustoma, and has the same function in several other dicotyledonous plants. Most flowers consist of sepals, petals, stamens and a carpel, whose identity establishment is explained by the ABC model. The establishment of the identity of petals is determined by the expression of class A and B genes in whorl 2. The aim of this study was to clarify whether the InMYB1 promoter functions by recognizing whorl position or petal identity by examining its activity in various mutant and transgenic Arabidopsis thaliana plants in which genes related to the ABC model have been modified. In plants defective in class C gene function, the InMYB1 promoter functioned not only in petals generated in whorl 2 but also in petaloid organs generated in whorl 3; while in the plants defective in class B gene function, the InMYB1 promoter did not function in the sepaloid organs generated in whorl 2. Plants overexpressing class A, B and E genes set flowers with petaloid sepals in whorl 1, i.e. the lateral parts were white and looked like petals, while the central parts were green and looked like sepals. The InMYB1 promoter functioned in the lateral white parts but not in the central green parts. These results show that the InMYB1 promoter functions by recognizing petal identity at the cellular level rather than the whorl position. The petal-specific function of the InMYB1 promoter could be used as a marker to identify petaloid cells. PMID:26858281

  15. A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet

    PubMed Central

    Stahl, Dietmar J; Kloos, Dorothee U; Hehl, Reinhard

    2004-01-01

    Background Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. Results Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. Conclusions This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed. PMID:15579211

  16. Development of functionalized nanodiamond fluorescence detection platform: Analysis the specific promoter regulated by p53

    NASA Astrophysics Data System (ADS)

    Wu, Diansyue; Chu, Hsueh-Liang; Chuang, Hung; Lu, Yu-Ning; Ho, Li-Ping; Li, Hsing-Yuan; Hsu, Ming-Hua; Chang, Chia-Ching

    2014-03-01

    Nanodiamond (ND) is one of the biocompatible nanomaterials with large tunable surface for chemical modification. It possesses unique mechanical, spectroscopy, and thermal properties. It is an excellent molecular vehicle to deliver specific molecules in biological system. The green fluorescent protein (GFP) is a protein that emits strong green fluorescence when it is excited by ultra-violet to blue light. It makes GFP a good indicator. By combining ND-GFP, a visible biocompatible delivery system will be developed. p53 is a tumor suppressor protein encoded by the TP53 gene. P53 plays an important role in apoptosis, genomic stability, and inhibition of angiogenesis by interacting with specific DNA sequence of promoter of related genes. In this study, a p53 functionalized ND-GFP will be developed. This complex can recognize the specific DNA sequence of promoter and the intermolecular interactions can be monitored directly by fluorescence and Raman spectroscopy both in vivo and in vitro.

  17. Peptides from regenerating central nervous system promote specific populations of macroglia.

    PubMed Central

    Giulian, D; Tomozawa, Y; Hindman, H; Allen, R L

    1985-01-01

    The regenerating central nervous system of goldfish contains peptides referred to as glia-promoting factors (GPFs) that stimulate the proliferation of mammalian macroglia. We find that, in vitro, GPF1 and GPF3 promote the appearance of oligodendroglia and GPF2 and GPF4 stimulate proliferation of astroglia. The activities of GPF1, GPF3, and GPF4 increase during regeneration of the goldfish visual system. These results suggest that brain peptides may play a role in the recovery of the injured central nervous system by regulating the growth and development of specific macroglial populations. Images PMID:3858882

  18. Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator

    PubMed Central

    Severinov, Konstantin; Minakhin, Leonid; Sekine, Shun-ichi; Lopatina, Anna; Yokoyama, Shigeyuki

    2014-01-01

    Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue. PMID:25105059

  19. Artemisia extracts activate PPARγ, promote adipogenesis, and enhance insulin sensitivity in adipose tissue of obese mice

    PubMed Central

    Richard, Allison J.; Burris, Thomas P.; Sanchez-Infantes, David; Wang, Yongjun; Ribnicky, David M.; Stephens, Jacqueline M.

    2014-01-01

    Objective Studies have shown that the inability of adipose tissue to properly expand during the obese state or respond to insulin can lead to metabolic dysfunction. Artemisia is a diverse group of plants that has a history of medicinal use. This study examines the ability of ethanolic extracts of Artemisia scoparia (SCO) and Artemisia santolinifolia (SAN) to modulate adipocyte development in cultured adipocytes and white adipose tissue (WAT) function in vivo using a mouse model of diet-induced obesity. Research Design & Procedures Adipogenesis was assessed using Oil Red O staining and immunoblotting. A nuclear receptor specificity assay was used to examine the specificity of SCO- and SAN-induced PPARγ activation. C57BL/6J mice, fed a high-fat diet, were gavaged with saline, SCO, or SAN for 2 weeks. Whole-body insulin sensitivity was examined using insulin tolerance tests. WAT depots were assessed via immunoblotting for markers of insulin action and adipokine production. Results We established that SCO and SAN were highly specific activators of PPARγ and did not activate other nuclear receptors. After a one-week daily gavage, SCO- and SAN-treated mice had lower insulin-induced glucose disposal rates than control mice. At the end of the 2-week treatment period, SCO- and SAN-treated mice had enhanced insulin-responsive Akt serine-473 phosphorylation and significantly decreased MCP-1 levels in visceral WAT relative to control mice; these differences were depot specific. Moreover, plasma adiponectin levels were increased following SCO treatment. Conclusion Overall, these studies demonstrate that extracts from two Artemisia species can have metabolically favorable effects on adipocytes and WAT. PMID:24985103

  20. Effective delivery of a nematode-repellent peptide using a root-cap-specific promoter.

    PubMed

    Lilley, Catherine J; Wang, Dong; Atkinson, Howard J; Urwin, Peter E

    2011-02-01

    The potential of the MDK4-20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode-repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root-specific β-glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED-E5 established that the MDK4-20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode-repellent peptide under the control of either AtMDK4-20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4-20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4-20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4-20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst-nematode defence. PMID:20602721

  1. Spectral unmixing of multi-color tissue specific in vivo fluorescence in mice

    NASA Astrophysics Data System (ADS)

    Zacharakis, Giannis; Favicchio, Rosy; Garofalakis, Anikitos; Psycharakis, Stylianos; Mamalaki, Clio; Ripoll, Jorge

    2007-07-01

    Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo. The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for specific applications.

  2. Tissue Microbiome Profiling Identifies an Enrichment of Specific Enteric Bacteria in Opisthorchis viverrini Associated Cholangiocarcinoma.

    PubMed

    Chng, Kern Rei; Chan, Sock Hoai; Ng, Amanda Hui Qi; Li, Chenhao; Jusakul, Apinya; Bertrand, Denis; Wilm, Andreas; Choo, Su Pin; Tan, Damien Meng Yew; Lim, Kiat Hon; Soetinko, Roy; Ong, Choon Kiat; Duda, Dan G; Dima, Simona; Popescu, Irinel; Wongkham, Chaisiri; Feng, Zhu; Yeoh, Khay Guan; Teh, Bin Tean; Yongvanit, Puangrat; Wongkham, Sopit; Bhudhisawasdi, Vajaraphongsa; Khuntikeo, Narong; Tan, Patrick; Pairojkul, Chawalit; Ngeow, Joanne; Nagarajan, Niranjan

    2016-06-01

    Cholangiocarcinoma (CCA) is the primary cancer of the bile duct system. The role of bile duct tissue microbiomes in CCA tumorigenesis is unestablished. To address this, sixty primary CCA tumors and matched normals, from both liver fluke (Opisthorchis viverrini) associated (OVa, n=28) and non-O. viverrini associated (non-OVa, n=32) cancers, were profiled using high-throughput 16S rRNA sequencing. A distinct, tissue-specific microbiome dominated by the bacterial families Dietziaceae, Pseudomonadaceae and Oxalobacteraceae was observed in bile duct tissues. Systemic perturbation of the microbiome was noted in tumor and paired normal samples (vs non-cancer normals) for several bacterial families with a significant increase in Stenotrophomonas species distinguishing tumors vs paired normals. Comparison of parasite associated (OVa) vs non-associated (non-OVa) groups identified enrichment for specific enteric bacteria (Bifidobacteriaceae, Enterobacteriaceae and Enterococcaceae). One of the enriched families, Bifidobacteriaceae, was found to be dominant in the O. viverrini microbiome, providing a mechanistic link to the parasite. Functional analysis and comparison of CCA microbiomes revealed higher potential for producing bile acids and ammonia in OVa tissues, linking the altered microbiota to carcinogenesis. These results define how the unique microbial communities resident in the bile duct, parasitic infections and the tissue microenvironment can influence each other, and contribute to cancer. PMID:27428430

  3. Adipose tissue depot specific differences of PLIN protein content in endurance trained rats.

    PubMed

    Ramos, Sofhia V; Turnbull, Patrick C; MacPherson, Rebecca E K

    2016-01-01

    Adipose tissue is classified as either white (WAT) or brown (BAT) and differs not only by anatomical location but also in function. WAT is the main source of stored energy and releases fatty acids in times of energy demand, whereas BAT plays a role in regulating non-shivering thermogenesis and oxidizes fatty acids released from the lipid droplet. The PLIN family of proteins has recently emerged as being integral in the regulation of fatty acid storage and release in adipose tissue. Previous work has demonstrated that PLIN protein content varies among adipose tissue depots, however an examination of endurance training-induced depot specific changes in PLIN protein expression has yet to be done. Male Sprague-dawley rats (n = 10) underwent 8-weeks of progressive treadmill training (18-25 m/min for 30-60 min at 10% incline) or remained sedentary as control. Following training, under isoflurane induced anesthesia epidydmal (eWAT), inguinal subcutaneous (iWAT) and intrascapular brown adipose tissue (BAT) was excised, and plasma was collected. Endurance training resulted in an increase in BAT PLIN5 and iWAT PLIN3 content, while there was no difference in PLIN protein content in endurance trained eWAT. Interestingly, endurance training resulted in a robust increase in ATGL and CGI-58 in eWAT alone. Together these results suggest the potential of a depot specific function of PLIN3 and PLIN5 in adipose tissue in response to endurance training. PMID:27386161

  4. Interference with virus and bacteria replication by the tissue specific expression of antibodies and interfering molecules.

    PubMed

    Enjuanes, L; Sola, I; Izeta, A; Sánchez-Morgado, J M; González, J M; Alonso, S; Escors, D; Sánchez, C M

    1999-01-01

    into mucosal areas either antibodies to provide immediate protection, or antigens to elicit immune responses in the enteric or respiratory surfaces in order to prevent virus infection. One strategy is based on the development of expression vectors using coronavirus derived defective RNA minigenomes, and the other relies on the development of transgenic animals providing virus neutralizing antibodies in the milk during lactation. Two types of expression vectors are being engineered based on transmissible gastroenteritis coronavirus (TGEV) defective minigenomes. The first one is a helper virus dependent expression system and the second is based on self-replicating RNAs including the information required to encode the TGEV replicase. The minigenomes expressing the heterologous gene have been improved by using a two-step amplification system based on cytomegalovirus (CMV) and viral promoters. Expression levels around 5 micrograms per 10(6) cells were obtained. The engineered minigenomes will be useful to understand the mechanism of coronavirus replication and for the tissue specific expression of antigen, antibody or virus interfering molecules. To protect from viral infections of the enteric tract, transgenic animals secreting virus neutralizing recombinant antibodies in the milk during lactation have been developed. Neutralizing antibodies with isotypes IgG1 or IgA were produced in the milk with titers of 10(6) in RIA that reduced virus infectivity by one million-fold. The recombinant antibodies recognized a conserved epitope apparently essential for virus replication. Antibody expression levels were transgene transgene copy number independent and were related to the transgene integration site. This strategy may be of general use since it could be applied to protect newborn animals against infections of the enteric tract by viruses or bacteria for which a protective MAb has been identified. Alternatively, the same strategy could be used to target the expression of

  5. Tissue-specific responses to the LRPPRC founder mutation in French Canadian Leigh Syndrome

    PubMed Central

    Sasarman, Florin; Nishimura, Tamiko; Antonicka, Hana; Weraarpachai, Woranontee; Shoubridge, Eric A.; Allen, Bruce; Burelle, Yan; Charron, Guy; Coderre, Lise; DesRosiers, Christine; Laprise, Catherine; Morin, Charles; Rioux, John; Shoubridge, Eric A.

    2015-01-01

    French Canadian Leigh Syndrome (LSFC) is an early-onset, progressive neurodegenerative disorder with a distinct pattern of tissue involvement. Most cases are caused by a founder missense mutation in LRPPRC. LRPPRC forms a ribonucleoprotein complex with SLIRP, another RNA-binding protein, and this stabilizes polyadenylated mitochondrial mRNAs. LSFC fibroblasts have reduced levels of LRPPRC and a specific complex IV assembly defect; however, further depletion of mutant LRPPRC results in a complete failure to assemble a functional oxidative phosphorylation system, suggesting that LRPPRC levels determine the nature of the biochemical phenotype. We tested this hypothesis in cultured muscle cells and tissues from LSFC patients. LRPPRC levels were reduced in LSFC muscle cells, resulting in combined complex I and IV deficiencies. A similar combined deficiency was observed in skeletal muscle. Complex IV was only moderately reduced in LSFC heart, but was almost undetectable in liver. Both of these tissues showed elevated levels of complexes I and III. Despite the marked biochemical differences, the steady-state levels of LRPPRC and mitochondrial mRNAs were extremely low, LRPPRC was largely detergent-insoluble, and SLIRP was undetectable in all LSFC tissues. The level of the LRPPRC/SLIRP complex appeared much reduced in control tissues by the first dimension blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis compared with fibroblasts, and even by second dimension analysis it was virtually undetectable in control heart. These results point to tissue-specific pathways for the post-transcriptional handling of mitochondrial mRNAs and suggest that the biochemical defects in LSFC reflect the differential ability of tissues to adapt to the mutation. PMID:25214534

  6. Tissue-specific expression of human arylsulfatase-C isozymes and steroid sulfatase.

    PubMed Central

    Munroe, D G; Chang, P L

    1987-01-01

    Steroid sulfatase (STS; E.C.3.1.6.2), which acts on 3-hydroxysteroid sulfates, and arylsulfatase-C (ARC; E.C.3.1.6.1), assayed with aromatic artificial substrates, are both membrane-bound, microsomal enzymes with alkaline pH optima. Although they copurify during preparation and their gene loci are mapped to the short arm of the human X chromosome where they appear to have escaped from X inactivation, it has not been settled whether STS and ARC are the same enzyme or not. Recent work from our laboratory has shown that ARC exists in two electrophoretically distinct forms in human fibroblasts. We now report that these two forms--the faster migrating (F) and more slowly migrating (S)--occur in human tissues. Each of 11 human tissue types from 10 subjects showed a consistent pattern of ARC isozymes. Thyroid, heart, spleen, skeletal muscle, and adrenal tissue mainly had the S form. In contrast, kidney, liver, and pancreas tissue had mainly the F form, while gonadal, lung, and intestinal tissue had both the S and the F forms. The question of escape of their gene locus from X-chromosome inactivation was examined by comparing the specific activities of ARC and STS in male-derived vis-à-vis female-derived tissues. The majority of the tissues did not show any significant difference in these activities between the sexes, the exceptions being heart muscle, gonadal, and kidney tissue. None showed the 1:2 ratio between male- and female-derived tissues expected of a locus that had escaped X inactivation. The question of identity between ARC and STS was examined by comparing the ratios of their activities in these tissue types: if the enzymes were identical, the ratios of their activities should have remained constant across the different tissue types. It was thus shown that ARC activity varied by as much as 100-fold, depending on the ARC isozymic pattern of the tissue. STS, measured as estrone sulfatase and dehydroepiandrosterone sulfatase, did not show similar variations. This

  7. Light-regulated, tissue-specific immunophilins in a higher plant.

    PubMed

    Luan, S; Albers, M W; Schreiber, S L

    1994-02-01

    In addition to their application in organ transplantation, immunosuppressive drugs are valuable tools for studying signal transduction in eukaryotic cells. Using affinity chromatography, we have purified immunosuppressive drug receptors (immunophilins) from fava bean. Proteins belonging to both major classes of the immunophilin family identified from animal sources [FK506- and rapamycin-binding proteins (FKBPs) and cyclophilins] were present in this higher plant. FKBP13, the most abundant FKBP family member in leaf tissues, was not detected in root tissues, whereas other FKBPs were present in both tissues. While the abundance of cyclophilin A in leaves was similar to that in roots, cyclophilin B/C was expressed at a much higher level in leaf tissues than in root tissues. Subcellular localization of immunophilins in mesophyll cells showed that chloroplasts contained FKBP13 and cyclophilin B/C but not other members, which explains the preferential expression of these two proteins in leaves over roots. The abundance of chloroplast-localized immunophilins, FKBP13 and cyclophilin B/C, was regulated by light. Although etiolated leaves produced detectable levels of cyclophilin B/C, they did not express FKBP13. Illumination of etiolated plants dramatically increased the expression of both FKBP13 and cyclophilin B/C. The light-induced expression of FKBP13 is closely correlated with the accumulation of chlorophyll in the leaf tissue. Our findings suggest that FKBP13 and cyclophilin B/C may play a specific role in chloroplasts. PMID:7508125

  8. Tissue specific characterisation of Lim-kinase 1 expression during mouse embryogenesis.

    PubMed

    Lindström, Nils O; Neves, Carlos; McIntosh, Rebecca; Miedzybrodzka, Zosia; Vargesson, Neil; Collinson, J Martin

    2011-01-01

    The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms' tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue-tissue interactions guiding multiple developmental processes. PMID:21167960

  9. Tissue-Specific Evolution of Protein Coding Genes in Human and Mouse

    PubMed Central

    Kryuchkova-Mostacci, Nadezda; Robinson-Rechavi, Marc

    2015-01-01

    Protein-coding genes evolve at different rates, and the influence of different parameters, from gene size to expression level, has been extensively studied. While in yeast gene expression level is the major causal factor of gene evolutionary rate, the situation is more complex in animals. Here we investigate these relations further, especially taking in account gene expression in different organs as well as indirect correlations between parameters. We used RNA-seq data from two large datasets, covering 22 mouse tissues and 27 human tissues. Over all tissues, evolutionary rate only correlates weakly with levels and breadth of expression. The strongest explanatory factors of purifying selection are GC content, expression in many developmental stages, and expression in brain tissues. While the main component of evolutionary rate is purifying selection, we also find tissue-specific patterns for sites under neutral evolution and for positive selection. We observe fast evolution of genes expressed in testis, but also in other tissues, notably liver, which are explained by weak purifying selection rather than by positive selection. PMID:26121354

  10. Cell-Type-Specific Genome-wide Expression Profiling after Laser Capture Microdissection of Living Tissue

    SciTech Connect

    Marchetti, F; Manohar, C F

    2005-02-09

    The purpose of this technical feasibility study was to develop and evaluate robust microgenomic tools for investigations of genome-wide expression of very small numbers of cells isolated from whole tissue sections. Tissues contain large numbers of cell-types that play varied roles in organ function and responses to endogenous and exogenous toxicants whether bacterial, viral, chemical or radiation. Expression studies of whole tissue biopsy are severely limited because heterogeneous cell-types result in an averaging of molecular signals masking subtle but important changes in gene expression in any one cell type(s) or group of cells. Accurate gene expression analysis requires the study of specific cell types in their tissue environment but without contamination from surrounding cells. Laser capture microdissection (LCM) is a new technology to isolate morphologically distinct cells from tissue sections. Alternative methods are available for isolating single cells but not yet for their reliable genome-wide expression analyses. The tasks of this feasibility project were to: (1) Develop efficient protocols for laser capture microdissection of cells from tissues identified by antibody label, or morphological stain. (2) Develop reproducible gene-transcript analyses techniques for single cell-types and determine the numbers of cells needed for reliable genome-wide analyses. (3) Validate the technology for epithelial and endothelial cells isolated from the gastrointestinal tract of mice.

  11. Pleiotropic Effects and Compensation Mechanisms Determine Tissue Specificity in Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

    PubMed Central

    Bykhovskaya, Yelena; Mengesha, Emebet; Fischel-Ghodsian, Nathan

    2007-01-01

    The tissue specificity of mitochondrial diseases is poorly understood. Recently, tissue-specific quantitative differences of the components of the mitochondrial translation system have been found to correlate with disease presentation in fatal hepatopathy caused by mutations in mitochondrial translation factor EFG1. MLASA is an autosomal recessive inherited progressive oxidative phosphorylation disorder that affects muscle and erythroid cells. The disease is caused by the homozygous point mutation C656T (R116W) in the catalytic domain of the pseudouridylate synthase 1 (PUS1) gene, which leads to a complete lack of pseudouridylation at the expected sites in mitochondrial and cytoplasmic tRNAs. Despite the presence of these altered tRNAs, most tissues are unaffected, and even in muscle and erythroid cells the disease phenotype only slowly emerges over the course of years. In order to elucidate intracellular pathways through which the homozygous mutation leads to tissue-restricted phenotype, we performed microarray expression analysis of EBV-transformed lymphoblasts from MLASA patients, heterozygous parents, and controls using human Beadchip microarray with 47,296 transcripts. Genes coding for proteins involved in DNA transcription and its regulation, and metal binding proteins, demonstrated major differences in expression between patients and all other individuals with normal phenotype. Genes coding for ribosomal proteins differed significantly between individual with at least one copy of the mutated PUS1 gene and controls. These findings indicate that the lack of tRNA pseudouridylation can be overcome by compensatory changes in levels of ribosomal proteins, and that the disease phenotype in affected tissues is likely due to pleiotropic effects of PUS1p on non-tRNA molecules involved in DNA transcription and iron metabolism. Similar combinations of mechanisms may play a role in the tissue specificity of other mitochondrial disorders. PMID:17374500

  12. miTALOS v2: Analyzing Tissue Specific microRNA Function.

    PubMed

    Preusse, Martin; Theis, Fabian J; Mueller, Nikola S

    2016-01-01

    MicroRNAs are involved in almost all biological processes and have emerged as regulators of signaling pathways. We show that miRNA target genes and pathway genes are not uniformly expressed across human tissues. To capture tissue specific effects, we developed a novel methodology for tissue specific pathway analysis of miRNAs. We incorporated the most recent and highest quality miRNA targeting data (TargetScan and StarBase), RNA-seq based gene expression data (EBI Expression Atlas) and multiple new pathway data sources to increase the biological relevance of the predicted miRNA-pathway associations. We identified new potential roles of miR-199a-3p, miR-199b-3p and the miR-200 family in hepatocellular carcinoma, involving the regulation of metastasis through MAPK and Wnt signaling. Also, an association of miR-571 and Notch signaling in liver fibrosis was proposed. To facilitate data update and future extensions of our tool, we developed a flexible database backend using the graph database neo4j. The new backend as well as the novel methodology were included in the updated miTALOS v2, a tool that provides insights into tissue specific miRNA regulation of biological pathways. miTALOS v2 is available at http://mips.helmholtz-muenchen.de/mitalos. PMID:26998997

  13. Automated tissue classification of pediatric brains from magnetic resonance images using age-specific atlases

    NASA Astrophysics Data System (ADS)

    Metzger, Andrew; Benavides, Amanda; Nopoulos, Peg; Magnotta, Vincent

    2016-03-01

    The goal of this project was to develop two age appropriate atlases (neonatal and one year old) that account for the rapid growth and maturational changes that occur during early development. Tissue maps from this age group were initially created by manually correcting the resulting tissue maps after applying an expectation maximization (EM) algorithm and an adult atlas to pediatric subjects. The EM algorithm classified each voxel into one of ten possible tissue types including several subcortical structures. This was followed by a novel level set segmentation designed to improve differentiation between distal cortical gray matter and white matter. To minimize the req uired manual corrections, the adult atlas was registered to the pediatric scans using high -dimensional, symmetric image normalization (SyN) registration. The subject images were then mapped to an age specific atlas space, again using SyN registration, and the resulting transformation applied to the manually corrected tissue maps. The individual maps were averaged in the age specific atlas space and blurred to generate the age appropriate anatomical priors. The resulting anatomical priors were then used by the EM algorithm to re-segment the initial training set as well as an independent testing set. The results from the adult and age-specific anatomical priors were compared to the manually corrected results. The age appropriate atlas provided superior results as compared to the adult atlas. The image analysis pipeline used in this work was built using the open source software package BRAINSTools.

  14. miTALOS v2: Analyzing Tissue Specific microRNA Function

    PubMed Central

    Preusse, Martin; Theis, Fabian J.; Mueller, Nikola S.

    2016-01-01

    MicroRNAs are involved in almost all biological processes and have emerged as regulators of signaling pathways. We show that miRNA target genes and pathway genes are not uniformly expressed across human tissues. To capture tissue specific effects, we developed a novel methodology for tissue specific pathway analysis of miRNAs. We incorporated the most recent and highest quality miRNA targeting data (TargetScan and StarBase), RNA-seq based gene expression data (EBI Expression Atlas) and multiple new pathway data sources to increase the biological relevance of the predicted miRNA-pathway associations. We identified new potential roles of miR-199a-3p, miR-199b-3p and the miR-200 family in hepatocellular carcinoma, involving the regulation of metastasis through MAPK and Wnt signaling. Also, an association of miR-571 and Notch signaling in liver fibrosis was proposed. To facilitate data update and future extensions of our tool, we developed a flexible database backend using the graph database neo4j. The new backend as well as the novel methodology were included in the updated miTALOS v2, a tool that provides insights into tissue specific miRNA regulation of biological pathways. miTALOS v2 is available at http://mips.helmholtz-muenchen.de/mitalos. PMID:26998997

  15. Shade-induced stem elongation in rice seedlings: Implication of tissue-specific phytohormone regulation.

    PubMed

    Liu, Huihui; Yang, Chuanwei; Li, Lin

    2016-07-01

    A better understanding of shade avoidance syndrome (SAS) is an urgent need because of its effect on energy reallocation. Leverage-related mechanism in crops is of potential economic interest for agricultural applications. Here we report the SAS phenotype at tissue level rice seedlings. Tissue-specific RNA-sequencing indicates auxin plays different roles between coleoptile and the first leaf. Phenotypes of wild type treated by gibberellin and brassinosteroid biosynthesis inhibitors and of related mutants suggest these two hormones positively regulate SAS. Our work reveals the diversity of hormone responses in different organs and different species in shade conditions. PMID:26888633

  16. Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion*

    PubMed Central

    Ramezani-Moghadam, Mehdi; Wang, Jianhua; Ho, Vikki; Iseli, Tristan J.; Alzahrani, Badr; Xu, Aimin; Van der Poorten, David; Qiao, Liang; George, Jacob; Hebbard, Lionel

    2015-01-01

    Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin. PMID:25575598

  17. The vesicular glutamate transporter-1 upstream promoter and first intron each support glutamatergic-specific expression in rat postrhinal cortex

    PubMed Central

    Zhang, Guo-rong; Li, Xu; Cao, Haiyan; Zhao, Hua; Geller, Alfred I.

    2011-01-01

    Multiple applications of direct gene transfer into neurons require restricting expression to glutamatergic neurons, or specific subclasses of glutamatergic neurons. Thus, it is desirable to develop and analyze promoters that support glutamatergic-specific expression. The three vesicular glutamate transporters (VGLUTs) are found in different populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported on a plasmid (amplicon) Herpes Simplex Virus vector that contains a VGLUT1 promoter. This vector supports long-term expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex, but does not support expression in VGLUT2-containing glutamatergic neurons in the ventral medial hypothalamus. This VGLUT1 promoter contains both the VGLUT1 upstream promoter and the VGLUT1 first intron. In this study, we begin to isolate and analyze the glutamatergic-specific regulatory elements in this VGLUT1 promoter. We show that the VGLUT1 upstream promoter and first intron each support glutamatergic-specific expression. We isolated a small, basal VGLUT1 promoter that does not support glutamatergic-specific expression. Next, we fused either the VGLUT1 upstream promoter or the first intron to this basal promoter. The VGLUT1 upstream promoter or the first intron, fused to the basal promoter, each supported glutamatergic-specific expression in POR cortex. PMID:21172319

  18. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

    PubMed Central

    Amin, Nirav M.; Greco, Todd M.; Kuchenbrod, Lauren M.; Rigney, Maggie M.; Chung, Mei-I; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

    2014-01-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  19. Evidence for frequent and tissue-specific sequence heteroplasmy in human mitochondrial DNA.

    PubMed

    Naue, Jana; Hörer, Steffen; Sänger, Timo; Strobl, Christina; Hatzer-Grubwieser, Petra; Parson, Walther; Lutz-Bonengel, Sabine

    2015-01-01

    Mitochondrial point heteroplasmy is a common event observed not only in patients with mitochondrial diseases but also in healthy individuals. We here report a comprehensive investigation of heteroplasmy occurrence in human including the whole mitochondrial control region from nine different tissue types of 100 individuals. Sanger sequencing was used as a standard method and results were supported by cloning, minisequencing, and massively parallel sequencing. Only 12% of all individuals showed no heteroplasmy, whereas 88% showed at least one heteroplasmic position within the investigated tissues. In 66% of individuals up to 8 positions were affected. The highest relative number of heteroplasmies was detected in muscle and liver (79%, 69%), followed by brain, hair, and heart (36.7%-30.2%). Lower percentages were observed in bone, blood, lung, and buccal cells (19.8%-16.2%). Accumulation of position-specific heteroplasmies was found in muscle (positions 64, 72, 73, 189, and 408), liver (position 72) and brain (partial deletion at position 71). Deeper analysis of these specific positions in muscle revealed a non-random appearance and position-specific dependency on age. MtDNA heteroplasmy frequency and its potential functional importance have been underestimated in the past and its occurrence is ubiquitous and dependent at least on age, tissue, and position-specific mutation rates. PMID:25526677

  20. Tissue specificity and variability of imprinted IGF2 expression in humans

    SciTech Connect

    Giannoukakis, N.; Rouleau, G.; Polychronakos, C.

    1994-09-01

    Parental genomic imprinting refers to the phenomenon where expression of a gene copy depends on the sex of the parent from which it is derived. The human insulin-like growth factor II gene, IGF2, is parentally imprinted with the paternal gene copy exclusively expressed in fetal and term placenta as well as in fetal kidney. In mice, imprinted IGF2 expression is tissue-specific. In a preliminary approach to investigate tissue-specific IGF2 imprinting in humans, we evaluated allele-specific expression in four samples of umbilical cord blood leukocytes of fetuses found to imprint IGF2 in placenta. IGF2 mRNA transcripts from the gene copy transmitted from each parent were distinguished using a transcribed ApaI polymorphism by performing reverse transcription-PCR on total RNA from cord blood leukocytes. Postnatal peripheral blood was examined using the same method. Of 77 informative individuals, 68 expressed both IGF2 copies, but 9 individuals showed unambiguous monoallelic expression. Two individuals from each category were screened again and the results were identical. These data indicate that imprinted IGF2 expression is tissue-specific and show variability of IGF2 imprinting among individuals. This variability may be genetic. We are in the process of screening large pedigrees to test this hypothesis.

  1. Specific repression of β-globin promoter activity by nuclear ferritin

    PubMed Central

    Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

    2001-01-01

    Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

  2. Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Kanofsky, Konstantin; Hanko, Erik K R; Ahrendt, Sarah; Wehrs, Maren; Machens, Fabian; Hehl, Reinhard

    2016-01-01

    Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA. PMID:25819608

  3. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    SciTech Connect

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn

    2013-08-23

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  4. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples

    USGS Publications Warehouse

    Gorelick, Daniel A.; Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki; Halpern, Marnie E.

    2014-01-01

    Background: Environmental endocrine disruptors (EED) are exogenous chemicals that mimic endogenous hormones, such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ER) in the larval heart compared to the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit similar tissue-specific effects as BPA and genistein or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of estrogen receptor genes by RNA in situ hybridization. Results: Selective patterns of ER activation were observed in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue-specificity in ER activation is due to differences in the expression of estrogen receptor subtypes. ERα is expressed in developing heart valves but not in the liver, whereas ERβ2 has the opposite profile. Accordingly, subtype-specific ER agonists activate the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish has revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero is associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.

  5. Extraction, Identification, and Quantification of Histones from Small Quantities of Specific Brain Tissue.

    PubMed

    Beldjoud, Hassiba; Messanvi, Fany; Nadif Kasri, Nael; Roozendaal, Benno

    2016-01-01

    Histone posttranslational modifications (PTMs), by their action on the chromatin state, play a central role in the regulation of gene expression. The discovery that some PTMs in the brain are dynamically regulated by experience and environmental factors makes them an important subject for the study of plasticity changes in learning and memory, addiction, and psychiatric disorders. Current histone isolation protocols, however, require large amounts of tissue, which limits their application for analyzing small tissue samples from a specific brain region. We describe here a step-by-step protocol for histone extraction and isolation from 1 mm(3) of tissue from brain punches, which allows reproducible and reliable results for histone PTM identification and quantification without losing anatomical precision. © 2016 by John Wiley & Sons, Inc. PMID:27367963

  6. Tissue-specific regulation of porcine prolactin receptor expression by estrogen, progesterone, and prolactin.

    PubMed

    Trott, Josephine F; Horigan, Katherine C; Gloviczki, Julia M; Costa, Kristen M; Freking, Bradley A; Farmer, Chantal; Hayashi, Kanako; Spencer, Thomas; Morabito, Joseph E; Hovey, Russell C

    2009-07-01

    Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue- and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. Abundance of pPRLR-long form (LF) mRNA increased in the mammary gland and endometrium during gestation while in other tissues it remained constant. There was a parallel increase in the abundance of the pPRLR-LF protein in the mammary gland and endometrium during gestation. We determined the hormonal regulation of pPRLR-LF mRNA expression in various tissues from ovariectomized, hypoprolactinemic gilts given combinations of the replacement hormones estrogen (E(2)), progestin (P), and/or haloperidol-induced PRL. Abundance of pPRLR-LF mRNA in kidney and liver was unaffected by hormone treatments. Expression of uterine pPRLR-LF mRNA was induced by E(2) whereas the effect of E(2) was abolished by co-administering P. The expression of pPRLR-LF mRNA in the mammary gland stroma was induced by PRL, whereas E(2) induced its expression in the epithelium. In contrast to these changes in pPRLR expression, pPRL expression was relatively constant and low during gestation in all tissues except the pituitary. Taken together, these data reveal that specific combinations of E(2), P, and PRL differentially regulate pPRLR-LF expression in the endometrium and mammary glands, and that the action of PRL on its target tissues is dependent upon pPRLR-LF abundance more so than the local PRL expression. PMID:19401343

  7. Tissue-specific accumulation of hepatic zinc metallothionein following parenteral iron loading

    SciTech Connect

    McCormick, C.C.

    1984-05-01

    The synthesis in various tissues of the unique metal-binding protein, metallothionein, can be influenced by the administration of certain trace elements. Zinc and cadmium, both of which bind to metallothionein, are most widely recognized as potent inducers. Preliminary results in our laboratory suggested that iron loading causes a marked accumulation of hepatic zinc metallothionein. In this report the effects of parenteral iron administration on metallothionein concentration in various tissues are presented. Male chicks (300-350 g) received (ip) either a single injection (+1 Fe) of iron (10 mg Fe/kg, as FeCl/sub 3/), two injections (+2 Fe) given 24-hr apart, three injections (+3 Fe) each given 24-hr apart, or an equivalent volume of 0.9% saline (control). Twenty-four hours following the final injection, chicks were killed and tissues analyzed for cytoplasmic zinc and metallothionein (Zn-MT). The parenteral administration of ferric iron, FeCl/sub 3/, resulted in a marked tissue-specific accumulation of zinc as metallothionein. In chicks given +2 Fe, hepatic Zn-MT increased more than 10-fold with a third injection (+3 Fe) causing no further change. The concentration of Zn-MT in renal and pancreatic tissue was unaffected by iron loading. An increase in hepatic Zn-MT was evident prior to detectable changes in total hepatic iron. The administration of other ferrous iron compounds at a similar rate produced comparable changes in hepatic Zn-MT. Feeding excess dietary iron, however, had no effect on liver Zn-MT levels even though similar hepatic iron concentrations were attained. Results indicated that parenteral administration, but not feeding, of various iron compounds causes a marked increase in zinc metallothionein, specifically in liver tissue.

  8. Analysis of tissue specific progenitor cell differentiation using FT-IR

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Kimura, Akinori; Kushibiki, Toshihiro; Awazu, Kunio

    2007-07-01

    Tissue specific progenitor cells and its differentiations have got a lot of attentions in regenerative medicine. The process of differentiations, the formation of tissues, has become better understood by the study using a lot of cell types progressively. These studies of cells and tissue dynamics at molecular levels are carried out through various approaches like histochemical methods, application of molecular biology and immunology. However, in case of using regenerative sources (cells, tissues and biomaterials etc.) clinically, they are measured and quality-controlled by non-contact and non-destructive methods from the view point of safety. Or the analysis with small quantities of materials could be possible if the quantities of materials are acceptable. A non-contact and non-destructive quality control method has been required. Recently, the use of Fourier Transform Infrared spectroscopy (FT-IR) has been used to monitor biochemical changes in cells, and has gained considerable importance. The changes in the cells and tissues, which are subtle and often not obvious in the histpathological studies, are shown to be well resolved using FT-IR. Moreover, although most techniques designed to detect one or a few changes, FT-IR is possible to identify the changes in the levels of various cellular biochemicals simultaneously under in vivo and in vitro conditions. The objective of this study is to establish the infrared spectroscopy of tissue specific progenitor cell differentiations as a quality control of cell sources for regenerative medicine. In the present study, as a basic study, we examine the adipose differentiation kinetics of preadipose cells (3T3-L1) and the osteoblast differentiation kinetics of mesenchymal stem cells (Kusa-A1) to analyze the infrared absorption spectra.

  9. Hypoxia-inducible factor prolyl hydroxylase 1 (PHD1) deficiency promotes hepatic steatosis and liver-specific insulin resistance in mice.

    PubMed

    Thomas, Amandine; Belaidi, Elise; Aron-Wisnewsky, Judith; van der Zon, Gerard C; Levy, Patrick; Clement, Karine; Pepin, Jean-Louis; Godin-Ribuot, Diane; Guigas, Bruno

    2016-01-01

    Obesity is associated with local tissue hypoxia and elevated hypoxia-inducible factor 1 alpha (HIF-1α) in metabolic tissues. Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. In the present study, we investigated the consequence of whole-body PHD1 gene (Egln2) inactivation on metabolic homeostasis in mice. At baseline, PHD1-/- mice exhibited higher white adipose tissue (WAT) mass, despite lower body weight, and impaired insulin sensitivity and glucose tolerance when compared to age-matched wild-type (WT) mice. When fed a synthetic low-fat diet, PHD1-/- mice also exhibit a higher body weight gain and WAT mass along with glucose intolerance and systemic insulin resistance compared to WT mice. PHD1 deficiency led to increase in glycolytic gene expression, lipogenic proteins ACC and FAS, hepatic steatosis and liver-specific insulin resistance. Furthermore, gene markers of inflammation were also increased in the liver, but not in WAT or skeletal muscle, of PHD1-/- mice. As expected, high-fat diet (HFD) promoted obesity, hepatic steatosis, tissue-specific inflammation and systemic insulin resistance in WT mice but these diet-induced metabolic alterations were not exacerbated in PHD1-/- mice. In conclusion, PHD1 deficiency promotes hepatic steatosis and liver-specific insulin resistance but does not worsen the deleterious effects of HFD on metabolic homeostasis. PMID:27094951

  10. Hypoxia-inducible factor prolyl hydroxylase 1 (PHD1) deficiency promotes hepatic steatosis and liver-specific insulin resistance in mice

    PubMed Central

    Thomas, Amandine; Belaidi, Elise; Aron-Wisnewsky, Judith; van der Zon, Gerard C.; Levy, Patrick; Clement, Karine; Pepin, Jean-Louis; Godin-Ribuot, Diane; Guigas, Bruno

    2016-01-01

    Obesity is associated with local tissue hypoxia and elevated hypoxia-inducible factor 1 alpha (HIF-1α) in metabolic tissues. Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. In the present study, we investigated the consequence of whole-body PHD1 gene (Egln2) inactivation on metabolic homeostasis in mice. At baseline, PHD1−/− mice exhibited higher white adipose tissue (WAT) mass, despite lower body weight, and impaired insulin sensitivity and glucose tolerance when compared to age-matched wild-type (WT) mice. When fed a synthetic low-fat diet, PHD1−/− mice also exhibit a higher body weight gain and WAT mass along with glucose intolerance and systemic insulin resistance compared to WT mice. PHD1 deficiency led to increase in glycolytic gene expression, lipogenic proteins ACC and FAS, hepatic steatosis and liver-specific insulin resistance. Furthermore, gene markers of inflammation were also increased in the liver, but not in WAT or skeletal muscle, of PHD1−/− mice. As expected, high-fat diet (HFD) promoted obesity, hepatic steatosis, tissue-specific inflammation and systemic insulin resistance in WT mice but these diet-induced metabolic alterations were not exacerbated in PHD1−/− mice. In conclusion, PHD1 deficiency promotes hepatic steatosis and liver-specific insulin resistance but does not worsen the deleterious effects of HFD on metabolic homeostasis. PMID:27094951

  11. Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.

    PubMed

    Krahn, Katy Nash; Bouten, Carlijn V C; van Tuijl, Sjoerd; van Zandvoort, Marc A M J; Merkx, Maarten

    2006-03-15

    Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods. PMID:16476406

  12. Denovo assembly and characterization of tissue-specific transcriptome in the endangered golden mahseer, Tor putitora

    PubMed Central

    Barat, Ashoktaru; Kumar, Rohit; Goel, Chirag; Singh, Atul Kumar; Sahoo, Prabhati Kumari

    2015-01-01

    The golden mahseer (Tor putitora) graces most of the Himalayan Rivers of India and neighboring South Asian countries. Despite its several importance as a research model, as food, and in sport fishing, knowledge on transcriptome database is nil. Therefore, it was targeted to develop reference transcriptome databases of the species using next-generation sequencing. In the present study, 100,540,130 high-quality paired-end reads were obtained from six cDNA libraries of spleen, liver, gill, kidney, muscle, and brain with 28.4 GB data using Illumina paired-end sequencing technology. Tissue-specific transcriptomes as well as complete transcriptome assembly were analyzed for concise representation of the study. In brief, the de novo assembly of individual tissue resulted in an average of 31,829 (18,512–46,348) contigs per sample, while combined transcriptome comprised 77,907 unique transcript fragments (unigenes) assembled from reads of six tissues. Approximately 75,407 (96.8%) unigenes could be annotated according to their homology matches in the nr, SwisseProt, GO, or KEGG databases. Comparative analysis showed that 84% of the unigenes have significant similarity to zebra fish RefSeq proteins. Tissue-specific-dominated genes were also identified to hypothesize their localization and expression in individual tissue. In addition, 2485 simple sequence repeats (SSRs) were detected from 77,907 transcripts in the combined transcriptome of the golden mahseer. This study has generated organ-specific transcriptome profiles, which will be helpful to understand the local adaptation, genome evolution, and also future functional studies on immune system of the golden mahseer. PMID:26702399

  13. Profile analysis and prediction of tissue-specific CpG island methylation classes

    PubMed Central

    2009-01-01

    Background The computational prediction of DNA methylation has become an important topic in the recent years due to its role in the epigenetic control of normal and cancer-related processes. While previous prediction approaches focused merely on differences between methylated and unmethylated DNA sequences, recent experimental results have shown the presence of much more complex patterns of methylation across tissues and time in the human genome. These patterns are only partially described by a binary model of DNA methylation. In this work we propose a novel approach, based on profile analysis of tissue-specific methylation that uncovers significant differences in the sequences of CpG islands (CGIs) that predispose them to a tissue- specific methylation pattern. Results We defined CGI methylation profiles that separate not only between constitutively methylated and unmethylated CGIs, but also identify CGIs showing a differential degree of methylation across tissues and cell-types or a lack of methylation exclusively in sperm. These profiles are clearly distinguished by a number of CGI attributes including their evolutionary conservation, their significance, as well as the evolutionary evidence of prior methylation. Additionally, we assess profile functionality with respect to the different compartments of protein coding genes and their possible use in the prediction of DNA methylation. Conclusion Our approach provides new insights into the biological features that determine if a CGI has a functional role in the epigenetic control of gene expression and the features associated with CGI methylation susceptibility. Moreover, we show that the ability to predict CGI methylation is based primarily on the quality of the biological information used and the relationships uncovered between different sources of knowledge. The strategy presented here is able to predict, besides the constitutively methylated and unmethylated classes, two more tissue specific methylation classes

  14. An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

    PubMed Central

    Slack, J L; Liska, D J; Bornstein, P

    1991-01-01

    Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2005897

  15. Identification of a 5-Methylcytosine Site that may Regulate C/EBPβ Binding and Determine Tissue-Specific Expression of the BPI Gene in Piglets

    PubMed Central

    Sun, Li; Wang, Jing; Yin, Xuemei; Sun, Shouyong; Zi, Chen; Zhu, Guoqiang; Wu, Shenglong; Bao, Wenbin

    2016-01-01

    Bactericidal/permeability-increasing protein (BPI) plays an important role in innate immune defense in mammals. A previous study showed that BPI gene expression correlates to gram-negative bacteria resistance. However, this gene showed tissue-specific expression in piglets and strongly expressed only in the digestive tract. To investigate the mechanisms governing the tissue-specificity, bisulfite sequencing PCR and next generation sequencing were used for high accuracy methylation quantitation of CpG islands of BPI gene upstream in 11 different tissues from weaned Yorkshire piglets. Additionally, qPCR was used to examine mRNA levels of BPI gene as well as transcription factor. We additionally analyzed transcriptional regulation by studying key 5-methylcytosine sites and transcription factors. Results showed that BPI mRNA levels significantly correlated with the overall methylation as well as methylation at mC-15 which was non-CpG site, no significant correlation could be found between the BPI and transcription factor mRNA levels, EMSA test showed that C/EBPβ could interact with BPI wild-type promoter DNA, but not methylated DNA. So we confirmed that methylation of mC-15 residue could inhibit the ability of C/EBPβ binding to the BPI promoter and affect the expression, and this mechanism probably plays a role in the tissue specificity of BPI gene expression in weaned piglets. PMID:27338589

  16. Age-dependent tissue-specific exposure of cell phone users

    NASA Astrophysics Data System (ADS)

    Christ, Andreas; Gosselin, Marie-Christine; Christopoulou, Maria; Kühn, Sven; Kuster, Niels

    2010-04-01

    The peak spatial specific absorption rate (SAR) assessed with the standardized specific anthropometric mannequin head phantom has been shown to yield a conservative exposure estimate for both adults and children using mobile phones. There are, however, questions remaining concerning the impact of age-dependent dielectric tissue properties and age-dependent proportions of the skull, face and ear on the global and local absorption, in particular in the brain tissues. In this study, we compare the absorption in various parts of the cortex for different magnetic resonance imaging-based head phantoms of adults and children exposed to different models of mobile phones. The results show that the locally induced fields in children can be significantly higher (>3 dB) in subregions of the brain (cortex, hippocampus and hypothalamus) and the eye due to the closer proximity of the phone to these tissues. The increase is even larger for bone marrow (>10 dB) as a result of its significantly high conductivity. Tissues such as the pineal gland show no increase since their distances to the phone are not a function of age. This study, however, confirms previous findings saying that there are no age-dependent changes of the peak spatial SAR when averaged over the entire head.

  17. NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

    SciTech Connect

    Zhao Guohua; Shi Lingfang; Qiu Daoming; Hu Hong; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2005-05-01

    NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

  18. Adiponectin Enhances Cold-Induced Browning of Subcutaneous Adipose Tissue via Promoting M2 Macrophage Proliferation.

    PubMed

    Hui, Xiaoyan; Gu, Ping; Zhang, Jialiang; Nie, Tao; Pan, Yong; Wu, Donghai; Feng, Tianshi; Zhong, Cheng; Wang, Yu; Lam, Karen S L; Xu, Aimin

    2015-08-01

    Adiponectin is an abundant adipokine with pleiotropic protective effects against a cluster of obesity-related cardiometabolic disorders. However, its role in adaptive thermogenesis has scarcely been explored. Here we showed that chronic cold exposure led to a markedly elevated production of adiponectin in adipocytes of subcutaneous white adipose tissue (scWAT), which in turn bound to M2 macrophages in the stromal vascular fraction. Chronic cold exposure-induced accumulation of M2 macrophages, activation of beige cells, and thermogenic program were markedly impaired in scWAT of adiponectin knockout (ADN KO) mice, whereas these impairments were reversed by replenishment with adiponectin. Mechanistically, adiponectin was recruited to the cell surface of M2 macrophages via its binding partner T-cadherin and promoted the cell proliferation by activation of Akt, consequently leading to beige cell activation. These findings uncover adiponectin as a key efferent signal for cold-induced adaptive thermogenesis by mediating the crosstalk between adipocytes and M2 macrophages in scWAT. PMID:26166748

  19. Chitosan-based hydrogel tissue scaffolds made by 3D plotting promotes osteoblast proliferation and mineralization.

    PubMed

    Liu, I-Hsin; Chang, Shih-Hsin; Lin, Hsin-Yi

    2015-06-01

    A 3D plotting system was used to make chitosan-based tissue scaffolds with interconnected pores using pure chitosan (C) and chitosan cross-linked with pectin (CP) and genipin (CG). A freeze-dried chitosan scaffold (CF/D) was made to compare with C, to observe the effects of structural differences. The fiber size, pore size, porosity, compression strength, swelling ratio, drug release efficacy, and cumulative weight loss of the scaffolds were measured. Osteoblasts were cultured on the scaffolds and their proliferation, type I collagen production, alkaline phosphatase activity, calcium deposition, and morphology were observed. C had a lower swelling ratio, degradation, porosity and drug release efficacy and a higher compressional stiffness and cell proliferation compared to CF/D (p < 0.05). Of the 3D-plotted samples, cells on CP exhibited the highest degree of mineralization after 21 d (p < 0.05). CP also had the highest swelling ratio and fastest drug release, followed by C and CG (p < 0.05). Both CP and CG were stiffer and degraded more slowly in saline solution than C (p < 0.05). In summary, 3D-plotted scaffolds were stronger, less likely to degrade and better promoted osteoblast cell proliferation in vitro compared to the freeze-dried scaffolds. C, CP and CG were structurally similar, and the different crosslinking caused significant changes in their physical and biological performances. PMID:25970802

  20. Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing.

    PubMed

    Balamurugan, Kuppareddi; Bombardi, Robin; Duncan, George; McCord, Bruce

    2014-11-01

    The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT1, USP49, DDX4, Hs_INSL6_03, Hs_ZC3H12D_05, and B_SPTB_03 were identified to contain tissue-specific differential methylation. Samples ranging from 9-21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR, and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_SPTB_03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied (p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample. PMID:24913642

  1. Metabolic profiling of the tissue-specific responses in mussel Mytilus galloprovincialis towards Vibrio harveyi challenge.

    PubMed

    Liu, Xiaoli; Ji, Chenglong; Zhao, Jianmin; Wang, Qing; Li, Fei; Wu, Huifeng

    2014-08-01

    Mussel Mytilus galloprovincialis is a marine aquaculture shellfish distributing widely along the coast in north China. In this work, we studied the differential metabolic responses induced by Vibrio harveyi in digestive gland and gill tissues from M. galloprovincialis using NMR-based metabolomics. The differential metabolic responses in the two tissue types were detected, except the similarly altered taurine and betaine. These metabolic responses suggested that V. harveyi mainly induced osmotic disruption and reduced energy demand via the metabolic pathways of glucose synthesis and ATP/AMP conversion in mussel digestive gland. In mussel gill tissues, V. harveyi basically caused osmotic stress and possible reduced energy demand as shown by the elevated phosphocholine that is involved in one of the metabolic pathways of ATP synthesis from ADP and phosphocholine. The altered mRNA expression levels of related genes (superoxide dismutase with copper and zinc, heat shock protein 90, defensin and lysozyme) suggested that V. harveyi induced clear oxidative and immune stresses in both digestive gland and gill tissues. However, the mRNA expression levels of both lysozyme and defensin in digestive gland were more significantly up-regulated than those in gill from V. harveyi-challenged mussel M. galloprovincialis, meaning that the immune organ, digestive gland, was more sensitive than gill. Overall, our results indicated that V. harveyi could induce tissue-specific metabolic responses in mussel M. galloprovincialis. PMID:24911264

  2. Identification of a midgut-specific promoter in the silkworm Bombyx mori.

    PubMed

    Jiang, Liang; Cheng, Tingcai; Dang, Yinghui; Peng, Zhengwen; Zhao, Ping; Liu, Shiping; Jin, Shengkai; Lin, Ping; Sun, Qiang; Xia, Qingyou

    2013-04-19

    The midgut is an important organ for digestion and absorption of nutrients and immune defense in the silkworm Bombyx mori. In an attempt to create a tool for midgut research, we cloned the 1080 bp P2 promoter sequence (P2P) of a highly expressed midgut-specific gene in the silkworm. The transgenic line (P2) was generated via embryo microinjection, in which the expression of EGFP was driven by P2P. There was strong green fluorescence only in the midgut of P2. RT-PCR and Western blot showed that P2P was a midgut-specific promoter with activity throughout the larval stage. A transgenic truncation experiment suggested that regions -305 to -214 and +107 to +181 were very important for P2P activity. The results of this study revealed that we have identified a midgut-specific promoter with a high level of activity in the silkworm that will aid future research and application of silkworm genes. PMID:23524268

  3. Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

    PubMed

    Danjo, Teruko; Eiraku, Mototsugu; Muguruma, Keiko; Watanabe, Kiichi; Kawada, Masako; Yanagawa, Yuchio; Rubenstein, John L R; Sasai, Yoshiki

    2011-02-01

    During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning. PMID:21289201

  4. Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

    PubMed

    Feng, Guihai; Tong, Man; Xia, Baolong; Luo, Guan-Zheng; Wang, Meng; Xie, Dongfang; Wan, Haifeng; Zhang, Ying; Zhou, Qi; Wang, Xiu-Jie

    2016-09-01

    How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features. PMID:27466324

  5. Intergenic Alu exonisation facilitates the evolution of tissue-specific transcript ends.

    PubMed

    Tajnik, Mojca; Vigilante, Alessandra; Braun, Simon; Hänel, Heike; Luscombe, Nicholas M; Ule, Jernej; Zarnack, Kathi; König, Julian

    2015-12-01

    The 3' untranslated regions (3' UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3' UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3' UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3' UTRs. PMID:26400176

  6. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics.

    PubMed

    Lundby, Alicia; Rossin, Elizabeth J; Steffensen, Annette B; Acha, Moshe Rav; Newton-Cheh, Christopher; Pfeufer, Arne; Lynch, Stacey N; Olesen, Søren-Peter; Brunak, Søren; Ellinor, Patrick T; Jukema, J Wouter; Trompet, Stella; Ford, Ian; Macfarlane, Peter W; Krijthe, Bouwe P; Hofman, Albert; Uitterlinden, André G; Stricker, Bruno H; Nathoe, Hendrik M; Spiering, Wilko; Daly, Mark J; Asselbergs, Folkert W; van der Harst, Pim; Milan, David J; de Bakker, Paul I W; Lage, Kasper; Olsen, Jesper V

    2014-08-01

    Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics. PMID:24952909

  7. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

    PubMed Central

    Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.; Rav Acha, Moshe; Newton-Cheh, Christopher; Pfeufer, Arne; Lynch, Stacey N.; Olesen, Søren-Peter; Brunak, Søren; Ellinor, Patrick T.; Jukema, J.Wouter; Trompet, Stella; Ford, Ian; Macfarlane, Peter W.; Krijthe, Bouwe P.; Hofman, Albert; Uitterlinden, Andre G.; Stricker, Bruno H.; Nathoe, Hendrik M.; Spiering, Wilko; Daly, Mark J.; Asselbergs, Folkert W.; van der Harst, Pim; Milan, David J.; de Bakker, Paul I.W.; Lage, Kasper; Olsen, Jesper V.

    2014-01-01

    Genome-wide association studies (GWAS) have identified thousands of loci associated wtih complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics. PMID:24952909

  8. Method for promoting specific alignment of short oligonucleotides on nucleic acids

    DOEpatents

    Studier, F. William; Kieleczawa, Jan; Dunn, John J.

    1996-01-01

    Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

  9. Genome-wide identification of human- and primate-specific core promoter short tandem repeats.

    PubMed

    Bushehri, A; Barez, M R Mashhoudi; Mansouri, S K; Biglarian, A; Ohadi, M

    2016-08-01

    Recent reports of a link between human- and primate-specific genetic factors and human/primate-specific characteristics and diseases necessitate genome-wide identification of those factors. We have previously reported core promoter short tandem repeats (STRs) of extreme length (≥6-repeats) that have expanded exceptionally in primates vs. non-primates, and may have a function in adaptive evolution. In the study reported here, we extended our study to the human STRs of ≥3-repeats in the category of penta and hexaucleotide STRs, across the entire human protein coding gene core promoters, and analyzed their status in several superorders and orders of vertebrates, using the Ensembl database. The ConSite software was used to identify the transcription factor (TF) sets binding to those STRs. STR specificity was observed at different levels of human and non-human primate (NHP) evolution. 73% of the pentanucleotide STRs and 68% of the hexanucleotide STRs were found to be specific to human and NHPs. AP-2alpha, Sp1, and MZF were the predominantly selected TFs (90%) binding to the human-specific STRs. Furthermore, the number of TF sets binding to a given STR was found to be a selection factor for that STR. Our findings indicate that selected STRs, the cognate binding TFs, and the number of TF set binding to those STRs function as switch codes at different levels of human and NHP evolution and speciation. PMID:27108803

  10. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

    PubMed

    Bonvallet, Paul P; Schultz, Matthew J; Mitchell, Elizabeth H; Bain, Jennifer L; Culpepper, Bonnie K; Thomas, Steven J; Bellis, Susan L

    2015-01-01

    Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these

  11. Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

    PubMed Central

    2004-01-01

    Cystathionine γ-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by λ genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice. PMID:15038791

  12. Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

    PubMed Central

    Bonvallet, Paul P.; Schultz, Matthew J.; Mitchell, Elizabeth H.; Bain, Jennifer L.; Culpepper, Bonnie K.; Thomas, Steven J.; Bellis, Susan L.

    2015-01-01

    Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these

  13. Seasonal, tissue-specific regulation of Akt/protein kinase B and glycogen synthase in hibernators.

    PubMed

    Hoehn, Kyle L; Hudachek, Susan F; Summers, Scott A; Florant, Gregory L

    2004-03-01

    Yellow-bellied marmots (Marmota flaviventris) exhibit a circannual cycle of hyperphagia and nutrient storage in the summer followed by hibernation in the winter. This annual cycle of body mass gain and loss is primarily due to large-scale accumulation of lipid in the summer, which is then mobilized and oxidized for energy during winter. The rapid and predictable change in body mass makes these animals ideal for studies investigating the molecular basis for body weight regulation. In the study described herein, we monitored seasonal changes in the protein levels and activity of a central regulator of anabolic metabolism, the serine-threonine kinase Akt-protein kinase B (Akt/PKB), during the months accompanying maximal weight gain and entry into hibernation (June-November). Interestingly, under fasting conditions, Akt/PKB demonstrated a tissue-specific seasonal activation. Specifically, although Akt/PKB levels did not change, the activity of Akt/PKB (isoforms 1/alpha and 2/beta) in white adipose tissue (WAT) increased significantly in July. Moreover, glycogen synthase, which lies downstream of Akt/PKB on a linear pathway linking the enzyme to the stimulation of glycogen synthesis, demonstrated a similar pattern of seasonal activation. By contrast, Akt/PKB activity in skeletal muscle peaked much later (i.e., September). These data suggest the existence of a novel, tissue-specific mechanism regulating Akt/PKB activation during periods of marked anabolism. PMID:14656767

  14. Generation and Characterization of a Tissue-Specific Centrosome Indicator Mouse Line.

    PubMed

    Hirai, Maretoshi; Chen, Ju; Evans, Sylvia M

    2016-05-01

    Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue-specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP-Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre-mediated excision, EGFP-Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real-time imaging, and showed that EGFP-Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP-Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286-296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc. PMID:26990996

  15. Semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

    PubMed

    Romano, Shannon N; Gorelick, Daniel A

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  16. Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

    PubMed Central

    Romano, Shannon N.; Gorelick, Daniel A.

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  17. Conservation and tissue-specific transcription patterns of long noncoding RNAs

    PubMed Central

    Ward, Melanie; McEwan, Callum; Mills, James D; Janitz, Michael

    2015-01-01

    Abstract Over the past decade, the focus of molecular biology has shifted from being predominately DNA and protein-centric to having a greater appreciation of RNA. It is now accepted that the genome is pervasively transcribed in tissue- and cell-specific manner, to produce not only protein-coding RNAs, but also an array of noncoding RNAs (ncRNAs). Many of these ncRNAs have been found to interact with DNA, protein and other RNA molecules where they exert regulatory functions. Long ncRNAs (lncRNAs) are a subclass of ncRNAs that are particularly interesting due to their cell-specific and species-specific expression patterns and unique conservation patterns. Currently, individual lncRNAs have been classified functionally; however, for the vast majority the functional relevance is unknown. To better categorize lncRNAs, an understanding of their specific expression patterns and evolutionary constraints are needed. PMID:27335896

  18. Specific binding of nuclear proteins to a bifunctional promoter element upstream of the H1/AC box of the testis-specific histone H1t gene.

    PubMed

    Wolfe, Steven A; Grimes, Sidney R

    2003-06-01

    The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes during spermatogenesis. Studies with transgenic mice show that 141 base pairs (bp) of the H1t proximal promoter accompanied with 800 bp of downstream sequence are sufficient for tissue-specific transcription. Nuclear proteins from testis and pachytene spermatocytes produce footprints spanning the region covering the repressor element (RE) from 100 to 125 nucleotides upstream of the H1t transcriptional initiation site. Only testis nuclear proteins bind to the 5'-end of the element and produce a unique, low-mobility complex in electrophoretic mobility shift assays. This testis complex is distinct from the complex formed by a repressor protein derived from several cell lines that binds to the 3'-end of the element. The testis complex band is formed when using nuclear proteins from primary spermatocytes, where the H1t gene is transcribed, and band intensity drops 70%-80% when using nuclear proteins from early spermatids, where H1t gene transcription ceases. Protein-DNA cross-linking experiments using testis nuclear proteins produce electrophoretic bands of 59, 52, and 50 kDa on SDS/PAGE gels. PMID:12606375

  19. P01.08LINEAGE-SPECIFIC SPLICING OF AN ALTERNATIVE EXON OF ANXA7 PROMOTES EGFR SIGNALING ACTIVATION AND TUMOR PROGRESSION IN GLIOBLASTOMA

    PubMed Central

    Ferrarese, R.; Bug, E.; Maticzka, D.; Reichardt, W.; Masilamani, A.P.; Dai, F.; Weyerbrock, A.; Prinz, M.; Bredel, M.; Carro, M.S.

    2014-01-01

    Tissue-specific alternative splicing is critical to the emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence signaling pathways to profound biological effect. In the brain, Annexin A7 isoform 1 (ANXA7-I1) is exclusively expressed in mature neurons, while isoform 2 (ANXA7-I2) in which exon 6 is skipped, is expressed in glial and progenitor cells. We show that lineage-specific splicing of the cassette exon 6 in the membrane-binding tumor suppressor ANXA7diminishes endosomal targeting and consequent signal termination of the EGFR oncoprotein during brain tumor progression. Splicing of this exon is mediated by Polypyrimidine Tract-Binding Protein 1 (PTBP1), a ribonucleoprotein normally repressed during neuronal development but which we found to be highly expressed also in glioblastomas through loss of a brain-enriched microRNA, miR-124, and gene amplification. Here, we show that the PTBP1-ANXA7 splicing-EGFR signal activation axis promotes in vitro cell migration and invasion, and tumor angiogenesis in vivo. In glioblastoma, ANXA7 splicing is likely inherited from a potential tumor-initiating ancestor but this trait is further exploited through accumulation of mutations that enhance EGFR signaling. Our data illustrate how lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates its tumor suppressor function and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can contribute to reprogramming normal development to oncogenesis.

  20. Hole Burning Imaging Studies of Cancerous and Analogous Normal Ovarian Tissues Utilizing Organelle Specific Dyes

    SciTech Connect

    Satoshi Matsuzaki

    2004-12-19

    Presented in this dissertation is the successful demonstration that nonphotochemical hole burning (NPWB) imaging can be used to study in vitro tissue cellular systems for discerning differences in cellular ultrastructures due to cancer development. This has been accomplished with the surgically removed cancerous ovarian and analogous normal peritoneal tissues from the same patient and the application of a fluorescent mitochondrion specific dye, Molecular Probe MitoFluor Far Red 680 (MF680), commonly known as rhodamine 800, that has been proven to exhibit efficient NPHB. From the results presented in Chapters 4 and 5 , and Appendix B, the following conclusions were made: (1) fluorescence excitation spectra of MF680 and confocal microscopy images of thin sliced tissues incubated with MF680 confirm the site-specificity of the probe molecules in the cellular systems. (2) Tunneling parameters, {lambda}{sub 0} and {sigma}{sub {lambda}}, as well as the standard hole burning parameters (namely, {gamma} and S), have been determined for the tissue samples by hole growth kinetics (HGK) analyses. Unlike the preliminary cultured cell studies, these parameters have not shown the ability to distinguish tissue cellular matrices surrounding the chromophores. (3) Effects of an external electric (Stark) field on the nonphotochemical holes have been used to determine the changes in permanent dipole moment (f{Delta}{mu}) for MF680 in tissue samples when burn laser polarization is parallel to the Stark field. Differences are detected between f{Delta}{mu}s in the two tissue samples, with the cancerous tissue exhibiting a more pronounced change (1.35-fold increase) in permanent dipole moment change relative to the normal analogs. It is speculated that the difference may be related to differences in mitochondrial membrane potentials in these tissue samples. (4) In the HGK mode, hole burning imaging (HBI) of cells adhered to coverslips and cooled to liquid helium temperatures in the

  1. Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways

    PubMed Central

    Swarbreck, David; Febrer, Melanie; Larson, Tony R.; Graham, Ian A.; Caccamo, Mario; Slabas, Antoni R.

    2012-01-01

    Background Storage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis. Methodology/Findings Storage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG. Conclusions/Significance Several of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic

  2. Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.

    PubMed

    Shu, Xiaomei; Livingston, David P; Franks, Robert G; Boston, Rebecca S; Woloshuk, Charles P; Payne, Gary A

    2015-09-01

    Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21-22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance. PMID:25469958

  3. Tissue structure-specific distribution of glycosaminoglycans in the human penis.

    PubMed

    Goulas, A; Papakonstantinou, E; Karakiulakis, G; Mirtsou-Fidani, V; Kalinderis, A; Hatzichristou, D G

    2000-09-01

    The aim of this work was to isolate and characterise the glycosaminoglycans present in the different tissue structures of the human penis in view of their potentially significant role in the physiology of erection. Penile tissue samples were obtained from patients who underwent penectomy and were subsequently dissected into individual tissue structures. Total glycosaminoglycans were isolated and purified from tunica albuginea, corpora cavernosa and corpus spongiosum, following tissue mincing, ultrasonication, lipid extraction, extensive digestion with pronase and DNase, treatment with alkali-borohydride and ethanol precipitation. Isolated glycosaminoglycans were separated by cellulose acetate electrophoresis and fractionated by anion exchange chromatography on DEAE Sephacel columns. Different glycosaminoglycan fractions were identified using glycosaminoglycan-degrading enzymes of known specificity. Gradient polyacrylamide gel electrophoresis was used to determine the average molecular mass of the glycosaminoglycans. The corpus cavernosum and the corpus spongiosum extracts contained almost twice the amount of glycosaminoglycan-associated uronic acids as compared to the tunical extracts (1.47+/-0.09, and 1.49+/-0.15 as opposed to 0.75+/-0.15 microg/mg dry defatted tissue, respectively; S.E.M., n=5). With the exception of hyaluronic acid, the relative amount of individual glycosaminoglycan types varied significantly among extracts of different origin. Heparan sulphate was more abundant in cavernosal, dermatan sulphate in tunical, and chondroitin-6-sulphate in corpus spongiosum extracts. No structure-specific differences were detected with respect to the molecular mass distribution of each glycosaminoglycan type. Our study shows that the different structures of the human penis produce distinct profiles of glycosaminoglycans, which are well suited to the individual functional characteristics of these structures. PMID:11084377

  4. Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression.

    PubMed

    Di, Wei-Li; Semenova, Ekaterina; Larcher, Fernando; Del Rio, Marcela; Harper, John I; Thrasher, Adrian J; Qasim, Waseem

    2012-01-01

    Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector-mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral-derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter-transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type-related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization. PMID:21895535

  5. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    PubMed

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation. PMID:25982944

  6. Expression of Tissue Factor by Melanoma Cells Promotes Efficient Hematogenous Metastasis

    NASA Astrophysics Data System (ADS)

    Mueller, Barbara M.; Reisfeld, Ralph A.; Edgington, Thomas S.; Ruf, Wolfram

    1992-12-01

    Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher levels of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence is insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

  7. Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster.

    PubMed

    Tsai, Yu-Cheng; Cooke, Nancy E; Liebhaber, Stephen A

    2016-06-01

    The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR 'loops' over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed 'hCS chromatin hub'. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster. PMID:26893355

  8. Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster

    PubMed Central

    Tsai, Yu-Cheng; Cooke, Nancy E.; Liebhaber, Stephen A.

    2016-01-01

    The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR ‘loops’ over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed ‘hCS chromatin hub’. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster. PMID:26893355

  9. Tissue-specific expression of the bovine aromatase-encoding gene uses multiple transcriptional start sites and alternative first exons.

    PubMed

    Fürbass, R; Kalbe, C; Vanselow, J

    1997-07-01

    Here we report on the genomic structure of the bovine aromatase cytochrome P450-encoding gene (Cyp19) and its tissue-specific transcript variants. The gene comprises at least 14 exons (1.1, 1.2a, 1.2b, 1.3,1.4, and 2-10) spanning more than 56 kilobases of genomic DNA. The coding area is confined to exons 2-10. Transcriptional start sites of Cyp19 were examined in granulosa cells, placenta, testis, adrenal gland, and brain, employing 5'-RACE (rapid amplification of complementary DNA ends) and primer extension. The analysis of 5'-RACE clones revealed six Cyp19 transcript variants that were different within their 5'-untranslated regions (5'-UTR). Yet, the coding region was identical in all clones. Although two of these 5'-UTR (the first 152 nucleotides of exon 2 and exon 1.4) are conserved among different species, four others (exons 1.1, 1.2a, 1.2b, and 1.3) did not show sequence homology to any other species. Transcription from exons 1.1 and 2 starts at several adjacent sites. In granulosa cells and placenta, but not in brain, a fraction of transcripts starting with exon 1.2a contains an additional untranslated exon, 1.2b, due to alternative splicing. Transcript variants comprising exon 1.1, 1.2a, 1.2b, or 1.3 were mainly found in the placenta, those with the 5'-UTR of exon 2 were predominant in granulosa cells, and transcripts with exon 1.4 prevailed in the brain. Estimates of Cyp19 transcript concentrations in six different tissues revealed high levels in granulosa cells and placenta, intermediate levels in testis and brain, and low levels in adrenal gland and liver. Our experiments demonstrate that six transcript variants of the bovine Cyp19 gene, including 9-11 exons, are expressed with tissue-specific preferences. These transcripts are presumably generated using five different promoter regions and tissue-specific alternative splicing. PMID:9202222

  10. The oil palm metallothionein promoter contains a novel AGTTAGG motif conferring its fruit-specific expression and is inducible by abiotic factors.

    PubMed

    Omidvar, Vahid; Abdullah, Siti Nor Akmar; Izadfard, Amir; Ho, Chai Ling; Mahmood, Maziah

    2010-09-01

    The 1,053-bp promoter of the oil palm metallothionein gene (so-called MSP1) and its 5' deletions were fused to the GUS reporter gene, and analysed in transiently transformed oil palm tissues. The full length promoter showed sevenfold higher activity in the mesocarp than in leaves and 1.5-fold more activity than the CaMV35S promoter in the mesocarp. The 1,053-bp region containing the 5' untranslated region (UTR) gave the highest activity in the mesocarp, while the 148-bp region was required for minimal promoter activity. Two positive regulatory regions were identified at nucleotides (nt) -953 to -619 and -420 to -256 regions. Fine-tune deletion of the -619 to -420 nt region led to the identification of a 21-bp negative regulatory sequence in the -598 to -577 nt region, which is involved in mesocarp-specific expression. Gel mobility shift assay revealed a strong interaction of the leaf nuclear extract with the 21-bp region. An AGTTAGG core-sequence within this region was identified as a novel negative regulatory element controlling fruit-specificity of the MSP1 promoter. Abscisic acid (ABA) and copper (Cu(2+)) induced the activity of the promoter and its 5' deletions more effectively than methyl jasmonate (MeJa) and ethylene. In the mesocarp, the full length promoter showed stronger inducibility in response to ABA and Cu(2+) than its 5' deletions, while in leaves, the -420 nt fragment was the most inducible by ABA and Cu(2+). These results suggest that the MSP1 promoter and its regulatory regions are potentially useful for engineering fruit-specific and inducible gene expression in oil palm. PMID:20635097

  11. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  12. Microstructural manipulation of electrospun scaffolds for specific bending stiffness for heart valve tissue engineering.

    PubMed

    Amoroso, Nicholas J; D'Amore, Antonio; Hong, Yi; Rivera, Christian P; Sacks, Michael S; Wagner, William R

    2012-12-01

    Biodegradable thermoplastic elastomers are attractive for application in cardiovascular tissue construct development due to their amenability to a wide range of physical property tuning. For heart valve leaflets, while low flexural stiffness is a key design feature, control of this parameter has been largely neglected in the scaffold literature where electrospinning is being utilized. This study evaluated the effect of processing variables and secondary fiber populations on the microstructure, tensile and bending mechanics of electrospun biodegradable polyurethane scaffolds for heart valve tissue engineering. Scaffolds were fabricated from poly(ester urethane) urea (PEUU) and the deposition mandrel was translated at varying rates in order to modify fiber intersection density. Scaffolds were also fabricated in conjunction with secondary fiber populations designed either for mechanical reinforcement or to be selectively removed following fabrication. It was determined that increasing fiber intersection densities within PEUU scaffolds was associated with lower bending moduli. Further, constructs fabricated with stiff secondary fiber populations had higher bending moduli whereas constructs with secondary fiber populations which were selectively removed had noticeably lower bending moduli. Insights gained from this work will be directly applicable to the fabrication of soft tissue constructs, specifically in the development of cardiac valve tissue constructs. PMID:22890285

  13. Tissue-specific transcriptome sequencing analysis expands the non-human primate reference transcriptome resource (NHPRTR)

    PubMed Central

    Peng, Xinxia; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Nishida, Andrew; Pipes, Lenore; Bozinoski, Marjan; Thomas, Matthew J.; Kelly, Sara; Weiss, Jeffrey M.; Raveendran, Muthuswamy; Muzny, Donna; Gibbs, Richard A.; Rogers, Jeffrey; Schroth, Gary P.; Katze, Michael G.; Mason, Christopher E.

    2015-01-01

    The non-human primate reference transcriptome resource (NHPRTR, available online at http://nhprtr.org/) aims to generate comprehensive RNA-seq data from a wide variety of non-human primates (NHPs), from lemurs to hominids. In the 2012 Phase I of the NHPRTR project, 19 billion fragments or 3.8 terabases of transcriptome sequences were collected from pools of ∼20 tissues in 15 species and subspecies. Here we describe a major expansion of NHPRTR by adding 10.1 billion fragments of tissue-specific RNA-seq data. For this effort, we selected 11 of the original 15 NHP species and subspecies and constructed total RNA libraries for the same ∼15 tissues in each. The sequence quality is such that 88% of the reads align to human reference sequences, allowing us to compute the full list of expression abundance across all tissues for each species, using the reads mapped to human genes. This update also includes improved transcript annotations derived from RNA-seq data for rhesus and cynomolgus macaques, two of the most commonly used NHP models and additional RNA-seq data compiled from related projects. Together, these comprehensive reference transcriptomes from multiple primates serve as a valuable community resource for genome annotation, gene dynamics and comparative functional analysis. PMID:25392405

  14. Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos.

    PubMed

    Vejares, Sandra González; Sabat, Pablo; Sanchez-Hernandez, Juan C

    2010-04-01

    Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil. PMID:20045489

  15. Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers

    PubMed Central

    Zhang, Yan; Nowicka, Aleksandra; Solley, Travis N.; Wei, Caimiao; Parikh, Aaroh; Court, Laurence; Burks, Jared K.; Andreeff, Michael; Woodward, Wendy A.; Dadbin, Ali; Kolonin, Mikhail G.; Lu, Karen H.; Klopp, Ann H.

    2015-01-01

    Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination. PMID:26317219

  16. Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

    PubMed Central

    Chan, Elsa C.; Kuo, Shyh-Ming; Kong, Anne M.; Morrison, Wayne A.; Dusting, Gregory J.; Mitchell, Geraldine M.

    2016-01-01

    Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo. PMID:26900837

  17. Ablation of eNOS does not promote adipose tissue inflammation.

    PubMed

    Jurrissen, Thomas J; Sheldon, Ryan D; Gastecki, Michelle L; Woodford, Makenzie L; Zidon, Terese M; Rector, R Scott; Vieira-Potter, Victoria J; Padilla, Jaume

    2016-04-15

    Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an important determinant of insulin resistance and cardiovascular disease; therefore, a better understanding of factors regulating AT inflammation is critical. It is well established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes arterial inflammation; however, the role of NO in modulating inflammation in AT remains disputed. In the present study, 10-wk-old C57BL6 wild-type and endothelial nitric oxide synthase (eNOS) knockout male mice were randomized to either a control diet (10% kcal from fat) or a Western diet (44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 wk (n= 7 or 8/group). In wild-type mice, Western diet-induced obesity led to increased visceral white AT expression of inflammatory genes (e.g., MCP1, TNF-α, and CCL5 mRNAs) and markers of macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs, and Mac-2 protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were not accompanied by decreases in eNOS phosphorylation at Ser-1177 or increases in eNOS phosphorylation at Thr-495. Also counter to expectations, eNOS knockout mice, independent of the diet, were leaner and did not exhibit greater white or brown AT inflammation compared with wild-type mice. Collectively, these findings do not support the hypothesis that reduced NO production from eNOS contributes to obesity-related AT inflammation. PMID:26864812

  18. Tissue Inhibitor of Metalloproteinase-3 (TIMP3) Promotes Endothelial Apoptosis via a Caspase-Independent Mechanism

    PubMed Central

    Qi, Jian Hua; Anand-Apte, Bela

    2015-01-01

    Tissue Inhibitor of Metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in endothelial cells (ECs) in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in endothelial cells expressing KDR (PAE/KDR), but not in endothelial cells expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway. PMID:25558000

  19. Sleep promotes branch-specific formation of dendritic spines after learning.

    PubMed

    Yang, Guang; Lai, Cora Sau Wan; Cichon, Joseph; Ma, Lei; Li, Wei; Gan, Wen-Biao

    2014-06-01

    How sleep helps learning and memory remains unknown. We report in mouse motor cortex that sleep after motor learning promotes the formation of postsynaptic dendritic spines on a subset of branches of individual layer V pyramidal neurons. New spines are formed on different sets of dendritic branches in response to different learning tasks and are protected from being eliminated when multiple tasks are learned. Neurons activated during learning of a motor task are reactivated during subsequent non-rapid eye movement sleep, and disrupting this neuronal reactivation prevents branch-specific spine formation. These findings indicate that sleep has a key role in promoting learning-dependent synapse formation and maintenance on selected dendritic branches, which contribute to memory storage. PMID:24904169

  20. Inter-specific coral chimerism: Genetically distinct multicellular structures associated with tissue loss in Montipora capitata

    USGS Publications Warehouse

    Work, Thierry M.; Forsman, Zac H.; Szabo, Zoltan; Lewis, Teresa D.; Aeby, Greta S.; Toonen, Robert J.

    2011-01-01

    Montipora white syndrome (MWS) results in tissue-loss that is often lethal to Montipora capitata, a major reef building coral that is abundant and dominant in the Hawai'ian Archipelago. Within some MWS-affected colonies in Kane'ohe Bay, Oahu, Hawai'i, we saw unusual motile multicellular structures within gastrovascular canals (hereafter referred to as invasive gastrovascular multicellular structure-IGMS) that were associated with thinning and fragmentation of the basal body wall. IGMS were in significantly greater densities in coral fragments manifesting tissue-loss compared to paired normal fragments. Mesenterial filaments from these colonies yielded typical M. capitata mitochondrial haplotypes (CO1, CR), while IGMS from the same colony consistently yielded distinct haplotypes previously only found in a different Montipora species (Montipora flabellata). Protein profiles showed consistent differences between paired mesenterial filaments and IGMS from the same colonies as did seven microsatellite loci that also exhibited an excess of alleles per locus inconsistent with a single diploid organism. We hypothesize that IGMS are a parasitic cellular lineage resulting from the chimeric fusion between M. capitata and M. flabellata larvae followed by morphological reabsorption of M. flabellata and subsequent formation of cell-lineage parasites. We term this disease Montiporaiasis. Although intra-specific chimerism is common in colonial animals, this is the first suspected inter-specific example and the first associated with tissue loss.

  1. VISTA Enhancer Browser--A Database of Tissue-Specific HumanEnhancers

    SciTech Connect

    Visel, Axel; Minovitsky, Simon; Dubchak, Inna; Pennacchio, Len A.

    2006-08-01

    Despite the known existence of distant-acting cis-regulatoryelements in the human genome, only a small fraction of these elements hasbeen identified and experimentally characterized in vivo. This paucity ofenhancer collections with defined activities has thus hinderedcomputational approaches for the genome-wide prediction of enhancers andtheir functions. To fill this void, we utilize comparative genomeanalysis to identify candidate enhancer elements in the human genomecoupled with the experimental determination of their in vivo enhanceractivity in transgenic mice (1). These data are available through theVISTA Enhancer Browser (http://enhancer.lbl.gov). This growing databasecurrently contains over 250 experimentally tested DNA fragments, of whichmore than 100 have been validated as tissue-specific enhancers. For eachpositive enhancer, we provide digital images of whole-mount embryostaining at embryonic day 11.5 and an anatomical description of thereporter gene expression pattern. Users can retrieve elements near singlegenes of interest, search for enhancers that target reporter geneexpression to a particular tissue, or download entire collections ofenhancers with a defined tissue specificity or conservation depth. Theseexperimentally validated training sets are expected to provide a basisfor a wide range of downstream computational and functional studies ofenhancer function.

  2. Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems.

    PubMed

    Suzuki, Y; Tsuda, M; Takiya, S; Hirose, S; Suzuki, E; Kameda, M; Ninaki, O

    1986-12-01

    Six cell-free extracts have been used to characterize the nature of DNA signals and trans-acting factors responsible for the transcription enhancement of the Bombyx mori fibroin gene. The upstream element of the fibroin gene involved in the enhancement can be divided into two regions. The proximal region, -72 to -32, is recognized as a common enhancing signal by all B. mori extracts from the posterior silk gland, the middle silk gland, the ovarian tissue, and an embryonic cell line. It is weakly recognized by an Antheraea silkworm cell line extract but not by a HeLa cell extract. The distal region, -238 to -73, appears to be a tissue-specific enhancing signal that is recognized more effectively by the posterior silk gland extract than by the middle silk gland extract. These observations suggest that the use of these cell-free systems can offer a means for the biochemical characterization of the trans-acting factors involved in the tissue-specific regulation of the fibroin gene. PMID:3467322

  3. Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways

    PubMed Central

    Mitsche, Matthew A; McDonald, Jeffrey G; Hobbs, Helen H; Cohen, Jonathan C

    2015-01-01

    Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol. DOI: http://dx.doi.org/10.7554/eLife.07999.001 PMID:26114596

  4. Structural disorder: a tool for housekeeping proteins performing tissue-specific interactions.

    PubMed

    Banerjee, Sanghita; De, Rajat K

    2016-09-01

    An interaction between a pair of proteins unique for a particular tissue is denoted as a tissue-specific interaction (TSI). Tissue-specific (TS) proteins always perform TSIs with a limited number of interacting partners. However, it has been claimed that housekeeping (HK) proteins frequently take part in TSIs. This is actually an unusual phenomenon. How a single HK protein mediates TSIs - remains an interesting yet an unsolved question. We have hypothesized that HK proteins have attained a high degree of structural flexibility to modulate TSIs efficiently. We have observed that HK proteins are selected to be intrinsically disordered compared to TS proteins. Therefore, the purposeful adaptation of structural disorder brings out special advantages for HK proteins compared to TS proteins. We have demonstrated that TSIs may play vital roles in shaping the molecular adaptation of disordered regions within HK proteins. We also have noticed that HK proteins, mediating a huge number of TSIs, have a greater portion of their interacting interfaces overlapped with the adjacent disordered segment. Moreover, these HK proteins, mediating TSIs, preferably adapt single domain (SD). We have concluded that HK proteins adapt a high degree of structural flexibility to mediate TSIs. Besides, having a SD along with structural flexibility is more economic than maintaining multiple domains with a rigid structure. This assists them in attaining various structural conformations upon binding to their partners, thereby designing an economically optimum molecular system. PMID:26375894

  5. Inter-Specific Coral Chimerism: Genetically Distinct Multicellular Structures Associated with Tissue Loss in Montipora capitata

    PubMed Central

    Work, Thierry M.; Forsman, Zac H.; Szabó, Zoltán; Lewis, Teresa D.; Aeby, Greta S.; Toonen, Robert J.

    2011-01-01

    Montipora white syndrome (MWS) results in tissue-loss that is often lethal to Montipora capitata, a major reef building coral that is abundant and dominant in the Hawai'ian Archipelago. Within some MWS-affected colonies in Kane'ohe Bay, Oahu, Hawai'i, we saw unusual motile multicellular structures within gastrovascular canals (hereafter referred to as invasive gastrovascular multicellular structure-IGMS) that were associated with thinning and fragmentation of the basal body wall. IGMS were in significantly greater densities in coral fragments manifesting tissue-loss compared to paired normal fragments. Mesenterial filaments from these colonies yielded typical M. capitata mitochondrial haplotypes (CO1, CR), while IGMS from the same colony consistently yielded distinct haplotypes previously only found in a different Montipora species (Montipora flabellata). Protein profiles showed consistent differences between paired mesenterial filaments and IGMS from the same colonies as did seven microsatellite loci that also exhibited an excess of alleles per locus inconsistent with a single diploid organism. We hypothesize that IGMS are a parasitic cellular lineage resulting from the chimeric fusion between M. capitata and M. flabellata larvae followed by morphological reabsorptio