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Sample records for fibroblast translocator cdna

  1. Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

    International Nuclear Information System (INIS)

    Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

    1987-01-01

    α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

  2. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    International Nuclear Information System (INIS)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-01-01

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of ∼ 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Further studies on the effect of chloroquine on the uptake, metabolism and intracellular translocation of [35S]cystine in cystinotic fibroblasts.

    Science.gov (United States)

    Danpure, C J; Jennings, P R; Fyfe, D A

    1986-03-14

    The present study uses the lysosomotropic drug chloroquine to investigate the mechanisms by which exogenous [35S]cystine is able to label the intracellular (intralysosomal) cystine pool(s) in cystinotic fibroblasts. When cystinotic fibroblasts were labelled for short periods of time (8 h or less), chloroquine (20 microM) inhibited the labelling of the intracellular cystine pool(s). However, when the cells were labelled for longer periods of time (24 h or more) chloroquine stimulated the labelling of the intracellular cystine pool(s). The short-term effect was selectively abolished when the cells were washed free of chloroquine, while the long-term effect was selectively abolished when the medium was depleted of cystine. Two routes of translocation of exogenous cystine to the lysosomes could be defined. One route was fast, had a low capacity, was inhibited by chloroquine and increased with increasing medium pH, while the other route was slow, had a high capacity, was stimulated by chloroquine and was more active at low pH. The former pathway probably consisted of plasma membrane transport of cystine into the cytosol followed by direct or indirect transport into the lysosomes. The latter route possibly consisted of pinocytosis with fusion of the cystine-containing pinosomes with lysosomes.

  5. Prolongation of the survival of breast cancer-bearing mice immunized with GM-CSF-secreting syngeneic/allogeneic fibroblasts transfected with a cDNA expression library from breast cancer cells.

    Science.gov (United States)

    Kim, Tae S; Jung, Mi Y; Cho, Daeho; Cohen, Edward P

    2006-10-30

    Breast cancer cells, like other types of neoplastic cells, form weakly immunogenic tumor-associated antigens. The antigenic properties of the tumor-associated antigens can be enhanced if they are expressed by highly immunogenic cells. In this study, a cancer vaccine was prepared by transfer of a cDNA expression library from SB5b breast carcinoma into mouse fibroblast cells of C3H/He mouse origin (H-2(k)), that had been previously modified to secrete GM-CSF and to express allogeneic class I-determinants (H-2(b)). The transfected syngeneic/allogeneic fibroblasts secreting GM-CSF were used as a vaccine in C3H/He mice. Robust cell-mediated immunity toward the breast cancer cells was generated in mice immunized with the cDNA-based vaccine. The immunity, mediated predominantly by CD8(+) T lymphocytes, was directed toward the breast cancer cells, but not against either of two other non-cross-reactive neoplasms of C3H/He mice. The immunity was sufficient to prolong the survival of mice with established breast cancer. Among other advantages, preparation of the vaccine by cDNA-transfer into a fibroblast cell line enabled the recipient cells to be modified in advance of DNA-transfer to augment their immunogenic properties. As the transferred DNA is replicated as the transfected cells divide, the vaccine could be prepared from microgram quantities of tumor tissue.

  6. Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8-beta-D-lactosylceramide.

    Science.gov (United States)

    Kim, Seong-Youl; Wang, Teng-ke; Singh, Raman Deep; Wheatley, Christine L; Marks, David L; Pagano, Richard E

    2009-09-01

    Plasma membrane (PM) microdomains, including caveolae and other cholesterol-enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D-erythro-octanoyl-lactosylceramide (C8-D-e-LacCer), stimulates endocytosis via caveolae and induces the appearance of micron-size microdomains on the PM. To further understand the effects of C8-D-e-LacCer treatment on PM microdomains, we used a detergent-free method to isolate microdomain-enriched membranes from fibroblasts treated +/-C8-D-e-LacCer, and performed 2-DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain-enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho-associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D-e-LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8-D-e-LacCer-treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8-L-threo-lactosylceramide, a glycosphingolipid with non-natural stereochemistry. This study identifies new microdomain-associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.

  7. Complete cDNA sequence coding for human docking protein

    Energy Technology Data Exchange (ETDEWEB)

    Hortsch, M; Labeit, S; Meyer, D I

    1988-01-11

    Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

  8. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    International Nuclear Information System (INIS)

    Wang Qin; Liu Xiaoqiu; Xu Chang; Du Liqing; Sun Zhijuan; Wang Yan; Liu Qiang; Song Li; Li Jin; Fan Feiyue

    2013-01-01

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  9. Nuclear translocation and retention of growth hormone

    DEFF Research Database (Denmark)

    Mertani, Hichem C; Raccurt, Mireille; Abbate, Aude

    2003-01-01

    We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use...... of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect...... the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm...

  10. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  11. Fibroblastic rheumatism

    Directory of Open Access Journals (Sweden)

    Jyoti Ranjan Parida

    2017-01-01

    Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

  12. Sequence of a cloned cDNA encoding human ribosomal protein S11

    Energy Technology Data Exchange (ETDEWEB)

    Lott, J B; Mackie, G A

    1988-02-11

    The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

  13. Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient

    NARCIS (Netherlands)

    Huizing, M.; Iacobazzi, V.; IJlst, L.; Savelkoul, P.; Ruitenbeek, W.; van den Heuvel, L.; Indiveri, C.; Smeitink, J.; Trijbels, F.; Wanders, R.; Palmieri, F.

    1997-01-01

    The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC

  14. Problem-elephant translocation: translocating the problem and the elephant?

    Directory of Open Access Journals (Sweden)

    Prithiviraj Fernando

    Full Text Available Human-elephant conflict (HEC threatens the survival of endangered Asian elephants (Elephas maximus. Translocating "problem-elephants" is an important HEC mitigation and elephant conservation strategy across elephant range, with hundreds translocated annually. In the first comprehensive assessment of elephant translocation, we monitored 16 translocations in Sri Lanka with GPS collars. All translocated elephants were released into national parks. Two were killed within the parks where they were released, while all the others left those parks. Translocated elephants showed variable responses: "homers" returned to the capture site, "wanderers" ranged widely, and "settlers" established home ranges in new areas soon after release. Translocation caused wider propagation and intensification of HEC, and increased elephant mortality. We conclude that translocation defeats both HEC mitigation and elephant conservation goals.

  15. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

    2017-06-15

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  16. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    International Nuclear Information System (INIS)

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-01-01

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  17. Simulations of polymer translocation

    NARCIS (Netherlands)

    Vocks, H.

    2008-01-01

    Transport of molecules across membranes is an essential mechanism for life processes. These molecules are often long, and the pores in the membranes are too narrow for the molecules to pass through as a single unit. In such circumstances, the molecules have to squeeze --- i.e., translocate ---

  18. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  19. An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

    Science.gov (United States)

    Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

    2012-07-01

    cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

  20. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  1. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    International Nuclear Information System (INIS)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III; Billheimer, J.T.

    1991-01-01

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP 2 ). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP 2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP 2 . The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A) + RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP 2 gene in the human genome or that the SCP 2 gene is very large. Coexpression of the SCP 2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP 2 plays a role in regulating steroidogenesis, among other possible functions

  2. Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human α-ketoacid dehydrogenase complexes

    International Nuclear Information System (INIS)

    Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

    1988-01-01

    cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues

  3. Phenotype change and migration of adventitial fibroblasts during postangioplasty

    International Nuclear Information System (INIS)

    Wang Yongli; Zhang Jiaxing; He Nengshu; Si Tongguo; Fan Hailun; Ge Xihong; Xu Rui

    2006-01-01

    Objective: To verify fibroblasts translocation from adventitia into neointima by labeling adventitia cells with bromodeoxyuridine (BrDU) after angioplasty, and to explore the relationship of adventitial fibroblast with restenosis. Methods: Vascular restenosis model was created by injured intima of common carotid artery (CCA) of mouse with guide wire, adventitial fibroblasts were labeled with BrDU, and dynamic distribution of myofibroblasts in adventitia, media and neoitima was observed at different times (3 d, 7 d, 14 d and 28 d) by means of single/double-label immunohistochemistry, light microscope, electronic microscope and image analysis system. Results: 1.Immunohistochemistry: More adventitial fibroblasts combined with BrDU could be found in adventitia on the 3rd day of postangioplasty, and the number of this kind of cells reached the peak on 7th day, and at the same time fibroblasts changed their phenotypes and became myofibroblasts, which produced α-actin and extracellular matrix (ECM). On 14th day, the number of the positive cells decreased in adventitia, increased in media and neointima associated with intima thickening; on 28th day, while the number of fibroblasts labeled by BrDU returned to the basic-line in adventitia, media and intima, nevertheless, intima thickening and vascular stenosis and intimal ELM precipitation were still present. There were significant differences in the number of fibroblasts labeled with BrDU located in three layers of artery (P<0.05). 2. Electronic microscope: After angioplasty, the plasm of fibroblasts became rich, mitochondrious and increase of Golgi apparatus; and the amount of rough endoplasmic reticulums rose with more secretory granules, together with a great amount of collagen synthesized forming the microfilaments; on days of 7th and 14th, the wide pseudopodia of myofibroblasts could be found extending into the windows on the external elastic lamina (ELL) and the internal elastic lamina (ILL); and showing the tendency

  4. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  5. Abdominal radiation causes bacterial translocation

    International Nuclear Information System (INIS)

    Guzman-Stein, G.; Bonsack, M.; Liberty, J.; Delaney, J.P.

    1989-01-01

    The purpose of this study was to determine if a single dose of radiation to the rat abdomen leads to bacterial translocation into the mesenteric lymph nodes (MLN). A second issue addressed was whether translocation correlates with anatomic damage to the mucosa. The radiated group (1100 cGy) which received anesthesia also was compared with a control group and a third group which received anesthesia alone but no abdominal radiation. Abdominal radiation lead to 100% positive cultures of MLN between 12 hr and 4 days postradiation. Bacterial translocation was almost nonexistent in the control and anesthesia group. Signs of inflammation and ulceration of the intestinal mucosa were not seen until Day 3 postradiation. Mucosal damage was maximal by Day 4. Bacterial translocation onto the MLN after a single dose of abdominal radiation was not apparently dependent on anatomical, histologic damage of the mucosa

  6. Electochemical detection of chromosome translocation

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Dimaki, Maria; Silahtaroglu, Asli

    2014-01-01

    Cytogenetics is a study of the cell structure with a main focus on chromosomes content and their structure. Chromosome abnormalities, such as translocations may cause various genetic disorders and heametological malignancies. Chromosome translocations are structural rearrangements of two...... chromosomes that results in formation of derivative chromosomes with a mixed DNA sequence. The method currently used for their detection is Fluorescent In Situ Hybridization, which requires a use of expensive, fluorescently labeled probes that target the derivative chromosomes. We present here a double...... hybridization approach developed for label-free detection of the chromosome translocations. For specific translocation detection it is necessary to determine that the two DNA sequences forming a derivative chromosome are connected, which is achieved by two subsequent hybridization steps. The electrochemical...

  7. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

    Science.gov (United States)

    Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

    2010-01-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

  8. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  9. Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

    OpenAIRE

    Tomasiewicz, H G; Cook-Deegan, R; Chikaraishi, D M

    1984-01-01

    We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

  10. Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jon M Carthy

    Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

  11. Radioactive cDNA microarray in neurospsychiatry

    International Nuclear Information System (INIS)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon

    2003-01-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  12. Radioactive cDNA microarray in neurospsychiatry

    Energy Technology Data Exchange (ETDEWEB)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon [Korea University Medical School, Seoul (Korea, Republic of)

    2003-02-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  13. Fibroblast growth factor 23

    African Journals Online (AJOL)

    Dr Olaleye

    Systemic phosphate homeostasis is maintained through several hormonal mechanisms which involve fibroblast growth factor 23 (FGF-23), α-klotho, vitamin D and parathyroid hormone. FGF-23 is known to be the major regulator of phosphate balance (Mirams et al., 2004). FGF-23 is a phosphaturic hormone, which is.

  14. Stability of the translocation frequency following whole-body irradiation measured in rhesus monkeys

    Science.gov (United States)

    Lucas, J. N.; Hill, F. S.; Burk, C. E.; Cox, A. B.; Straume, T.

    1996-01-01

    Chromosome translocations are persistent indicators of prior exposure to ionizing radiation and the development of 'chromosome painting' to efficiently detect translocations has resulted in a powerful biological dosimetry tool for radiation dose reconstruction. However, the actual stability of the translocation frequency with time after exposure must be measured before it can be used reliably to obtain doses for individuals exposed years or decades previously. Human chromosome painting probes were used here to measure reciprocal translocation frequencies in cells from two tissues of 8 rhesus monkeys (Macaca mulatta) irradiated almost three decades previously. Six of the monkeys were exposed in 1965 to whole-body (fully penetrating) radiation and two were unexposed controls. The primates were irradiated as juveniles to single doses of 0.56, 1.13, 2.00, or 2.25 Gy. Blood lymphocytes (and skin fibroblasts from one individual) were obtained for cytogenetic analysis in 1993, near the end of the animals' lifespans. Results show identical dose-response relationships 28 y after exposure in vivo and immediately after exposure in vitro. Because chromosome aberrations are induced with identical frequencies in vivo and in vitro, these results demonstrate that the translocation frequencies induced in 1965 have not changed significantly during the almost three decades since exposure. Finally, our emerging biodosimetry data for individual radiation workers are now confirming the utility of reciprocal translocations measured by FISH in radiation dose reconstruction.

  15. Isolation of a cDNA for a Growth Factor of Vascular Endothelial Cells from Human Lung Cancer Cells: Its Identity with Insulin‐like Growth Factor II

    Science.gov (United States)

    Hagiwara, Koichi; Kobayashi, Tatsuo; Tobita, Masato; Kikyo, Nobuaki; Yazaki, Yoshio

    1995-01-01

    We have found growth‐promoting activity for vascular endothelial cells in the conditioned medium of a human lung cancer cell line, T3M‐11. Purification and characterization of the growth‐promoting activity have been carried out using ammonium sulfate precipitation and gel‐exclusion chromatography. The activity migrated as a single peak just after ribonuclease. It did not bind to a heparin affinity column. These results suggest that the activity is not a heparin‐binding growth factor (including fibroblast growth factors) or a vascular endothelial growth factor. To identify the molecule exhibiting the growth‐promoting activity, a cDNA encoding the growth factor was isolated through functional expression cloning in COS‐1 cells from a cDNA library prepared from T3M‐11 cells. The nucleotide sequence encoded by the cDNA proved to be identical with that of insulin‐like growth factor II. PMID:7730145

  16. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

    International Nuclear Information System (INIS)

    Gronwald, R.G.K.; Grant, F.J.; Haldeman, B.A.; Hart, C.E.; O'Hara, P.J.; Hagen, F.S.; Ross, R.; Bowen-Pope, D.F.; Murray, M.J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A) + RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

  17. Differential Gene Expression of Fibroblasts: Keloid versus Normal

    Directory of Open Access Journals (Sweden)

    Michael F. Angel

    2002-11-01

    Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

  18. Tumor-produced, active Interleukin-1 β regulates gene expression in carcinoma-associated fibroblasts

    International Nuclear Information System (INIS)

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-01-01

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-β. PDL fibroblasts possess receptor for IL1-β, and its expression is increased 4.56-times in the presence of SCC-25 tumor cells. IL1-β receptor expression in

  19. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  20. Bacterial translocation: impact of probiotics

    OpenAIRE

    Jeppsson, Bengt; Mangell, Peter; Adawi, Diya; Molin, Göran

    2004-01-01

    There is a considerable amount of data in humans showing that patients who cannot take in nutrients enterally have more organ failure in the intensive care unit, a less favourable prognosis, and a higher frequency of septicaemia, in particular involving bacterial species from the intestinal tract. However, there is little evidence that this is connected with translocation of bacterial species in humans. Animal data more uniformly imply the existence of such a connection. The main focus of thi...

  1. Further studies on the possible relationship between radiation-induced reciprocal translocations and intrinsic radiosensitivity of human tumor cells

    International Nuclear Information System (INIS)

    Virsik-Peuckert, P.; Rave-Fraenk, M.; Schmidberger, H.

    1996-01-01

    Background and purpose. The aim of the present study was to estimate yields of radiation-induced translocations in surviving cells of several human tumor cell lines and in normal diploid human fibroblasts, and to compare these yields with corresponding intrinsic radiosensitivities determined by standard colony-formation assay. Material and methods. The yields of radiation-induced reciprocal translocations were investigated by fluorescence in situ hybridization. Chromosomes no. 1 and no. 4 were 'painted' with fluorescent hybridization probes for whole chromosomes. Translocation yields and cell survival were determined for different doses up to 6 Gy of 200 kV X-rays. Results. We observed a higher frequency of reciprocal translocations in the radiosensitive cells MCF-7 and MDA-MB-436 than in the radioresistant cells CaSki, WiDr, A549 and normal skin fibroblasts. For primary squamous cell carcinoma cells, ZMK-1, an intermediate radiosensitivity and an intermediate translocation yield were observed. The dose-dependence of translocation yields involving chromosomes no. 1 or no. 4 varied in different cell lines: it was linear or linear with a plateau at higher doses. Conclusions. A comparison of the data obtained with chromosomes no. 1 and no. 4 in the investigated cell types, indicates that intrinsic radiosensitivity of different tumor cells observed at the survival level, is correlated with different translocation yields, respectively. This correlation was observed for all cell types investigated, independent of the number of copies of the painted chromosome per cell or the radiation dose. However, for low doses (under 1 Gy), the yields of translocations determined for the individual chromosomes seem to be too low for a discrimination between radioresistant or radiosensitive cells

  2. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  3. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    the sequence of the cDNA for the porcine CPE gene including all the coding region and the 3'-UTR region was generated. Comparisons with bovine, human, mouse, and rat CPE cDNA sequences showed that the coding regions of the gene are highly conserved both at the nucleotide and at the amino acid level. A very low...

  4. Infectious Maize rayado fino virus from cloned cDNA

    Science.gov (United States)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  5. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Namhai Chua; Kush, A.

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids.

  6. Suitability of amphibians and reptiles for translocation.

    Science.gov (United States)

    Germano, Jennifer M; Bishop, Phillip J

    2009-02-01

    Translocations are important tools in the field of conservation. Despite increased use over the last few decades, the appropriateness of translocations for amphibians and reptiles has been debated widely over the past 20 years. To provide a comprehensive evaluation of the suitability of amphibians and reptiles for translocation, we reviewed the results of amphibian and reptile translocation projects published between 1991 and 2006. The success rate of amphibian and reptile translocations reported over this period was twice that reported in an earlier review in 1991. Success and failure rates were independent of the taxonomic class (Amphibia or Reptilia) released. Reptile translocations driven by human-wildlife conflict mitigation had a higher failure rate than those motivated by conservation, and more recent projects of reptile translocations had unknown outcomes. The outcomes of amphibian translocations were significantly related to the number of animals released, with projects releasing over 1000 individuals being most successful. The most common reported causes of translocation failure were homing and migration of introduced individuals out of release sites and poor habitat. The increased success of amphibian and reptile translocations reviewed in this study compared with the 1991 review is encouraging for future conservation projects. Nevertheless, more preparation, monitoring, reporting of results, and experimental testing of techniques and reintroduction questions need to occur to improve translocations of amphibians and reptiles as a whole.

  7. Markovian description of unbiased polymer translocation

    International Nuclear Information System (INIS)

    Mondaini, Felipe; Moriconi, L.

    2012-01-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations which provide compelling evidence in favor of a general Markovian framework for unbiased three-dimensional polymer translocation. Our statistical analysis consists of careful evaluations of (i) two-point correlation functions of the translocation coordinate and (ii) the empirical probabilities of complete polymer translocation (taken as a function of the initial number of monomers on a given side of the membrane). We find good agreement with predictions derived from the Markov chain approach recently addressed in the literature by the present authors. -- Highlights: ► We investigate unbiased polymer translocation through membrane pores. ► Large statistical ensembles have been produced with the help of cloud computing resources. ► We evaluate the two-point correlation function of the translocation coordinate. ► We evaluate empirical probabilities for complete polymer translocation. ► Unbiased polymer translocation is described as a Markov stochastic process.

  8. Markovian description of unbiased polymer translocation

    Energy Technology Data Exchange (ETDEWEB)

    Mondaini, Felipe [Instituto de Física, Universidade Federal do Rio de Janeiro, C.P. 68528, 21945-970 Rio de Janeiro, RJ (Brazil); Centro Federal de Educação Tecnológica Celso Suckow da Fonseca, UnED Angra dos Reis, Angra dos Reis, 23953-030, RJ (Brazil); Moriconi, L., E-mail: moriconi@if.ufrj.br [Instituto de Física, Universidade Federal do Rio de Janeiro, C.P. 68528, 21945-970 Rio de Janeiro, RJ (Brazil)

    2012-10-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations which provide compelling evidence in favor of a general Markovian framework for unbiased three-dimensional polymer translocation. Our statistical analysis consists of careful evaluations of (i) two-point correlation functions of the translocation coordinate and (ii) the empirical probabilities of complete polymer translocation (taken as a function of the initial number of monomers on a given side of the membrane). We find good agreement with predictions derived from the Markov chain approach recently addressed in the literature by the present authors. -- Highlights: ► We investigate unbiased polymer translocation through membrane pores. ► Large statistical ensembles have been produced with the help of cloud computing resources. ► We evaluate the two-point correlation function of the translocation coordinate. ► We evaluate empirical probabilities for complete polymer translocation. ► Unbiased polymer translocation is described as a Markov stochastic process.

  9. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  10. Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

    International Nuclear Information System (INIS)

    Tomkinson, B.; Jonsson, A-K

    1991-01-01

    Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

  11. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...library.zip File URL: ftp://ftp.biosciencedbc.jp/archive/kaiko-cdna/LATEST/kaiko_cdna_library.zip File size:... 4.8 KB Simple search URL http://togodb.biosciencedbc.jp/togodb/view/kaiko_cdna_l

  12. ISOLASI cDNA SUCROSE TRANSPORTER (SUT DARI BATANG TANAMAN TEBU (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    - Slameto

    2010-09-01

    Full Text Available Sucrose Transporter (SUT is kind of protein transporter that control in sucrose translocation. Sucrose Transporter is intermediate in translocation of sucrose from apoplasmic to simplasmic. SUT facilitates sucrose transportation from vascular tissues to parenchyma cells toward in node sugarcane stem. This research was purposed to isolate cDNA SUT from sugarcane stem, and cloned in Escherichia coli strain DH5α. Total RNA of sugarcane stem was isolated by single step method, then add with oligo dT in order to obtain the first strand of SUT cDNA then used as template for PCR. The primer used for PCR is 5’ –ggg ctg att gtg gcc atg tc- ‘3 (SUT-F and 5’ –tgc cct ttg tct ccg gaa cc- ‘3 (SUT-R. PCR was programmed as follow denaturation at 94°C for 2 minutes and 30 second, annealing at 54°C for 30 s, extension at 72°C 2 min and 7 min, and storage at 4°C for unlimited, It was for 30 cycles. Complementary DNA SUT from PCR ligalized to pTOPO bunt-end, then it cloned in to E. coli strain DH5α. The cloning resulted then be sequenced in order to observe the homologues with other nucleotides sequences of some plant using BLASTn program in GENE BANK NCBI and the level of homology determined by Genetyx program. The concentrated of total RNA isolated was 5,024 μg/μl, with purity of 1,85. Complementary DNA SUT fragment from PCR with size 2037 bp appropriated to the both of primer was used. Complementary DNA SUT fragment showed by analyzed some of restriction enzyme e.g. EcoRI, PstI and BamHI. Homologues of this cDNA SUT fragment was 100% to SoSUT 2A of sugarcane stem and 84% to OsSUT of rice plant (Casu et al ., 2003.

  13. Evodiamine attenuates TGF-β1-induced fibroblast activation and endothelial to mesenchymal transition.

    Science.gov (United States)

    Wu, Qing-Qing; Xiao, Yang; Jiang, Xiao-Han; Yuan, Yuan; Yang, Zheng; Chang, Wei; Bian, Zhou-Yan; Tang, Qi-Zhu

    2017-06-01

    The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

  14. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals......-treated fibroblasts. Transcriptional differences in basal and radiation-induced gene expression profiles were investigated using 15K cDNA microarrays, and results analyzed by both SAM and PAM. RESULTS: Sixty differentially expressed genes were identified by applying SAM on 10 patients with the highest risk of RIF...

  15. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  16. Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

    Directory of Open Access Journals (Sweden)

    Chung-Hsun Chang

    2014-11-01

    Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

  17. Phase 2 study of dovitinib in patients with relapsed or refractory multiple myeloma with or without t(4;14) translocation

    NARCIS (Netherlands)

    C. Scheid (Christof); D. Reece (Donna); M. Beksaç (Meral); A. Spencer (Andrew); N. Callander (Natalie); P. Sonneveld (Pieter); G. Kalimi (Ghulam); C. Cai (Can); M. Shi (Michael); J.W. Scott (Jeffrey W.); A.K. Stewart (Keith)

    2015-01-01

    textabstractObjectives: Approximately 15% of patients with multiple myeloma (MM) exhibit a t(4;14) translocation, which often results in constitutive activation of the receptor tyrosine kinase (RTK) fibroblast growth factor receptor 3 (FGFR3). This study evaluated the efficacy and safety of

  18. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation

    DEFF Research Database (Denmark)

    Barres, Romain; Grémeaux, Thierry; Gual, Philippe

    2006-01-01

    a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma m...

  19. Sequence of human protamine 2 cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Domenjoud, L; Fronia, C; Uhde, F; Engel, W [Universitaet Goettingen (West Germany)

    1988-08-11

    The authors report the cloning and sequencing of a cDNA clone for human protamine 2 (hp2), isolated from a human testis cDNA library cloned in the vector {lambda}-gt11. A 66mer oligonucleotide, that corresponds to an amino acid sequence which is highly conserved between hp2 and mouse protamine 2 (mp2) served as hybridization probe. The homology between the amino acid sequence deduced from our cDNA and the published amino acid sequence for hp2 is 100%.

  20. Translocations affecting human immunoglobulin heavy chain locus

    Directory of Open Access Journals (Sweden)

    Sklyar I. V.

    2014-03-01

    Full Text Available Translocations involving human immunoglobulin heavy chain (IGH locus are implicated in different leukaemias and lymphomas, including multiple myeloma, mantle cell lymphoma, Burkitt’s lymphoma and diffuse large B cell lymphoma. We have analysed published data and identified eleven breakpoint cluster regions (bcr related to these cancers within the IgH locus. These ~1 kbp bcrs are specific for one or several types of blood cancer. Our findings could help devise PCR-based assays to detect cancer-related translocations, to identify the mechanisms of translocations and to help in the research of potential translocation partners of the immunoglobulin locus at different stages of B-cell differentiation.

  1. Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components

    Czech Academy of Sciences Publication Activity Database

    Steringer, J. P.; Lange, S.; Čujová, Sabína; Šachl, Radek; Poojari, C.; Lolicato, F.; Beutel, O.; Müller, H.-M.; Unger, S.; Coskun, U.; Honigmann, A.; Vattulainen, I.; Hof, Martin; Freund, Ch.; Nickel, W.

    2017-01-01

    Roč. 6, č. 2017 (2017), č. článku e28985. ISSN 2050-084X R&D Projects: GA ČR(CZ) GC14-03141J Institutional support: RVO:61388955 Keywords : Unconventional protein secretion * Fibroblast Growth Factor 2 * Protein translocation across membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 7.725, year: 2016

  2. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  3. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    of medicine, animal husbandry, fish farming and animal ..... northern pike (Esox lucius) growth hormone; Mol. Mar. Biol. ... prolactin 1-luciferase fusion gene in African catfish and ... 1988 Cloning and sequencing of cDNA that encodes goat.

  4. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  5. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  6. The hallmarks of fibroblast ageing.

    Science.gov (United States)

    Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

    2014-06-01

    Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Translocality in Global Software Development

    DEFF Research Database (Denmark)

    Bjørn, Pernille; Søderberg, Anne-Marie; Krishna, S.

    2017-01-01

    . We explored how agile processes in global outsourcing impacts work conditions of the Indian IT developers, and were surprised to find that agile methodologies, even after 3 years of implementation, created a stressful and inflexible work environment negatively impacting their personal lives. Many......What happens when agile methods are introduced in global outsourcing set-ups? Agile methods are designed to empower IT developers in decision-making through self-managing collocated teams. We studied how agile methods were introduced into global outsourcing from the Indian IT vendor’s perspective...... of the negative aspects of work, which agile methodologies were developed to reduce, were evident in the global agile outsourcing set-up. We propose translocality to repudiate the dichotomy of global/local reminding us that methodologies and technologies must be understood as immediately localized and situated...

  8. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available of data contents Results of homology search to cDNA clones in the KOME. Data file File name: rpd_cdna.zip F...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...//togodb.biosciencedbc.jp/togodb/view/rpd_cdna#en Data acquisition method - Data

  9. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  10. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

    Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

    2017-06-01

    The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

  11. Cloning and characterization of the human colipase cDNA

    International Nuclear Information System (INIS)

    Lowe, M.E.; Rosenblum, J.L.; McEwen, P.; Strauss, A.W.

    1990-01-01

    Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a λgt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH 2 -terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. The authors report, for the first time, a cDNA for colipase. The cDNA predicts a human procolipase an suggests that there may also be processing at the COOH-terminus. The regions of identity with colipase from other species will aid in defining the interaction with lipase and lipids through site-specific mutagenesis

  12. Characterization of a human MSX-2 cDNA and its fragment isolated as a transformation suppressor gene against v-Ki-ras oncogene.

    Science.gov (United States)

    Takahashi, C; Akiyama, N; Matsuzaki, T; Takai, S; Kitayama, H; Noda, M

    1996-05-16

    A cDNA (termed CT124) encoding a carboxyl-terminal fragment of the human homeobox protein MSX-2 was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in NIH3T3 cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a near full-length human MSX-2 cDNA and tested its activities in two cell systems, i.e. fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated CT124 cDNA interfered with the transforming activities of v-Ki-ras oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and CT124 was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that CT124 may act as a dominant suppressor of MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

  13. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  14. cDNA microarray screening in food safety

    International Nuclear Information System (INIS)

    Roy, Sashwati; Sen, Chandan K.

    2006-01-01

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

  15. Cloning and expression of cDNA coding for bouganin.

    Science.gov (United States)

    den Hartog, Marcel T; Lubelli, Chiara; Boon, Louis; Heerkens, Sijmie; Ortiz Buijsse, Antonio P; de Boer, Mark; Stirpe, Fiorenzo

    2002-03-01

    Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

  16. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  17. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    1993-02-16

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a pu GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  18. Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

    Science.gov (United States)

    Galetzka, D; Weis, E; Rittner, G; Schindler, D; Haaf, T

    2008-01-01

    Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development. (c) 2008 S. Karger AG, Basel.

  19. Carbon and nitrogen translocation between seagrass ramets

    NARCIS (Netherlands)

    Marbà, N.; Hemminga, M.A.; Mateo, M.A.; Duarte, C.M.; Maas, Y.E.M.; Terrados, J.; Gacia, E.

    2002-01-01

    The spatial scale and the magnitude of carbon and nitrogen translocation was examined in 5 tropical (Cymodocea serrulata, Halophila stipulacea, Halodule uninervis, Thalassodendron ciliatum, Thalassia hemprichii) and 3 temperate (Cymodocea nodosa, Posidonia oceanica, Zostera noltii) seagrass species

  20. Dudleya Variegata Translocation - San Diego [ds654

    Data.gov (United States)

    California Department of Resources — At Mission Trails Regional Park, a translocation project of Dudleya variegata was conducted in efforts to save the population from a private property undergoing...

  1. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Dicty_cDB cDNA library information Data detail Data name cDNA library information DOI 10.189...s Data item Description cDNA library name Names of cDNA libraries (AF, AH, CF, CH, FC, FC-IC, FCL, SF, SH, S...(C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to construct cDNA library...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library... construction protocol Link to the webpage describing the protocol for generating cDNA library Size

  2. Cloning of the anhidrotic ectodermal dysplasia gene: Identification of cDNAs associated with CpG islands mapped near translocation breakpoint in two female patients

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, A.K.; Schlessinger, D. [Washington Univ. School of Medicine, St. Louis, MO (United States); Kere, J. [Univ. of Helsinki (Finland)] [and others

    1994-09-01

    The gene for the X chromosomal developmental disorder anhidrotic ectodermal dysplasia (EDA) has been mapped to Xq12-q13 by linkage analysis and is expressed in a few females with chromosomal translocations involving band Xq12-q13. A yeast artificial chromosome (YAC) contig (2.0 Mb) spanning two translocation breakpoints has been assembled by sequence-tagged site (STS)-based chromosomal walking. The two translocation breakpoints (X:autosome translocations from the affected female patients) have been mapped less than 60 kb apart within a YAC contig. Unique probes and intragenic STSs (mapped between the two translocations) have been developed and a somatic cell hybrid carrying the translocated X chromosome from the AK patient has been analyzed by isolating unique probes that span the breakpoint. Several STSs made from intragenic sequences have been found to be conserved in mouse, hamster and monkey, but we have detected no mRNAs in a number of tissues tested. However, a probe and STS developed from the DNA spanning the AK breakpoint is conserved in mouse, hamster and monkey, and we have detected expressed sequences in skin cells and cDNA libraries. In addition, unique sequences have been obtained from two CpG islands in the region that maps proximal to the breakpoints. cDNAs containing these sequences are being studied as candidates for the gene affected in the etiology of EDA.

  3. Cloning and expression of human deoxycytidine kinase cDNA

    International Nuclear Information System (INIS)

    Chottiner, E.G.; Shewach, D.S.; Datta, N.S.; Ashcraft, E.; Gribbin, D.; Ginsburg, D.; Fox, I.H.; Mitchell, B.S.

    1991-01-01

    Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, the authors have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine

  4. MOLECULAR CHARACTERIZATION OF ENDOCRINE DISRUPTION IN FISH USING CDNA ARRAYS.

    Science.gov (United States)

    We are developing cDNA macroarrays to measure the induction of gene expression in sheepshead minnows and largemouth bass exposed to anthropogenic chemicals that can mimic the action of endogenous hormones. For sheepshead minnows exposed in aqua, we observed similar genetic profil...

  5. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  6. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    Science.gov (United States)

    Normal Nasal Gene Expression Levels Using cDNA Array Technology. The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  7. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  8. Microprobe analysis of human fibroblasts

    International Nuclear Information System (INIS)

    Allan, G.L.; Zhu, J.; Legge, G.J.F.

    1985-01-01

    The Melbourne Proton Microprobe has been used to study the copper content in human skin fibroblast cells derived from patients with the genetic disease Menkes Syndrome. Both normal and diseased cells have been studied to investigate any elemental differences occurring between the two cell types. This paper details the preparatory techniques necessary for individual cell analysis and presents the elemental information with a new three dimensional contour mapping technique. These maps are used to highlight elemental differences between normal and mutant fibroblasts. The work also confirms the expected copper excess found in the Menkes cell and indicates that the microprobe can be used for rapid identification of a Menkes carrier

  9. Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

    Directory of Open Access Journals (Sweden)

    Wang Jun

    2012-10-01

    Full Text Available Abstract Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4 antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB, which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1, compared with transforming growth factor-β1 (TGF-β1. Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial

  10. Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

    Science.gov (United States)

    Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

    2012-10-17

    Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of

  11. Case report 511: Fibroblastic rheumatism

    International Nuclear Information System (INIS)

    Hernandez, R.J.; Martel, W.; Headington, J.T.; Kaufman, R.A.; Cincinnati Univ., OH

    1989-01-01

    We report a ten-year-old child with the newly described entity of fibroblastic rheumatism. This child developed rapid, progressive, symmetrical polyarthritis, similar to the radiographic appearance of juvenile rheumatoid arthritis, except for the rapidity of progression. The polyarthritis was preceded by the development of skin nodules with characteristic histological changes. (orig./GDG)

  12. Stochastic resonance during a polymer translocation process

    International Nuclear Information System (INIS)

    Mondal, Debasish; Muthukumar, M.

    2016-01-01

    We have studied the occurrence of stochastic resonance when a flexible polymer chain undergoes a single-file translocation through a nano-pore separating two spherical cavities, under a time-periodic external driving force. The translocation of the chain is controlled by a free energy barrier determined by chain length, pore length, pore-polymer interaction, and confinement inside the donor and receiver cavities. The external driving force is characterized by a frequency and amplitude. By combining the Fokker-Planck formalism for polymer translocation and a two-state model for stochastic resonance, we have derived analytical formulas for criteria for emergence of stochastic resonance during polymer translocation. We show that no stochastic resonance is possible if the free energy barrier for polymer translocation is purely entropic in nature. The polymer chain exhibits stochastic resonance only in the presence of an energy threshold in terms of polymer-pore interactions. Once stochastic resonance is feasible, the chain entropy controls the optimal synchronization conditions significantly.

  13. [Preparation of the cDNA microarray on the differential expressed cDNA of senescence-accelerated mouse's hippocampus].

    Science.gov (United States)

    Cheng, Xiao-Rui; Zhou, Wen-Xia; Zhang, Yong-Xiang

    2006-05-01

    Alzheimer' s disease (AD) is the most common form of dementia in the elderly. AD is an invariably fatal neurodegenerative disorder with no effective treatment. Senescence-accelerated mouse prone 8 (SAMP8) is a model for studying age-related cognitive impairments and also is a good model to study brain aging and one of mouse model of AD. The technique of cDNA microarray can monitor the expression levels of thousands of genes simultaneously and can be used to study AD with the character of multi-mechanism, multi-targets and multi-pathway. In order to disclose the mechanism of AD and find the drug targets of AD, cDNA microarray containing 3136 cDNAs amplified from the suppression subtracted cDNA library of hippocampus of SAMP8 and SAMR1 was prepared with 16 blocks and 14 x 14 pins, the housekeeping gene beta-actin and G3PDH as inner conference. The background of this microarray was low and unanimous, and dots divided evenly. The conditions of hybridization and washing were optimized during the hybridization of probe and target molecule. After the data of hybridization analysis, the differential expressed cDNAs were sequenced and analyzed by the bioinformatics, and some of genes were quantified by the real time RT-PCR and the reliability of this cDNA microarray were validated. This cDNA microarray may be the good means to select the differential expressed genes and disclose the molecular mechanism of SAMP8's brain aging and AD.

  14. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

    CSIR Research Space (South Africa)

    Van den Berg, N

    2004-11-01

    Full Text Available Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression...

  15. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ontents List of cDNA in locus Data file File name: astra_cdna.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_cdn...a.zip File size: 3.3 MB Simple search URL http://togodb.biosciencedbc.jp/togodb/view/astra_cdna...n, Department of Molecular Genetics, National Institute of Agrobiological Sciences (Kikuchi et al., 2003; ftp://cdna

  16. Molecular cloning of growth hormone encoding cDNA of Indian

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  17. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  18. The cDNA sequence of a neutral horseradish peroxidase.

    Science.gov (United States)

    Bartonek-Roxå, E; Eriksson, H; Mattiasson, B

    1991-02-16

    A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

  19. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  20. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  1. Brain cDNA clone for human cholinesterase

    International Nuclear Information System (INIS)

    McTiernan, C.; Adkins, S.; Chatonnet, A.; Vaughan, T.A.; Bartels, C.F.; Kott, M.; Rosenberry, T.L.; La Du, B.N.; Lockridge, O.

    1987-01-01

    A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase

  2. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  3. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  4. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  5. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin B; Friborg, Christel R; Schneider, Linda

    2005-01-01

    Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687...... accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol......: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation....

  6. Translocations used to generate chromosome segment duplications ...

    Indian Academy of Sciences (India)

    a duplication (Dp) of the translocated segment and four inviable (white, W) ascospores with .... of this work, namely, the definition of breakpoint junction sequences of 12 ..... then our results would place supercontig 10.9 in distal. LG VIR. A third ...

  7. Familial cryptic translocation in Angelman syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Weyerts, L.K.; Wiley, J.E.; Loud, K.M. [ECU School of Medicine, Greenville, NC (United States)] [and others

    1994-09-01

    The majority of patients with Angelman syndrome have been shown to have a cytogenetic or molecular deletion on the maternally derived chromosome 15. We report on a case of Angelman syndrome in which this deletion occurs as an unbalanced cryptic translocation involving chromosomes 14 and 15. The proband was diagnosed clinically as having Angelman syndrome. Multiple cytogenetic studies were done without detecting any deletion. When DNA probes (Oncor) specific for the Prader Willi/Angelman locus became available, the patient was restudied and found to be deleted for {open_quotes}region A{close_quotes} (D15S11) but not for {open_quotes}region B{close_quotes} (GABRB3). No other abnormality was detected. The proband`s mother was then studied. The chromosome 15 marker probe and D15S11 were detected on different chromosomes. Using alpha-satellite probes, a cryptic 14;15 translocation was uncovered. This balanced translocation was also found to be carried by the sister of the proband. This case, along with a case presented at the 1993 ASHG meeting, illustrates the need for using acrocentric probes when studying Angelman syndrome patients. The proband was studied using additional probes specific for this region and found to be deleted for SNRPN but not for D15S10. The breakpoint of the translocation in this patient delineates the smallest deletion of the Angelman syndrome region reported to date and therefore may represent the specific gene involved.

  8. Nitrogen uptake and translocation by Chara

    NARCIS (Netherlands)

    Vermeer, C.P.; Escher, M.; Portielje, R.; Klein, de J.J.M.

    2003-01-01

    The potential for above-ground and below-ground uptake and subsequent internal translocation of ammonium (NH4+) and nitrate (NO3-) by the macroalga Chara spp. was investigated. In a two compartment experimental set-up separating above-ground and below-ground algal parts, the charophytes were exposed

  9. 11C-methionine translocation in barley

    International Nuclear Information System (INIS)

    Nakanishi, Hiromi; Bughio, Naimatullah; Shigeta Ishioka, Noriko

    2000-01-01

    11 C-methionine was supplied to barley plants through a single leaf or via the roots and real time 11 C movement was monitored using a PETIS (positron emitting tracer imaging system). In Fe-deficient plants, 11 C-methionine was translocated from the tip of the absorbing leaf to the discrimination center' at the basal part of the shoot and then retranslocated to all the chlorotic leaves, while a negligible amount was retranslocated to the roots. In Fe-sufficient plants, methionine was translocated from the absorbing leaf to the discrimination center and then only to the newest leaf on the main shoot. A negligible amount was also retranslocated to the roots. Although, in Fe-sufficient plants, methionine translocation was observed from absorbing roots to shoots, in Fe-deficient plants, only a little amount was translocated from roots to shoots. In conclusion, methionine from the upper portion of a plant is not used as a precursor of mugineic acid under Fe-deficiency conditions. (author)

  10. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  11. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  12. Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

    Science.gov (United States)

    Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

    2018-03-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

  13. Translocation heterozygosity in southern African species of Viscum

    Directory of Open Access Journals (Sweden)

    D. Wiens

    1980-11-01

    Full Text Available Sex-associated and floating translocation complexes are characteristic of dioecious species of  Viscum,  but are virtually absent in monoecious species. The majority of dioecious species has fixed sex-associated translocation complexes with the male being the heterozygous sex. The sex-associated multivalent is usually O4 (ring-of-four or O6 , rarely O8 . Dioecious species without sex-associated translocations are much less common. Most of the dioecious species are also polymorphic for floating translocations, producing one or more additional multivalents ranging from O4 to O12. Floating translocations may be more frequent in species that do not have sex-associated translocations. Supernumerary chromosomes are also present in several species. Sex ratios are at unity in most dioecious species, but female-biased ratios may occur in some species. The high correlation between dioecy and translocation heterozygosity suggests that translocations are primarily associated with the origin and establishment of dioecy. Any róle in the maintenance of biased sex ratios through meiotic drive is probably secondary. Sex-associated translocations may serve to stabilize dioecy by bringing the sex factors into close linkage. Subsequent structural rearrangements within a sex-associated translocation complex may bring the sex factors together in one chromosome pair, releasing floating translocations. The high frequencies of floating translocation heterozygosity in some species indicate that such heterozygosity also has adaptive value.

  14. Cloning and analysis of the mouse Fanconi anemia group a cDNA and an overlapping penta zinc finger cDNA

    NARCIS (Netherlands)

    Wong, JCY; Alon, N; Norga, K; Kruyt, FAE; Youssoufian, H; Buchwald, M

    2000-01-01

    Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA

  15. Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

    Science.gov (United States)

    Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

    2011-01-01

    Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

  16. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  17. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  18. Obstructive jaundice promotes bacterial translocation in humans.

    Science.gov (United States)

    Kuzu, M A; Kale, I T; Cöl, C; Tekeli, A; Tanik, A; Köksoy, C

    1999-01-01

    Significant bacterial translocation was demonstrated following experimental biliary obstruction, however very little is known about the importance and the prevalence of gut-origin sepsis in obstructive jaundice patients. Therefore, the aim of this study was to investigate the concept of gut-origin sepsis in obstructive jaundiced patients and its clinical importance. Twenty-one patients requiring laparotomy for obstructive jaundice (group I) and thirty patients operated on electively mainly for chronic cholecystitis (group II) were studied. Peritoneal swab, mesenteric lymph node, portal venous blood, liver wedge biopsy and bile were sampled for culture immediately after opening the peritoneum. Additionally, peripheral blood samples were taken pre- and post-operatively from all patients. Post-operatively, patients were monitored for infectious complications. The mean serum bilirubin concentration, gamma glutamyl transferase and alkaline phosphatase levels in jaundiced patients before therapeutic intervention were significantly higher than in control patients. Five patients demonstrated bacterial translocation in group I (24%), whereas only one did so in group II (3.5%, p jaundice significantly promotes bacterial translocation in humans, however, its clinical importance has yet to be defined.

  19. Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros.

    Science.gov (United States)

    Ishibashi, J; Saido-Sakanaka, H; Yang, J; Sagisaka, A; Yamakawa, M

    1999-12-01

    A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH2 (Ala22-Lys30-NH2), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH2, ALLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2. These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria. The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH2.

  20. Enhance and Maintain Chondrogenesis of Synovial Fibroblasts by Cartilage Extracellular Matrix Protein Matrilins

    Science.gov (United States)

    Pei, Ming; Luo, Junming; Chen, Qian

    2008-01-01

    Summary Objective Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN) 1 and 3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Methods Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time RT-PCR, while the protein levels of Col II and matrilins were determined by western blot analysis. Results SFBs underwent chondrogenesis after incubation with TGF-β1 for three days; however, this process was attenuated during the subsequent incubation period. Expression of a MATN1 or 3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of glycosaminoglycans, and stimulated expression of chondrogenic markers. Conclusion Our findings suggest a novel function for MATN1 and 3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-β. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation. PMID:18282772

  1. Enhancing and maintaining chondrogenesis of synovial fibroblasts by cartilage extracellular matrix protein matrilins.

    Science.gov (United States)

    Pei, M; Luo, J; Chen, Q

    2008-09-01

    Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN)1 and MATN3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans (GAG) with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time reverse transcription polymerase chain reaction, while the protein levels of Col II and MATNs were determined by western blot analysis. SFBs underwent chondrogenesis after incubation with transforming growth factor-beta1 (TGF-beta1) for 3 days; however, this process was attenuated during the subsequent incubation period. Expression of a Matn1 or Matn3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of GAG, and stimulated expression of chondrogenic markers. Our findings suggest a novel function for MATN1 and MATN3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-beta. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation.

  2. cDNA sequences of two apolipoproteins from lamprey

    International Nuclear Information System (INIS)

    Pontes, M.; Xu, X.; Graham, D.; Riley, M.; Doolittle, R.F.

    1987-01-01

    The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the high-density lipoprotein fraction of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point

  3. Infectious Maize rayado fino virus from Cloned cDNA.

    Science.gov (United States)

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  4. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  5. Radiation induced reciprocal translocations and inversions in anopheles albimanus

    International Nuclear Information System (INIS)

    Kaiser, P.E.; Seawright, J.A.; Benedict, M.Q.; Narang, S.

    1982-01-01

    Reciprocal translocations and inversions were induced in Anopheles albimanus Wiedemann by irradiation of males with X rays. A total of 1669 sperm were assayed, and 175 new aberrations were identified as follows: 102 reciprocal translocations (67 autosomal and 35 sex-linked), 45 pericentric inversions, and 28 paracentric inversions. Eleven of the translocations were nearly whole-arm interchanges, and these were selected for the construction of 'capture systems' for compound chromosomes. Two double-heterozygous translocation strains and four homozygous translocation strains were established. Anopheles albimanus females were irradiated, and a pseudolinkage scheme involving mutant markers was employed to identify reciprocal translocations. The irradiation of females was very inefficient; only one translocation was recovered from 1080 ova tested

  6. Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

    Science.gov (United States)

    Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

    2018-01-15

    Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. IMPORTANCE A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in

  7. Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

    Directory of Open Access Journals (Sweden)

    Min Ji Choi

    2015-06-01

    Full Text Available Transforming growth factor-β1 (TGF-β1 has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD. The mothers against decapentaplegic homolog 7 (SMAD7 is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator and induced the expression of poly (ADP-ribose polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

  8. Radiosensitivity in cultured human fibroblasts

    International Nuclear Information System (INIS)

    Cox, R.; Masson, W.K.

    1980-01-01

    Caution is urged in the use of freshly isolated cultures of human diploid fibroblasts for quantitative studies of radiosensitivity. The distribution of x ray sensitivities of 'normal' human fibroblast cultures of foetal origin (10 subjects, skin or lung biopsy) and post-foetal origin (34 subjects, skin biopsy) are compared with the distribution in 12 patients with ataxia telangiectasia (probability of including any one of these in a normal post-foetal distribution is 0.01%). Cultures from nominally normal subjects showed a broad distribution of D 0 range of 98 +- 160 rad and assuming normal distribution, a mean +- one standard deviation of 122 +- 17 rad. Mean D 0 values for foetal origin cultures were 117 +- 12; values for post-foetal cultures D 0 were 124 +- 18. No systematic variation in D 0 was observed for age of donor, number of cell divisions in culture or for cloning efficiency. For ataxia telangiectasia D 0 values were 46 +- 7 rad. (U.K.)

  9. Financial costs of large carnivore translocations--accounting for conservation.

    Directory of Open Access Journals (Sweden)

    Florian J Weise

    Full Text Available Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars. Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23, and $2,108 per leopard (n = 6. One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%, followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4% of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown with a strong species bias. Four leopards (66.7% were successfully translocated but only eight of the 20 cheetahs (40.0% with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  10. Financial costs of large carnivore translocations--accounting for conservation.

    Science.gov (United States)

    Weise, Florian J; Stratford, Ken J; van Vuuren, Rudolf J

    2014-01-01

    Human-carnivore conflict continues to present a major conservation challenge around the world. Translocation of large carnivores is widely implemented but remains strongly debated, in part because of a lack of cost transparency. We report detailed translocation costs for three large carnivore species in Namibia and across different translocation scenarios. We consider the effect of various parameters and factors on costs and translocation success. Total translocation cost for 30 individuals in 22 events was $80,681 (US Dollars). Median translocation cost per individual was $2,393, and $2,669 per event. Median cost per cheetah was $2,760 (n = 23), and $2,108 per leopard (n = 6). One hyaena was translocated at a cost of $1,672. Tracking technology was the single biggest cost element (56%), followed by captive holding and feeding. Soft releases, prolonged captivity and orphaned individuals also increased case-specific costs. A substantial proportion (65.4%) of the total translocation cost was successfully recovered from public interest groups. Less than half the translocations were confirmed successes (44.4%, 3 unknown) with a strong species bias. Four leopards (66.7%) were successfully translocated but only eight of the 20 cheetahs (40.0%) with known outcome met these strict criteria. None of the five habituated cheetahs was translocated successfully, nor was the hyaena. We introduce the concept of Individual Conservation Cost (ICC) and define it as the cost of one successfully translocated individual adjusted by costs of unsuccessful events of the same species. The median ICC for cheetah was $6,898 and $3,140 for leopard. Translocations are costly, but we demonstrate that they are not inherently more expensive than other strategies currently employed in non-lethal carnivore conflict management. We conclude that translocation should be one available option for conserving large carnivores, but needs to be critically evaluated on a case-by-case basis.

  11. A rapid method for screening arrayed plasmid cDNA library by PCR

    International Nuclear Information System (INIS)

    Hu Yingchun; Zhang Kaitai; Wu Dechang; Li Gang; Xiang Xiaoqiong

    1999-01-01

    Objective: To develop a PCR-based method for rapid and effective screening of arrayed plasmid cDNA library. Methods: The plasmid cDNA library was arrayed and screened by PCR with a particular set of primers. Results: Four positive clones were obtained through about one week. Conclusion: This method can be applied to screening not only normal cDNA clones, but also cDNA clones-containing small size fragments. This method offers significant advantages over traditional screening method in terms of sensitivity, specificity and efficiency

  12. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  13. Measurement of background translocation frequencies in individuals with clones

    Energy Technology Data Exchange (ETDEWEB)

    Wade, Marcelle J. [California State Univ. (CalState), Hayward, CA (United States)

    1996-08-01

    In the leukemia case the unseparated B and T lymphocytes had a high translocation frequency even after 0.0014, respectively. After purging all clones from the data, the translocation frequencies for Bio 8 and Bio 23 were 0.00750.0014 and 0.0073 metaphases were scored for chromosomal aberrations,, specifically reciprocal translocations, using fluorescence in situ hybridization (FISH). Metaphase spreads were used from two healthy, unexposed individuals (not exposed to radiation, chemotherapy or radiotherapy) and one early B- precursor acute lymphocytic leukemia (ALL) patient (metaphase spreads from both separated T lymphocytes and unseparated B and T lymphocytes were scored). All three individuals had an abnormally high translocation frequency. The high translocation frequencies resulted from clonal expansion of specific translocated chromosomes. I show in this thesis that by purging (discounting or removing) clones from the data of unexposed individuals, one can obtain true background translocation frequencies. In two cases, Bio 8 and Bio 23, the measured translocation frequency for chromosomes 1, 2 and 4 was 0.0124 purging all of the clones from the data. This high translocation frequency may be due to a low frequency of some clones and may not be recognized. The separated T lymphocytes had a higher translocation frequency than expected.

  14. Signal Transduction Pathways (MAPKs, NF-κB, and C/EBP) Regulating COX-2 Expression in Nasal Fibroblasts from Asthma Patients with Aspirin Intolerance

    Science.gov (United States)

    Garcia-Garcia, Francesc Josep; Mullol, Joaquim; Perez-Gonzalez, Maria; Pujols, Laura; Alobid, Isam

    2012-01-01

    Background Recent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-κB and C/EBP) are involved in COX-2 regulation. Objective To investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA). Methods Fibroblasts were isolated from specimens of nasal mucosa (NM, N = 5) and nasal polyps (NP, N = 5). After IL-1β (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK’s role in IL-1β-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1–10 µM) prior to IL-1β exposure. NF-κB and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1β (10 ng/ml) exposure. Results No differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1β-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1β on NF-κB and C/EBP subunits’ nuclear translocation was similar between NM and NP-AIA fibroblasts. Conclusions These results suggest that p38 MAPK is the only MAPK involved in IL-1β-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-κB and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation. PMID:23240010

  15. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  16. Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie’s Plaque

    Directory of Open Access Journals (Sweden)

    Dong Hyuk Kang

    2018-05-01

    Full Text Available Purpose: Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs in fibroblasts isolated from plaque tissue of Peyronie’s disease (PD or normal tunica albuginea (TA and to examine the anti-fibrotic effect of small interfering RNA (siRNA-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque. Materials and Methods: For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11 in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL. Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation. Results: The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein. Conclusions: These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

  17. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  18. Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Harper, J.F.; Surowy, T.K.; Sussman, M.R.

    1989-01-01

    In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H + -ATPase) that produces an electric potential and pH gradient. The authors isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H + -ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plant contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops

  19. Impact of matrix stiffness on fibroblast function

    Energy Technology Data Exchange (ETDEWEB)

    El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

    2017-05-01

    Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

  20. Longing Itineraries: Building the Translocal Community

    Directory of Open Access Journals (Sweden)

    Gustavo López Angel

    2017-06-01

    Full Text Available Migration has reshaped social practices, the sense of belonging has been rethought, and the membership is renegotiated and contended; this is why strategies for their sustainability have been generated. The translocal community operates through multilocated relationships that reveal the ways in which migrants are adapting to the new demands of the community. We emphasize the emotional impulse of nostalgia as one of the vehicles of sustainability for the community. The community is redefined and understood in a set of socio-cultural relationships its members generate, and where the locality is not central, but the connection. A new dimension of the social community space is not just the community gathered in a specific place, but also that agreements, commitments, and acknowledgments are exhibited and settled in the cyberspace; this cyberspace gives cohesion and brings a dynamic element to preserve the community, despite the fact that it is even less concrete than the spatial notion of territory. Facebook, YouTube and a blog are the web platforms of the virtual space where "neighbors, compatriots and citizens" (categories of ascription from the migration get together, where there is a reproduction of social practices (even the most ancient and fundamental ones, to give a new dimension to a translocal, multilocated and ciberlocated community.

  1. Another reptile translocation to a national park

    Directory of Open Access Journals (Sweden)

    W.R. Branch

    1990-10-01

    Full Text Available On 4 May 1988 a sub-adult (50 mm snout-vent length, 42 mm tail Jones' girdled lizard Cordylus tropidosternum jonesi was collected in a pile of wood being off-loaded at the new restcamp in the Karoo National Park, Beaufort West. The wood had been transported by lorry from the Kruger National Park. The specimen is deposited in the herpetological collection of the Port Elizabeth Museum (PEM R 4584. Jones' girdled lizard is a small, arboreal cordylid that shelters under tree bark and in hollow logs. It is common and widely-distributed in the Kruger National Park (Pienaar, Haacke & Jacobsen 1983, The Reptiles of the Kruger National Park, 3rd edition. Pretoria: National Parks Board and adjacent lowveld, being replaced in northern Zimbabwe and East Africa by the nominate race. Hewitt & Power (1913, Transactions of the Royal Society of South Africa 3: 147-176, 1913 reported a similar translocation of the species to Kimberley in association with timber brought to the diamond mining camps. One of us noted recently the ease and danger of the unwitting spread of commensal reptile species into conservation areas (Branch 1978, Koedoe 30: 165, and this is confirmed by this additional example. We recommend that should similar shipments of wood be considered essential, then they be fumigated to prevent the translocation of other alien organisms that may potentially have more dangerous consequences.

  2. Arsenic Uptake and Translocation in Plants.

    Science.gov (United States)

    Li, Nannan; Wang, Jingchao; Song, Won-Yong

    2016-01-01

    Arsenic (As) is a highly toxic metalloid that is classified as a non-threshold class-1 carcinogen. Millions of people worldwide suffer from As toxicity due to the intake of As-contaminated drinking water and food. Reducing the As concentration in drinking water and food is thus of critical importance. Phytoremediation of soil contaminated with As and the reduction of As contamination in food depend on a detailed understanding of As uptake and transport in plants. As transporters play essential roles in As uptake, translocation and accumulation in plant cells. In this review, we summarize the current understanding of As transport in plants, with an emphasis on As uptake, mechanisms of As resistance and the long-distance translocation of As, especially the accumulation of As in grains through phloem-mediated transport. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

    Science.gov (United States)

    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  4. Factors affecting translocation and sclerotial formation in Morchella esculenta

    International Nuclear Information System (INIS)

    Amir, R.; Levanon, D.; Hadar, Y.; Chet, I.

    1995-01-01

    Amir, R., Levanon, D., Hadar, Y., and Chet, I. 1995. Factors affecting translocation and sclerotial formation in Morchella esculenta. Experimental Mycology 19, 61-70. Morchella esculenta was grown on square split plates, forming sclerotia on one side and mycelium on the other. After the fungus ceased to colonize and before sclerotial initials appeared, [ 14 C]3-O-methyl glucose was added to the edge of the plate on the mycelial side. The effect of various activities in the mycelium (source) and sclerotia (sink) on sclerotial formation and translocation were examined using inhibitors and water potential changes of the media. Sodium azide or cycloheximide applied separately to both sides inhibited both sclerotial formation and translocation, showing that processes in the source and sink depend on metabolic activities as well as protein synthesis. The use of nikkomycin inhibited sclerotial formation, without affecting translocation to the sclerotia. Since the hyphal tips swelled and burst, the translocated compounds were lost to the media. In a strain defective in sclerotial formation, used as a control, no translocation took place, showing that there is a connection between sclerotial formation and translocation. Reversal of the water potential gradient between the two media (lower on the mycelial side), reduced the formation of sclerotia and translocation to them. Translocation to Morchella sclerotia takes place via turgor driven mass flow, but is nevertheless affected by activities in both the source and the sink. (author)

  5. Label Free Chromosome Translocation Detection with Silicon nanowires

    DEFF Research Database (Denmark)

    Kwasny, Dorota; Andersen, Karsten Brandt; Frøhling, Kasper Bayer

    HROMOSOME translocation, which is a rearrangement of arms between two chromosomes, is a major group of chromosome abnormalities leading to cancer. As a result, two derivative chromosomes with sequences coming from both chromosomes are formed. The current translocation detection method is a Fluore......HROMOSOME translocation, which is a rearrangement of arms between two chromosomes, is a major group of chromosome abnormalities leading to cancer. As a result, two derivative chromosomes with sequences coming from both chromosomes are formed. The current translocation detection method...

  6. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

    1989-01-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K m , low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  7. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  8. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

  9. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Science.gov (United States)

    Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  10. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-05-01

    Full Text Available In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  11. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  12. [Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

    Science.gov (United States)

    Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

    2007-02-01

    To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

  13. Fibroblast cultures in duchenne muscular dystrophy

    International Nuclear Information System (INIS)

    Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

    1977-01-01

    Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

  14. Translocation of cell-penetrating peptides into Candida fungal pathogens.

    Science.gov (United States)

    Gong, Zifan; Karlsson, Amy J

    2017-09-01

    Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF) 3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF) 3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens. © 2017 The Protein Society.

  15. Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

    International Nuclear Information System (INIS)

    Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana

    2005-01-01

    Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH 2 -terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process

  16. Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

    Science.gov (United States)

    Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

    2014-05-02

    The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

  17. Fibroblasts in fibrosis: novel roles and mediators

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Kendall

    2014-05-01

    Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

  18. Aryl hydrocarbon receptor–ligand axis mediates pulmonary fibroblast migration and differentiation through increased arachidonic acid metabolism

    International Nuclear Information System (INIS)

    Su, Hsiang-Han; Lin, Hsin-Ting; Suen, Jau-Ling; Sheu, Chau Chyun; Yokoyama, Kazunari K.; Huang, Shau-Ku; Cheng, Chih Mei

    2016-01-01

    Pulmonary fibroblast migration and differentiation are critical events in fibrogenesis; meanwhile, fibrosis characterizes the pathology of many respiratory diseases. The role of aryl hydrocarbon receptor (AhR), a unique cellular chemical sensor, has been suggested in tissue fibrosis, but the mechanisms through which the AhR-ligand axis influences the fibrotic process remain undefined. In this study, the potential impact of the AhR-ligand axis on pulmonary fibroblast migration and differentiation was analyzed using human primary lung fibroblasts HFL-1 and CCL-202 cells. Boyden chamber-based cell migration assay showed that activated AhR in HFL-1cells significantly enhanced cell migration in response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), and a known AhR antagonist, CH223191, inhibited its migratory activity. Furthermore, the calcium mobilization and subsequent upregulated expression of arachidonic acid metabolizing enzymes, including cyclooxygenase2 (COX-2) and 5-lipoxygenase (5-LOX), were observed in TCDD-treated HFL-1 cells, concomitant with elevated levels of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) secretion. Also, significantly increased expression of α-smooth muscle actin α-SMA), a fibroblast differentiation marker, was also noted in TCDD-treated HFL-1 cells (p < 0.05), resulting in a dynamic change in cytoskeleton protein levels and an increase in the nuclear translocation of the myocardin-related transcription factor. Moreover, the enhanced levels of α-SMA expression and fibroblast migration induced by TCDD, PGE2 and LTB4 were abrogated by selective inhibitors for COX-2 and 5-LOX. Knockdown of AhR by siRNA Completely diminished intracellular calcium uptake and reduced α-SMA protein verified by promoter-reporter assays and chromatin immunoprecipitation. Taken together, our results suggested the importance of the AhR-ligand axis in fibroblast migration and differentiation through its capacity in enhancing arachidonic acid metabolism.

  19. An Updated View of Translocator Protein (TSPO

    Directory of Open Access Journals (Sweden)

    Nunzio Denora

    2017-12-01

    Full Text Available Decades of study on the role of mitochondria in living cells have evidenced the importance of the 18 kDa mitochondrial translocator protein (TSPO, first discovered in the 1977 as an alternative binding site for the benzodiazepine diazepam in the kidneys. This protein participates in a variety of cellular functions, including cholesterol transport, steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and cell proliferation. Thus, TSPO has become an extremely attractive subcellular target for the early detection of disease states that involve the overexpression of this protein and the selective mitochondrial drug delivery. This special issue was programmed with the aim of summarizing the latest findings about the role of TSPO in eukaryotic cells and as a potential subcellular target of diagnostics or therapeutics. A total of 9 papers have been accepted for publication in this issue, in particular, 2 reviews and 7 primary data manuscripts, overall describing the main advances in this field.

  20. CDNA library from the Latex of Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Wilaiwan Chotigeat

    2010-12-01

    Full Text Available Latex from Hevea brasiliensis contains 30-50% (w/w of natural rubber (cis-1,4-polyisoprene, the important rawmaterial for many rubber industries. We have constructed a cDNA library from the latex of H. brasiliensis to investigate theexpressed genes and molecular events in the latex. We analyzed 412 expressed sequence tags (ESTs. More than 90% of theEST clones showed homology to previously described sequences in public databases. Functional classification of the ESTsshowed that the largest category were proteins of unknown function (30.1%, 11.4% of ESTs encoded for rubber synthesisrelatedproteins (RS and 8.5% for defense or stress related proteins (DS. Those with no significant homology to knownsequences (NSH accounted for 8.7%, primary metabolism (PM and gene expression and RNA metabolism were 7.8% and6.6%, respectively. Other categories included, protein synthesis-related proteins (6.6%, chromatin and DNA metabolism(CDM 3.9%, energy metabolism (EM 3.4%, cellular transport (CT 3.2%, cell structure (CS 3.2%, signal transduction (ST2.2%, secondary metabolism (SM 1.7%, protein fate (PF 2.2%, and reproductive proteins (RP 0.7%.

  1. Delayed reproduction of translocated red-cockaded woodpeckers

    Science.gov (United States)

    James R. McCormick; Richard N. Conner; Daniel Saenz; Brent Burt

    2001-01-01

    Twelve pairs of Red-cockaded Woodpeckers were translocated to the Angelina National Forest from 21 October 1998 to 17 December 1998. Five breeding pairs (consisting of at least one trnnslocated bird) produced eggs/nestlings within the first breeding season after translocation. Clutch initiation dates for all five pairs were later than those of resident breeders. The...

  2. Chromosomal Translocations: Chicken or Egg? | Center for Cancer Research

    Science.gov (United States)

    Many tumor cells have abnormal chromosomes. Some of these abnormalities are caused by chromosomal translocations, which occur when two chromosomes break and incorrectly rejoin, resulting in an exchange of genetic material. Translocations can activate oncogenes, silence tumor suppressor genes, or result in the creation of completely new fusion gene products. While there is

  3. Multistep Current Signal in Protein Translocation through Graphene Nanopores

    KAUST Repository

    Bonome, Emma Letizia

    2015-05-07

    © 2015 American Chemical Society. In nanopore sensing experiments, the properties of molecules are probed by the variation of ionic currents flowing through the nanopore. In this context, the electronic properties and the single-layer thickness of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics methods with a bioinformatic structural analysis. To obtain a qualitative picture of the translocation process and to identify salient features we performed unsupervised structural clustering on translocation conformations. This allowed us to identify some specific and robust translocation intermediates, characterized by significantly different ionic current flows. We found that the ion current strictly anticorrelates with the amount of pore occupancy by thioredoxin residues, providing a putative explanation of the multilevel current scenario observed in recently published translocation experiments.

  4. Fiscal 2000 report on result of the full-length cDNA structure analysis; 2000 nendo kanzen cho cDNA kozo kaiseki seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    This paper explains the results of research on full-length cDNA structure analysis for the period from April, 2000 to March, 2001. The outline of human genome sequence was published in June, 2000. In Japan, human gene analysis was such that, as the basic technology of the bio industry, a millennium project was decided in the budget of fiscal 2000. The full-length cDNA structure analysis is the core of the project. The libraries of cDNA were prepared using full-length and more than 4-5kbp-long cDNAs by oligo-capping method. It began from determining partial sequence data at end cDNA, and then, with new clones selected therefrom, full-length human cDNA sequence data were determined. The partial sequence data determined by fiscal 2000 were 1,035,000 clones while the full-length sequence data were 12,144 clones. The sequence data obtained were analyzed by homology search and translated into amino acid coding sequences, with predictions conducted on protein functions. A clustering method was examined that selects new clones from partial sequences. Database was constructed on gene expression profiles and disease-related gene sequence data. (NEDO)

  5. RICD: A rice indica cDNA database resource for rice functional genomics

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  6. Mechanisms underlying stage-1 TRPL channel translocation in Drosophila photoreceptors.

    Directory of Open Access Journals (Sweden)

    Minh-Ha Lieu

    Full Text Available TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere, TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels.We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content.Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a light-dependent anchor in the rhabdomere.

  7. Construction and characterization of cDNA library for IRM-2 mice

    International Nuclear Information System (INIS)

    Wang Qin; Li Jin; Song Li; Liu Qiang; Yue Jingyin; Mu Chuanjie; Tang Weisheng; Fan Feiyue

    2010-01-01

    Objective: To screen and isolate the radioresistance related genes of IRM-2 mice. Methods: cDNA library of IRM-2 mice was constructed by SMART technique. Total RNA was isolated from spleens of IRM-2 male mice. The first-strand cDNA was synthesized by using PowerScript reverse transcriptase, and double-strand cDNA was synthesized and amplified by long PCR. The PCR products were purified, digested with restriction enzyme Sfi I. The ds-cDNA fragment less than 500 bp was fractionated and ligated to the Sfi I-digested pDNR-LIB vector. The ligation mixture was transformed into E. coil DH5 α by electroporation transformation to generate the unamplified cDNA library. The quality of cDNA library was identified by PCR technique. 130 clones from cDNA library were sequenced and compared with GenBank database. Results: The cDNA library contained 2.25 x 10 6 independent clones with an average insert size of 1.2 kb. The ratio of recombination and full-length was 95% and 55%, respectively. 21 pieces of EST sequences from cDNA library were not the same as the known mice genes and registered into GenBank EST database, with registered number DW474856-DW474876. Conclusions: cDNA library of IRM-2 mice has been constructed successfully. 21 pieces of EST implies that radioresistance correlative genes may be in IRM-2 mice, which will lay a foundation for isolating and identifying radioresistance related genes in further study. (authors)

  8. Minimizing the cost of translocation failure with decision-tree models that predict species' behavioral response in translocation sites.

    Science.gov (United States)

    Ebrahimi, Mehregan; Ebrahimie, Esmaeil; Bull, C Michael

    2015-08-01

    The high number of failures is one reason why translocation is often not recommended. Considering how behavior changes during translocations may improve translocation success. To derive decision-tree models for species' translocation, we used data on the short-term responses of an endangered Australian skink in 5 simulated translocations with different release conditions. We used 4 different decision-tree algorithms (decision tree, decision-tree parallel, decision stump, and random forest) with 4 different criteria (gain ratio, information gain, gini index, and accuracy) to investigate how environmental and behavioral parameters may affect the success of a translocation. We assumed behavioral changes that increased dispersal away from a release site would reduce translocation success. The trees became more complex when we included all behavioral parameters as attributes, but these trees yielded more detailed information about why and how dispersal occurred. According to these complex trees, there were positive associations between some behavioral parameters, such as fight and dispersal, that showed there was a higher chance, for example, of dispersal among lizards that fought than among those that did not fight. Decision trees based on parameters related to release conditions were easier to understand and could be used by managers to make translocation decisions under different circumstances. © 2015 Society for Conservation Biology.

  9. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

  10. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    International Nuclear Information System (INIS)

    Cai Xuwei; Fu Xiaolong; Yang Jian; Song Houyan

    2005-01-01

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- X TM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-X TM genome, which was transformed into E. coli to get recombinant Adeno-X TM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 10 11 TCID 50 /ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-X TM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

  11. N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

    LENUS (Irish Health Repository)

    Burke, John P

    2012-02-01

    BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

  12. Forced Translocation of Polymer through Nanopore: Deterministic Model and Simulations

    Science.gov (United States)

    Wang, Yanqian; Panyukov, Sergey; Liao, Qi; Rubinstein, Michael

    2012-02-01

    We propose a new theoretical model of forced translocation of a polymer chain through a nanopore. We assume that DNA translocation at high fields proceeds too fast for the chain to relax, and thus the chain unravels loop by loop in an almost deterministic way. So the distribution of translocation times of a given monomer is controlled by the initial conformation of the chain (the distribution of its loops). Our model predicts the translocation time of each monomer as an explicit function of initial polymer conformation. We refer to this concept as ``fingerprinting''. The width of the translocation time distribution is determined by the loop distribution in initial conformation as well as by the thermal fluctuations of the polymer chain during the translocation process. We show that the conformational broadening δt of translocation times of m-th monomer δtm^1.5 is stronger than the thermal broadening δtm^1.25 The predictions of our deterministic model were verified by extensive molecular dynamics simulations

  13. [Clinical characteristics and preimplantation genetic diagnosis for male Robertsonian translocations].

    Science.gov (United States)

    Huang, Jin; Lian, Ying; Qiao, Jie; Liu, Ping

    2012-08-18

    To explore the clinical characteristics and the preimplantation genetic diagnosis (PGD) for male Robertsonian translocations. From Jan 2005 to Oct 2011, 96 PGD cycles of 80 male Robertsonian translocations were performed at the Center of Reproductive Medicine of Peking University Third Hospital, Beijing. All the couples were involved in assisted reproductive therapy because of oligozoospermia or repeated abortions. Pregnancy results and clinical characteristics were analyzed in this study. Of all the 80 Robertsonian translocation couples, 62 (77.50%, 62/80) couples suffered from primary infertility due to severe oligoospermia and 8 (10%, 8/80) couples suffered from secondary infertility due to oligoospermia. Moreover, 10 (12.50%, 10/80) couples had recurrent spontaneous abortion. Of all the 80 male Robertsonian translocations, 50 were (13; 14) translocations and 15 (14; 21) translocations. The study showed that 79 PGD cycles had the balanced embryos to transfer and 25 cycles resulted in clinical pregnancies. The clinical pregnancy rate per transfer cycle was 31.65% (25 of 79). Now, 18 couples had 21 viable infants and 3 were ongoing pregnant. Oligozoospermia is the main factor for the infertility of the male Robertsonian translocations. Artificial reproductive techniques can solve their reproductive problems. Moreover, PGD will decrease the risk of recurrent spontaneous abortion and the malformations.

  14. Genetic outcomes from the translocations of the critically endangered woylie

    Directory of Open Access Journals (Sweden)

    Carlo PACIONI, Adrian F.WAYNE, Peter B.S.SPENCER

    2013-06-01

    Full Text Available Translocations are an important conservation strategy for many species. However simply observing demographic growth of a translocated population is not sufficient to infer species recovery. Adequate genetic representation of the source population(s and their long-term viability should also be considered. The woylie Bettongia penicillata ogilbyi has been subject to more formal translocations for conservation than any other marsupial that, up until recently, has resulted in one of the most successful species recoveries in Australia. We used mitochondrial and nuclear DNA markers to assess the genetic outcomes of translocated woylie populations. These populations have lost genetic variability, differentiated from their source population and the supplementation program on two island populations appears to have failed. We discuss the conservation implications that our results have for managing threatened species, outline some general recommendations for the management of present and future translocations and discuss the appropriate sampling design for the establishment of new populations or captive breeding programs that may mitigate the genetic ‘erosion’ seen in our study species. This research provides some practical outcomes and a pragmatic understanding of translocation biology. The findings are directly applicable to other translocation programs [Current Zoology 59 (3: 294-310, 2013].

  15. Mode of ATM-dependent suppression of chromosome translocation

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Motohiro, E-mail: motoyama@nagasaki-u.ac.jp [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  16. Translocation of threatened plants as a conservation measure in China.

    Science.gov (United States)

    Liu, Hong; Ren, Hai; Liu, Qiang; Wen, XiangYing; Maunder, Michael; Gao, JiangYun

    2015-12-01

    We assessed the current status of plant conservation translocation efforts in China, a topic poorly reported in recent scientific literature. We identified 222 conservation translocation cases involving 154 species, of these 87 were Chinese endemic species and 101 (78%) were listed as threatened on the Chinese Species Red List. We categorized the life form of each species and, when possible, determined for each case the translocation type, propagule source, propagule type, and survival and reproductive parameters. A surprisingly large proportion (26%) of the conservation translocations in China were conservation introductions, largely implemented in response to large-scale habitat destruction caused by the Three-Gorge Dam and another hydropower project. Documentation and management of the translocations varied greatly. Less than half the cases had plant survival records. Statistical analyses showed that survival percentages were significantly correlated with plant life form and the type of planting materials. Thirty percent of the cases had records on whether or not individuals flowered or fruited. Results of information theoretic model selection indicated that plant life form, translocation type, propagule type, propagule source, and time since planting significantly influenced the likelihood of flowering and fruiting on the project level. We suggest that the scientific-based application of species conservation translocations should be promoted as part of a commitment to species recovery management. In addition, we recommend that the common practice of within and out of range introductions in nature reserves to be regulated more carefully due to its potential ecological risks. We recommend the establishment of a national office and database to coordinate conservation translocations in China. Our review effort is timely considering the need for a comprehensive national guideline for the newly announced nation-wide conservation program on species with extremely

  17. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  18. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....

  19. Development of Gene Expression Fingerprints for Identification of Environmental Contaminants Using cDNA Arrays

    National Research Council Canada - National Science Library

    Inouye, L

    2004-01-01

    ...) to develop cDNA array-based assays that map gene expression from contaminant exposures. Results substantiate that distinct gene expression profiles exist for major contaminant classes such as PARs, PCBs, and PCDD/Fs...

  20. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    Science.gov (United States)

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  1. Mechanical design of translocating motor proteins.

    Science.gov (United States)

    Hwang, Wonmuk; Lang, Matthew J

    2009-01-01

    Translocating motors generate force and move along a biofilament track to achieve diverse functions including gene transcription, translation, intracellular cargo transport, protein degradation, and muscle contraction. Advances in single molecule manipulation experiments, structural biology, and computational analysis are making it possible to consider common mechanical design principles of these diverse families of motors. Here, we propose a mechanical parts list that include track, energy conversion machinery, and moving parts. Energy is supplied not just by burning of a fuel molecule, but there are other sources or sinks of free energy, by binding and release of a fuel or products, or similarly between the motor and the track. Dynamic conformational changes of the motor domain can be regarded as controlling the flow of free energy to and from the surrounding heat reservoir. Multiple motor domains are organized in distinct ways to achieve motility under imposed physical constraints. Transcending amino acid sequence and structure, physically and functionally similar mechanical parts may have evolved as nature's design strategy for these molecular engines.

  2. Absorption and translocation of phosphorus-32 in guava leaves

    International Nuclear Information System (INIS)

    Natale, William

    1997-01-01

    Phosphorus is easily absorbed by the leaves and translocated. The objective of this work was to evaluate the absorption and translocation of P by guava leaves, with time. When a solution containing 2% MAP and specific activity 0.15 μCi/ml was applied. MAP labelled with 32 P was applied in the 3 rd pair of leaves. These and other leaves, roots and stem were collected separately and analyzed accordingly. The results showed that 20 days after application 12% of the applied P was absorbed by the guava leaves. The translocation of P started immediately after its absorption reaching 20% 2fter 20 days. (author). 19 refs., 4 tabs

  3. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  4. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  5. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  6. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  7. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  8. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  9. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  10. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    International Nuclear Information System (INIS)

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-01-01

    The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

  11. LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

    Science.gov (United States)

    Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

    2017-09-15

    Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

  12. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    Science.gov (United States)

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-02

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  13. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

    Full Text Available Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24 in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24. Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.

  14. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Directory of Open Access Journals (Sweden)

    Ju-Yeon Yoon

    2014-03-01

    Full Text Available Chrysanthemum stunt viroid (CSVd, a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1 were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

  15. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1989-01-01

    We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage

  16. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

  17. BCR translocation to derivative chromosome 2, a new case of chronic myeloid leukemia with complex variant translocation and Philadelphia chromosome

    International Nuclear Information System (INIS)

    Al-Achkar, W.; Wafa, A.; Al-Medani, S.

    2011-01-01

    The well-known typical fusion gene BCR/ABL can be observed in connection with a complex translocation event in only 5-8% of cases with chronic myeloid leukemia (CML). Herein we report an exceptional CML case with complex chromosomal aberrations not observed before, translocated BCR to the derivative chromosome 2 [der(2)], additional to involving a four chromosomes translocation implying chromosomal regions such as 1p32 and 2q21 besides 9q34 and 22q11.2. Which were characterized by molecular cytogenetics. (author)

  18. Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA.

    Science.gov (United States)

    Wong, J C; Alon, N; Norga, K; Kruyt, F A; Youssoufian, H; Buchwald, M

    2000-08-01

    Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA. Copyright 2000 Academic Press.

  19. Role of periodontal ligament fibroblasts in osteoclastogenesis: a review

    NARCIS (Netherlands)

    Sokos, D.; Everts, V.; de Vries, T.J.

    2015-01-01

    During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract

  20. A case of posttraumatic splenic translocation into the thorax

    International Nuclear Information System (INIS)

    Sosnowski, P.; Sikorski, L.; Ziemianski, A.

    1993-01-01

    A case of the left diaphragmatic hernia due to blunt thoracic and abdominal trauma is presented. Characteristic radiological signs of splenic translocation into the thorax contributed to quick diagnosis and immediate surgical intervention. (author)

  1. Multistep Current Signal in Protein Translocation through Graphene Nanopores

    KAUST Repository

    Bonome, Emma Letizia; Lepore, Rosalba; Raimondo, Domenico; Cecconi, Fabio; Tramontano, Anna; Chinappi, Mauro

    2015-01-01

    of graphene constitute a major advantage for molecule characterization. Here we analyze the translocation pathway of the thioredoxin protein across a graphene nanopore, and the related ionic currents, by integrating two nonequilibrium molecular dynamics

  2. Microbial translocation and cardiometabolic risk factors in HIV infection

    DEFF Research Database (Denmark)

    Trøseid, Marius; Manner, Ingjerd W; Pedersen, Karin K

    2014-01-01

    of microbial translocation are closely associated with several cardiovascular risk factors such as dyslipidemia, insulin resistance, hypertension, coagulation abnormalities, endothelial dysfunction, and carotid atherosclerosis. Future studies should investigate whether associations between microbial...

  3. Carbon translocation in zooanthaellae-coelenterate symbioses

    International Nuclear Information System (INIS)

    Battey, J.F.

    1985-01-01

    When host and algal triglycerides synthesized in the symbiotic sea anemone Condylactis gigantea during light and dark incubations in 14 C-bicarbonate and 14 C-acetate were deacylated, more then 80% of the radioactivity was found in the fatty acid moiety. In contrast, triglycerides isolated from zooxanthellae and host incubated in 14 C-glycerol in the dark were found to have more then 95% of their radioactivity in the glycerol moiety. During 14 C-glycerol incubations in the light, radioactivity in the fatty acid moiety of zooxanthellae triglyceride fatty acid moiety stayed below 5% during 14 C-glycerol incubations in the light. These results show neither the zooxanthellae nor host can rapidly convert glycerol to fatty acid. Radioactivity from 14 C-glycerol that does eventually appear in host lipid may have been respired to 14 CO 2 then photosynthetically fixed by the zooxanthellae and synthesized into lipid fatty acid. The isolated zooxanthellae of C. gigantea contained 3.62 +/- 0.33 mM glycerol, which was 26x the 0.141 +/- 0.02 mM found in the coelenterate tissue. Aposymbiotic coelenterate tissue contained 0.169 +/- 0.05 mM glycerol. The metabolic inhibitors, sodium cyanide, aminooxyacetic acid and cerulenin were used to try and uncouple the production of glycerol by the zooxanthellae from its utilization by the coelenterate host. 10 -5 M NaCN increased the ratio of cross photosynthesis to respiration in both intact tentacles and isolated zooxanthellae, increased translocation from 17.7 +/- 3.5% of total fixed carbon in controls to 43.5 +/- 5.79%, and doubled the amount of photosynthetically fixed carbon accumulating in the coelenterate host over that in controls

  4. Bacterial translocation in clinical intestinal transplantation.

    Science.gov (United States)

    Cicalese, L; Sileri, P; Green, M; Abu-Elmagd, K; Kocoshis, S; Reyes, J

    2001-05-27

    Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after small bowel transplantation (SBTx). Bacterial overgrowth, alteration of the mucosal barrier function as a consequence of preservation injury or acute rejection (AR), and potent immunosuppression are all associated with BT. The aim of this study was to evaluate and quantify the correlation of BT with these events. Fifty pediatric SBTx recipients on tacrolimus and prednisone immunosuppression were analyzed. Blood, stool, and liver biopsies and peritoneal fluid were cultured (circa 4000 total specimens) when infection was clinically suspected or as part of follow-up. BT episodes were considered when microorganisms were found simultaneously in blood or liver biopsy and stool. BT (average of 2.0 episodes/patient) was evident in 44% of patients and was most frequently caused by Enterococcus, Staphylococcus, Enterobacter, and Klebsiella. The presence of a colon allograft was associated with a higher rate of BT (75% vs. 33.3%). Furthermore, the transplantation procedure (colon vs. no colon) affected the rate of BT: SBTx=40% vs. 25%, combined liver and SBTx=100% vs. 30%, multivisceral transplantation=25% vs. 50%. AR was associated with 39% of BT episodes. BT followed AR in 9.6% of the cases. In 5.2% of the cases, positive blood cultures without stool confirmation of the bacteria were seen. Prolonged cold ischemia time (CIT) affected BT rate significantly (CIT>9 hr 76% vs. CIT<9 hr 20.8%). This study shows that 1) a substantial percentage of, but not all, BT is associated with AR, 2) the presence of a colon allograft increases the risk for BT, and 3) a long CIT is associated with a high incidence of BT after SBTx.

  5. Meiotic delay of translocation carrying spermatocytes responsible for reduced transmission

    International Nuclear Information System (INIS)

    Buul, P.P.W. van

    1991-01-01

    Using in vivo pulse labelling of spermatocytes from mice irradiated with different doses of X-rays (6 and 7 Gy). The authors demonstrated that cells having translocations derived from irradiated stem cells tend to spend longer time at the meiotic prophase than normal cells. At the 2 Gy level this effect is much less pronounced. The recorded delay forms a good explanation for the reduced transmission of translocations to the next generation observed by others. (author)

  6. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

  7. Uptake, translocation, and debromination of polybrominated diphenyl ethers in maize

    Institute of Scientific and Technical Information of China (English)

    Moming Zhao; Shuzhen Zhang; Sen Wang; Honglin Huang

    2012-01-01

    Uptake,translocation and debromination of three polybrominated diphenyl ethers(PBDEs),BDE-28,-47 and-99,in maize were studied in a hydroponic experiment.Roots took up most of the PBDEs in the culture solutions and more highly brominated PBDEs had a stronger uptake capability.PBDEs were detected in the stems and leaves of maize after exposure but rarely detected in the blank control plants.Furthermore,PBDE concentrations decreased from roots to stems and then to leaves,and a very clear decreasing gradient was found in segments upwards along the stem.These altogether provide substantiating evidence for the acropetal translocation of PBDEs in maize.More highly brominated PBDEs were translocated with more difficulty.Radial translocation of PBDEs from nodes to sheath inside maize was also observed.Both acropetal and radial translocations were enhanced at higher transpiration rates,suggesting that PBDE transport was probably driven by the transpiration stream.Debromination of PBDEs occurred in all parts of the maize,and debromination patterns of different parent PBDEs and in different parts of a plant were similar but with some differences.This study for the first time provides direct evidence for the acropetal translocation of PBDEs within plants,elucidates the process of PBDE transport and clarifies the debromination products of PBDEs in maize.

  8. The Unexplored Mechanisms and Regulatory Functions of Ribosomal Translocation

    Science.gov (United States)

    Alejo, Jose Luis

    In every cell, protein synthesis is carried out by the ribosome, a complex macromolecular RNA-protein assembly. Decades of structural and kinetic studies have increased our understanding of ribosome initiation, decoding, translocation and termination. Yet, the underlying mechanism of these fundamental processes has yet to be fully delineated. Hence, the molecular basis of regulation remains obscure. Here, single-molecule fluorescence methods are applied to decipher the mechanism and regulatory roles of the multi-step process of directional substrate translocation on the ribosome that accompanies every round of protein synthesis. In Chapter 1, single-molecule fluorescence resonance energy transfer (smFRET) is introduced as a tool for studying bacterial ribosome translocation. Chapter 2 details the experimental methods. In Chapter 3, the elongation factor G(EF-G)-catalyzed movement of substrates through the ribosome is examined from several perspectives or signals reporting on various degrees of freedom of ribosome dynamics. Two ribosomal states interconvert in the presence of EF-G(GDP), displaying novel head domain motions, until relocking takes place. In Chapter 4, in order to test if the mentioned fluctuations leading to relocking are correlated to the engagement of the P-site by the peptidyl-tRNA, the translocation of miscoded tRNAs is studied. Severe defects in the relocking stages of translocation reveal the correlation between this new stage of translocation and P-site tRNA engagement.

  9. Fibroblast growth factor 23 - et fosfatregulerende hormon

    DEFF Research Database (Denmark)

    Beck-Nielsen, Signe; Pedersen, Susanne Møller; Kassem, Moustapha

    2010-01-01

    Fibroblast growth factor 23 (FGF23) er et nyligt identificeret fosfatonin. FGF23's fysiologiske hovedfunktion er at opretholde normalt serumfosfat og at virke som et D-vitaminmodregulatorisk hormon. Sygdomme, der er koblet til forhøjet serum FGF23, er hypofosfatæmisk rakitis, fibrøs dysplasi og t...

  10. Cryopreservation of canine ovarian and testicular fibroblasts.

    Science.gov (United States)

    Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

    2009-01-01

    To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

  11. Matrix metalloproteinase (MMP-9 in cancer-associated fibroblasts (CAFs is suppressed by omega-3 polyunsaturated fatty acids in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Ayumi Taguchi

    Full Text Available Cancer associated fibroblasts (CAFs are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

  12. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Cecilia Prata

    2017-01-01

    Full Text Available Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance.

  13. Glycosides from Stevia rebaudiana Bertoni Possess Insulin-Mimetic and Antioxidant Activities in Rat Cardiac Fibroblasts

    Science.gov (United States)

    Prata, Cecilia; Zambonin, Laura; Rizzo, Benedetta; Vieceli Dalla Sega, Francesco

    2017-01-01

    Stevia rebaudiana Bertoni is a shrub having a high content of sweet diterpenoid glycosides in its leaves, mainly stevioside and rebaudioside A, which are used as noncaloric, natural sweeteners. The aim of this study was to deepen the knowledge about the insulin-mimetic effect exerted by four different mixtures of steviol glycosides, rich in stevioside and rebaudioside A, in neonatal rat cardiac fibroblasts. The potential antioxidant activity of these steviol glycosides was also assessed, as oxidative stress is associated with diabetes. Likewise the insulin effect, steviol glycosides caused an increase in glucose uptake into rat fibroblasts by activating the PI3K/Akt pathway, thus inducing Glut4 translocation to the plasma membrane. The presence of S961, an insulin antagonist, completely abolished these effects, allowing to hypothesize that steviol glycosides could act as ligands of the same receptor engaged by insulin. Moreover, steviol glycosides counteracted oxidative stress by increasing reduced glutathione intracellular levels and upregulating expression and activity of the two antioxidant enzymes superoxide dismutase and catalase. The present work unravels the insulin-mimetic effect and the antioxidant property exerted by steviol glycosides, suggesting their potential beneficial role in the cotreatment of diabetes and in health maintenance. PMID:28947927

  14. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  15. Conflict bear translocation: investigating population genetics and fate of bear translocation in Dachigam National Park, Jammu and Kashmir, India.

    Science.gov (United States)

    Mukesh; Sharma, Lalit Kumar; Charoo, Samina Amin; Sathyakumar, Sambandam

    2015-01-01

    The Asiatic black bear population in Dachigam landscape, Jammu and Kashmir is well recognized as one of the highest density bear populations in India. Increasing incidences of bear-human interactions and the resultant retaliatory killings by locals have become a serious threat to the survivorship of black bears in the Dachigam landscape. The Department of Wildlife Protection in Jammu and Kashmir has been translocating bears involved in conflicts, henceforth 'conflict bears' from different sites in Dachigam landscape to Dachigam National Park as a flagship activity to mitigate conflicts. We undertook this study to investigate the population genetics and the fate of bear translocation in Dachigam National Park. We identified 109 unique genotypes in an area of ca. 650 km2 and observed bear population under panmixia that showed sound genetic variability. Molecular tracking of translocated bears revealed that mostly bears (7 out of 11 bears) returned to their capture sites, possibly due to homing instincts or habituation to the high quality food available in agricultural croplands and orchards, while only four bears remained in Dachigam National Park after translocation. Results indicated that translocation success was most likely to be season dependent as bears translocated during spring and late autumn returned to their capture sites, perhaps due to the scarcity of food inside Dachigam National Park while bears translocated in summer remained in Dachigam National Park due to availability of surplus food resources. Thus, the current management practices of translocating conflict bears, without taking into account spatio-temporal variability of food resources in Dachigam landscape seemed to be ineffective in mitigating conflicts on a long-term basis. However, the study highlighted the importance of molecular tracking of bears to understand their movement patterns and socio-biology in tough terrains like Dachigam landscape.

  16. Conflict bear translocation: investigating population genetics and fate of bear translocation in Dachigam National Park, Jammu and Kashmir, India.

    Directory of Open Access Journals (Sweden)

    Mukesh

    Full Text Available The Asiatic black bear population in Dachigam landscape, Jammu and Kashmir is well recognized as one of the highest density bear populations in India. Increasing incidences of bear-human interactions and the resultant retaliatory killings by locals have become a serious threat to the survivorship of black bears in the Dachigam landscape. The Department of Wildlife Protection in Jammu and Kashmir has been translocating bears involved in conflicts, henceforth 'conflict bears' from different sites in Dachigam landscape to Dachigam National Park as a flagship activity to mitigate conflicts. We undertook this study to investigate the population genetics and the fate of bear translocation in Dachigam National Park. We identified 109 unique genotypes in an area of ca. 650 km2 and observed bear population under panmixia that showed sound genetic variability. Molecular tracking of translocated bears revealed that mostly bears (7 out of 11 bears returned to their capture sites, possibly due to homing instincts or habituation to the high quality food available in agricultural croplands and orchards, while only four bears remained in Dachigam National Park after translocation. Results indicated that translocation success was most likely to be season dependent as bears translocated during spring and late autumn returned to their capture sites, perhaps due to the scarcity of food inside Dachigam National Park while bears translocated in summer remained in Dachigam National Park due to availability of surplus food resources. Thus, the current management practices of translocating conflict bears, without taking into account spatio-temporal variability of food resources in Dachigam landscape seemed to be ineffective in mitigating conflicts on a long-term basis. However, the study highlighted the importance of molecular tracking of bears to understand their movement patterns and socio-biology in tough terrains like Dachigam landscape.

  17. 25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ichikawa, Tomohiro [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Sugiura, Hisatoshi, E-mail: sugiura@rm.med.tohoku.ac.jp [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan); Koarai, Akira; Kikuchi, Takashi; Hiramatsu, Masataka; Kawabata, Hiroki; Akamatsu, Keiichiro; Hirano, Tsunahiko; Nakanishi, Masanori; Matsunaga, Kazuto; Minakata, Yoshiaki [Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8509 (Japan); Ichinose, Masakazu [Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574 (Japan)

    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.

  18. Radiation enhances silica translocation to the pulmonary interstitium and increases fibrosis in mice

    International Nuclear Information System (INIS)

    Adamson, I.Y.R.

    1992-01-01

    The effects of whole body irradiation (WBR) on particle clearance and the development of pulmonary fibrosis have been investigated. Using carbon, clearance is accomplished by polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM), and only a few particles reach the interstitum. However, in preirradiated mice, the usual eflux of inflammatory cells is much delayed so that more free carbon remains in the alveoli, and by 1 week, many particles cross the epithelium to be phagocytized by interstitial macrophages. Carbon is found in the peribronchiolar interstitium 6 months later with no evidence of fibrosis. In the present study, mice received 1 mg silica intratracheally 2 days after 6.5 Gy WBR when the white blood cell count was low. A much-reduced Am and PMN response was found in the following 2 weeks compared to the reaction to silica alone, and many silica particles reached interstitial macrophages. In this case, macrophage activation by silica was associated with fibroblast proliferation, and by 16 weeks, much more pulmonary fibrosis was produced than after silica or irradiation only. This was measured biochemically and correlated with a large increase in retained silica in the irradiation-silica group. The results indicate that radiation inhibits the inflammatory response to particle instillation, resulting in greater translocation of free particles to the pulmonary interstitium. In the case of silica, the greater, prolonged interaction with interstitial macrophages leads to a much exaggerated fibrotic reaction. 17 refs., 11 figs

  19. Transcuticular translocation of radionuclides on plant leaf surfaces

    International Nuclear Information System (INIS)

    Matsumoto, Ken-ichi; Watanabe, Tadakazu; Ambe, Shizuko; Yamaguchi, Isamu

    1996-01-01

    The cuticle covering all the outermost surfaces of the aerial parts of plants could play a selective role in uptake and translocation of radionuclides from air into plants. In this study, we investigated the transcuticular uptake and translocation behavior via water droplets of various radionuclides in red clover, orchard grass, Japanese radish and mung bean. Ten μl of an aqueous solution of the multitracer generated from Au was applied to the upper surface of the 2nd leaf of the plants at the 5th leaf stage. The plants were then grown for 14 days at 25degC and 70% RH under illumination of artificial solar lights. The transcuticular uptake and translocation throughout the plant were periodically assayed by determining the radioactivity in the surface residue, the cuticle layer beneath the applied site, the leaf area outside the applied site, the other aerial parts and the root of the plant, using an HPGe detector. The applied radionuclides were absorbed into, in turn, the cuticle layer beneath the applied site and then translocated through the cuticle to the inner tissue and eventually to the other aerial parts and finally to the roots, of the plant. The distribution and accumulation in the plant seems to depend upon the characteristics of each radionuclide and plant species. Ca * and Te * tended to remain on leaf surfaces without being absorbed into the cuticle. On the other hand, Sc * , Co * , Zn * , Se * , Rb * , and Eu * were easily absorbed and translocated to every part of the plant including the root. The other radionuclides such as Be * , Mn * , Sr * , Y * , Ba * , Ce * , Pm * , Gd * , Hf * , Yb * , Lu * , Os * , Ir * , and Pt * remained in the region close to the site of their application. The above results possibly indicate the existence of mechanisms common to these plants for selective transcuticular uptake and translocation of radionuclides within plant tissues, though their translocation was considerably influenced by the plant species. (author)

  20. High-speed detection of DNA translocation in nanopipettes

    Science.gov (United States)

    Fraccari, Raquel L.; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

    2016-03-01

    We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface. Electronic supplementary information (ESI) available: Gel electrophoresis confirming lengths and purity of DNA samples, comparison between Axopatch 200B and custom-built setup, comprehensive low-noise amplifier characterization, representative I-V curves of nanopipettes used, typical scatter plots of τ vs. peak amplitude for the four LDNA's used, table of most probable τ values, a comparison between different fitting models for the DNA translocation time distribution, further details on the stochastic numerical simulation of the scaling statistics and the derivation of the extended

  1. Role of non-equilibrium conformations on driven polymer translocation.

    Science.gov (United States)

    Katkar, H H; Muthukumar, M

    2018-01-14

    One of the major theoretical methods in understanding polymer translocation through a nanopore is the Fokker-Planck formalism based on the assumption of quasi-equilibrium of polymer conformations. The criterion for applicability of the quasi-equilibrium approximation for polymer translocation is that the average translocation time per Kuhn segment, ⟨τ⟩/N K , is longer than the relaxation time τ 0 of the polymer. Toward an understanding of conditions that would satisfy this criterion, we have performed coarse-grained three dimensional Langevin dynamics and multi-particle collision dynamics simulations. We have studied the role of initial conformations of a polyelectrolyte chain (which were artificially generated with a flow field) on the kinetics of its translocation across a nanopore under the action of an externally applied transmembrane voltage V (in the absence of the initial flow field). Stretched (out-of-equilibrium) polyelectrolyte chain conformations are deliberately and systematically generated and used as initial conformations in translocation simulations. Independent simulations are performed to study the relaxation behavior of these stretched chains, and a comparison is made between the relaxation time scale and the mean translocation time (⟨τ⟩). For such artificially stretched initial states, ⟨τ⟩/N K polymers including single stranded DNA (ssDNA), double stranded DNA (dsDNA), and synthetic polymers. Even when these data are rescaled assuming a constant effective velocity of translocation, it is found that for flexible (ssDNA and synthetic) polymers with N K Kuhn segments, the condition ⟨τ⟩/N K polymers such as ssDNA, a crossover from quasi-equilibrium to non-equilibrium behavior would occur at N K ∼ O(1000).

  2. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    International Nuclear Information System (INIS)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S.; Rocchi, M.

    1991-01-01

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH 2 -terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH 2 -terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids

  3. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    International Nuclear Information System (INIS)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

  4. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  5. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    Directory of Open Access Journals (Sweden)

    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  6. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  7. Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion*

    Science.gov (United States)

    Steringer, Julia P.; Bleicken, Stephanie; Andreas, Helena; Zacherl, Sonja; Laussmann, Mareike; Temmerman, Koen; Contreras, F. Xabier; Bharat, Tanmay A. M.; Lechner, Johannes; Müller, Hans-Michael; Briggs, John A. G.; García-Sáez, Ana J.; Nickel, Walter

    2012-01-01

    Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate. PMID:22730382

  8. Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

    Science.gov (United States)

    Sena-Esteves, M; Camp, S M; Alroy, J; Breakefield, X O; Kaye, E M

    2000-03-20

    Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

  9. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    Science.gov (United States)

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with

  10. Sorting genomes by reciprocal translocations, insertions, and deletions.

    Science.gov (United States)

    Qi, Xingqin; Li, Guojun; Li, Shuguang; Xu, Ying

    2010-01-01

    The problem of sorting by reciprocal translocations (abbreviated as SBT) arises from the field of comparative genomics, which is to find a shortest sequence of reciprocal translocations that transforms one genome Pi into another genome Gamma, with the restriction that Pi and Gamma contain the same genes. SBT has been proved to be polynomial-time solvable, and several polynomial algorithms have been developed. In this paper, we show how to extend Bergeron's SBT algorithm to include insertions and deletions, allowing to compare genomes containing different genes. In particular, if the gene set of Pi is a subset (or superset, respectively) of the gene set of Gamma, we present an approximation algorithm for transforming Pi into Gamma by reciprocal translocations and deletions (insertions, respectively), providing a sorting sequence with length at most OPT + 2, where OPT is the minimum number of translocations and deletions (insertions, respectively) needed to transform Pi into Gamma; if Pi and Gamma have different genes but not containing each other, we give a heuristic to transform Pi into Gamma by a shortest sequence of reciprocal translocations, insertions, and deletions, with bounds for the length of the sorting sequence it outputs. At a conceptual level, there is some similarity between our algorithm and the algorithm developed by El Mabrouk which is used to sort two chromosomes with different gene contents by reversals, insertions, and deletions.

  11. A somatic origin of homologous Robertsonian translocations and isochromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A. (Univ. of Zurich (Switzerland)); Basaran, S.; Yueksel-Apak, M. (Univ. of Istanbul (Turkey)); Neri, G. (Universita Cattolica, Rome (Italy)); Serville, F. (Hopital d' Enfants Pellegrin, Bordeaux (France)); Balicek, P.; Haluza, R. (Univ. Hospital of Hradeck Kralove, Hradec Kralove (Czech Republic)); Farah, L.M.S. (Escuola Paulista de Medicina, Sao Paulo (Brazil)) (and others)

    1994-02-01

    One t(14q 14q), three t(15q 15q), two t(21q21q), and two t(22q22q) nonmosaic, apparently balanced, de novo Robertsonian translocation cases were investigated with polymorphic markers to establish the origin of the translocated chromosomes. Four cases had results indicative of an isochromosome: one t(14q14q) case with mild mental retardation and maternal uniparental disomy (UPD) for chromosome 14, one t(15q15q) case with the Prader-Willi syndrome and UPD(15), a phenotypically normal carrier of t(22q22q) with maternal UPD(22), and a phenotypically normal t(21q21q) case of paternal UPD(21). All UPD cases showed complete homozygosity throughout the involved chromosome, which is supportive of a postmeiotic origin. In the remaining four cases, maternal and paternal inheritance of the involved chromosome was found, which unambiguously implies a somatic origin. One t(15q15q) female had a child with a ring chromosome 15, which was also of probable postmeiotic origin as recombination between grandparental haplotypes had occurred prior to ring formation. UPD might be expected to result from de novo Robertsonian translocations of meiotic origin; however, all de novo homologous translocation cases, so far reported, with UPD of chromosomes 14, 15, 21, or 22 have been isochromosomes. These data provide the first direct evidence that nonmosaic Robertsonian translocations, as well as isochromosomes, are commonly the result of a mitotic exchange. 75 refs., 1 fig., 4 tabs.

  12. Electrostatics of polymer translocation events in electrolyte solutions.

    Science.gov (United States)

    Buyukdagli, Sahin; Ala-Nissila, T

    2016-07-07

    We develop an analytical theory that accounts for the image and surface charge interactions between a charged dielectric membrane and a DNA molecule translocating through the membrane. Translocation events through neutral carbon-based membranes are driven by a competition between the repulsive DNA-image-charge interactions and the attractive coupling between the DNA segments on the trans and the cis sides of the membrane. The latter effect is induced by the reduction of the coupling by the dielectric membrane. In strong salt solutions where the repulsive image-charge effects dominate the attractive trans-cis coupling, the DNA molecule encounters a translocation barrier of ≈10 kBT. In dilute electrolytes, the trans-cis coupling takes over image-charge forces and the membrane becomes a metastable attraction point that can trap translocating polymers over long time intervals. This mechanism can be used in translocation experiments in order to control DNA motion by tuning the salt concentration of the solution.

  13. Nonabsorbable Antibiotics Reduce Bacterial and Endotoxin Translocation in Hepatectomised Rats

    Directory of Open Access Journals (Sweden)

    S. K. Kakkos

    1997-01-01

    Full Text Available There is increasing evidence that septic complications, occurring after major hepatectomies, may be caused by gram negative bacteria, translocating from the gut. We investigated in rats, the effect of extended hepatectomy on the structure and morphology of the intestinal mucosa as well as on the translocation of intestinal bacteria and endotoxins. We also examined the effect of nonabsorbable antibiotics on reducing the intestinal flora and consequently the phenomenon of translocation by administering neomycin sulphate and cefazoline. Hepatectomy was found to increase translocation, while administration of nonabsorbable antibiotics decreased it significantly. In addition, hepatectomy increased the aerobic cecal bacterial population, which normalised in the group receiving antibiotics. Among the histological parameters evaluated, villus height demonstrated a significant reduction after hepatectomy, while the number of villi per cm and the number of mitoses per crypt, remained unchanged. Our results indicate that administration of nonabsorbable antibiotics presents a positive effect on bacterial and endotoxin translocation after extended hepatectomy, and this may be related to reduction of colonic bacterial load as an intraluminal effect of antibiotics.

  14. Styl RFLP recognized by a human IRBP cDNA localized to chromosome 10

    Energy Technology Data Exchange (ETDEWEB)

    Chin, K S; Mathew, C G.P.; Fong, S L; Bridges, C D; Ponder, B A.J.

    1988-02-25

    A 2184 bp cDNA (H.4 IRBP) encoding human interstitial retinol-biding protein isolated from a human retina cDNA library in lambdagt10 by screening with a bovine IRBP cDNA probe. Styl identifies a 2-allele polymorphism with bands at 2.3 kb (Cl) and 1.95 kb (C2) and invariant bands at 1.1, 1.0 and 0.8kb. Codominant segregation was observed in two informative families. The RFLP was mapped to chromosome 10 using somatic cell hybrids. In situ hybridization suggests regional assignments near p11.2 -q11.2 with a secondary site of hybridization at q24-25.

  15. Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

    Science.gov (United States)

    Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

    2003-10-01

    A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

  16. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  17. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

  18. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis.

    Science.gov (United States)

    Pardo, Annie; Selman, Moisés

    2016-12-01

    Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible lung disease of unknown etiology, elusive pathogenesis, and very limited therapeutic options. The hallmarks of IPF are aberrant activation of alveolar epithelial cells and accumulation of fibroblasts and myofibroblasts along with excessive production of extracellular matrix. The linkage of aging with this disorder is uncertain, but a number of changes associated with aging, including telomere attrition, cell senescence, and mitochondrial dysfunction, have been revealed in IPF lungs. Also, aging seems to confer a profibrotic phenotype upon fibroblasts and to increase the severity of the fibrogenic response in non-IPF fibrotic lung disorders. Better knowledge of the pathophysiological mechanisms linking aging to IPF will advance understanding of its pathogenesis and may provide new therapeutic windows to treatment of this devastating disease.

  19. cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

    International Nuclear Information System (INIS)

    Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.; Tanaka, K.

    1986-01-01

    MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the β-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded [ 32 P]labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting

  20. Display of a Maize cDNA library on baculovirus infected insect cells

    Directory of Open Access Journals (Sweden)

    Jones Ian M

    2008-08-01

    Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  1. Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

    DEFF Research Database (Denmark)

    Nielsen, M-B; Christensen, Søren Tvorup; Hoffmann, E K

    2008-01-01

    Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts...... in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most...... likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments...

  2. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

    2013-01-01

    Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput...... of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR...

  3. The nucleotide sequence of human transition protein 1 cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Luerssen, H; Hoyer-Fender, S; Engel, W [Universitaet Goettingen (West Germany)

    1988-08-11

    The authors have screened a human testis cDNA library with an oligonucleotide of 81 mer prepared according to a part of the published nucleotide sequence of the rat transition protein TP 1. They have isolated a cDNA clone with the length of 441 bp containing the coding region of 162 bp for human transition protein 1. There is about 84% homology in the coding region of the sequence compared to rat. The human cDNA-clone encodes a polypeptide of 54 amino acids of which 7 are different to that of rat.

  4. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end.......PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  5. Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Watson, M D; Lambert, N; Delauney, A; Yarwood, J N; Croy, R R; Gatehouse, J A; Wright, D J; Boulter, D

    1988-01-01

    A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was puri...

  6. Gene targeting in adult rhesus macaque fibroblasts

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2008-03-01

    Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

  7. Mechanism for translocation of fluoroquinolones across lipid membranes

    DEFF Research Database (Denmark)

    Cramariuc, O.; Rog, T.; Javanainen, M.

    2012-01-01

    Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone...... antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins....... Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach...

  8. Biological mechanisms and translocation kinetics of particulate plutonium

    International Nuclear Information System (INIS)

    Bruenger, F.W.; Stevens, W.; Atherton, D.R.; Roswell, R.L.; Smith, J.M.

    1981-01-01

    The dissolution and elimination of particulate 239 Pu from its initial sites of deposition in phagocytic organs (the liver, spleen, and lung), as well as its translocation and redeposition in soft tissue organs and skeleton have been investigated. Beagles were injected intravenously with particulate Pu and sacrificed sequentially at times ranging from 33 to 830 days after injection. Equations that describe the overall retention of Pu in liver, spleen, lung, and bone were calculated. Plutonium mobilized from these organs either re-entered the blood stream and redeposited in the skeleton and liver parenchyma or was excreted. The protracted translocation of Pu to bone surfaces potentially exposes all cells involved in osteogenesis to continuous α-radiation, a situation that could enhance the hazard of developing osteosarcoma. A kinetic model that describes the translocation of Pu from the phagocytic compartments to blood and its subsequent redistribution to bone, liver, and other organs was formulated

  9. Evaluating descriptors for the lateral translocation of membrane proteins.

    Science.gov (United States)

    Domanova, Olga; Borbe, Stefan; Mühlfeld, Stefanie; Becker, Martin; Kubitz, Ralf; Häussinger, Dieter; Berlage, Thomas

    2011-01-01

    Microscopic images of tissue sections are used for diagnosis and monitoring of therapy, by analysis of protein patterns correlating to disease states. Spatial protein distribution is influenced by protein translocation between different membrane compartments and quantified by comparison of microscopic images of biological samples. Cholestatic liver diseases are characterized by translocation of transport proteins, and quantification of their dislocation offers new diagnostic options. However, reliable and unbiased tools are lacking. The nowadays used manual method is slow, subjective and error-prone. We have developed a new workflow based on automated image analysis and improved it by the introduction of scale-free descriptors for the translocation quantification. This fast and unbiased method can substitute the manual analysis, and the suggested descriptors perform better than the earlier used statistical variance.

  10. Crystallographic snapshot of cellulose synthesis and membrane translocation.

    Science.gov (United States)

    Morgan, Jacob L W; Strumillo, Joanna; Zimmer, Jochen

    2013-01-10

    Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

  11. Scintigraphic visualization of bacterial translocation in experimental strangulated intestinal obstruction

    International Nuclear Information System (INIS)

    Galeev, Yu.M.; Popov, M.V.; Salato, O.V.; Lishmanov, Yu.B.; Grigorev, E.G.; Aparcin, K.A.

    2009-01-01

    The purpose of this study was to obtain scintigraphic images depicting translocation of 99m Tc-labelled Escherichia coli bacteria through the intestinal barrier and to quantify this process using methods of nuclear medicine. Thirty male Wistar rats (including 20 rats with modelled strangulated intestinal obstruction and 10 healthy rats) were used for bacterial scintigraphy. 99m Tc-labelled E. coli bacteria ( 99m Ts-E. coli) with an activity of 7.4-11.1 MBq were administered into a section of the small intestine. Scintigraphic visualization of bacterial translocation into organs and tissues of laboratory animals was recorded in dynamic (240 min) and static (15 min) modes. The number of labelled bacteria, which migrated through the intestinal barrier, was quantified by calculating the translocation index (TI). Control indicated no translocation of 99m Ts-E. coli administered into the intestine through the parietes of the small intestine's distal part in healthy animals. Animals with strangulated obstruction demonstrated different migration strength and routes of labelled bacteria from strangulated and superior to strangulation sections of the small intestine. 99m Ts-E. coli migrated from the strangulated loop into the peritoneal cavity later causing systemic bacteraemia through peritoneal resorption. The section of the small intestine, which was superior to the strangulation, demonstrated migration of labelled bacteria first into the portal and then into the systemic circulation. The strangulated section of the small intestine was the main source of bacteria dissemination since the number of labelled bacteria, which migrated from this section significantly, exceeded that of the area superior to the strangulation section of the small intestine (p = 0.0003). Bacterial scintigraphy demonstrated the possibility of visualizing migration routes of labelled bacteria and quantifying their translocation through the intestinal barrier. This approach to study bacterial

  12. The Genetics of a Probable Insertional Translocation in SORDARIA BREVICOLLIS.

    Science.gov (United States)

    Bond, D J

    1979-05-01

    A chromosome rearrangement has been isolated and characterized in Sordaria brevicollis. Crosses to spore color mutants on each of the seven linkage groups have enabled the breakpoints to be mapped. The simplest hypothesis to account for the results is that a piece of linkage group VI has been translocated to linkage group V and inserted 2.7 map units from its centromere. Previous reports of markers on this linkage group with centromere distances greater than 2.7 units make it unlikely that the translocation is quasiterminal.

  13. Free energy evaluation in polymer translocation via Jarzynski equality

    Energy Technology Data Exchange (ETDEWEB)

    Mondaini, Felipe, E-mail: fmondaini@if.ufrj.br [Centro Federal de Educação Tecnológica Celso Suckow da Fonseca, Petrópolis, 25.620-003, RJ (Brazil); Moriconi, L., E-mail: moriconi@if.ufrj.br [Instituto de Física, Universidade Federal do Rio de Janeiro, C.P. 68528, 21945-970, Rio de Janeiro, RJ (Brazil)

    2014-05-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations, which provide free energy estimates for unbiased three-dimensional polymer translocation. We employ the Jarzynski equality in its rigorous setting, to compute the variation of the free energy in single monomer translocation events. In our three-dimensional Langevin simulations, the excluded-volume and van der Waals interactions between beads (monomers and membrane atoms) are modeled through a repulsive Lennard-Jones (LJ) potential and consecutive monomers are subject to the Finite-Extension Nonlinear Elastic (FENE) potential. Analysing data for polymers with different lengths, the free energy profile is noted to have interesting finite-size scaling properties.

  14. Free energy evaluation in polymer translocation via Jarzynski equality

    International Nuclear Information System (INIS)

    Mondaini, Felipe; Moriconi, L.

    2014-01-01

    We perform, with the help of cloud computing resources, extensive Langevin simulations, which provide free energy estimates for unbiased three-dimensional polymer translocation. We employ the Jarzynski equality in its rigorous setting, to compute the variation of the free energy in single monomer translocation events. In our three-dimensional Langevin simulations, the excluded-volume and van der Waals interactions between beads (monomers and membrane atoms) are modeled through a repulsive Lennard-Jones (LJ) potential and consecutive monomers are subject to the Finite-Extension Nonlinear Elastic (FENE) potential. Analysing data for polymers with different lengths, the free energy profile is noted to have interesting finite-size scaling properties.

  15. The action spectrum in chloroplast translocation in multilayer leaf cells

    Directory of Open Access Journals (Sweden)

    Zbigniew Lechowski

    2015-01-01

    Full Text Available By measurement of light transmittance through a leaf as criterion of chloroplast translocation, the action spectrum of Ajuga reptans was established. In the spectrum obtained, a correction was introduced for leaf autoabsorption calculated on the basis of the Beer-Lambert law. The action spectrum has two maxima: at λ= 375 nm and λ= 481 nm. The range above 502 nm has no significant effect on chloroplast translocation. Comparison with other objects examined demonstrated that in multilayer leaf cells riboflavin seems also to be a photoreceptor active in this process.

  16. Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

    Science.gov (United States)

    Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

    2010-04-09

    Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

  17. Isolation and characterization of a cDNA encoding phytochrome A in the non-photosynthetic parasitic plant, Orobanche minor Sm.

    Science.gov (United States)

    Trakulnaleamsai, Chitra; Okazawa, Atsushi; An, Chung-Il; Kajiyama, Shin'ichiro; Fukusaki, Ei'ichiro; Yoneyama, Koichi; Takeuchi, Yasutomo; Kobayashi, Akio

    2005-01-01

    In this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.

  18. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available east_seq_qual.zip File URL: ftp://ftp.biosciencedbc.jp/archive/yeast_cdna/LATEST/...yeast_seq_qual.zip File size: 59.9MB Simple search URL http://togodb.biosciencedbc.jp/togodb/view/budding_yeast_cdna

  19. Analysis of FBN1 allele expression by dermal fibroblasts from Marfan syndrome patients

    Energy Technology Data Exchange (ETDEWEB)

    Putman, E.A.; Cao, S.N.; Milewicz, D.M. [Univ. of Texas Medical School, Houston, TX (United States)

    1994-09-01

    Screening for mutations in the FBN1 cDNA from Marfan patient cell strains has detected mutations in only 10-15% of patients. In an attempt to explain this poor detection rate, we examined FBN1 allele expression and fibrillin synthesis by 26 cell strains from Marfan patients. DNA from the patients and 10 controls was assessed for the presence of a polymorphic Rsa I restriction site in the 3{prime} untranslated region of the FBN1 gene. Twelve of 26 patient and 5 of 10 control DNAs were heterozygous. Fibroblast RNA from the heterozygous cell strains was reverse-transcribed and subsequently PCR amplified using a [{sup 32}P]-labelled primer, digested with Rsa I and analyzed. Although 3 samples showed no transcript from one allele by ethidium bromide staining, a Betagen scanner detected low levels (10-15%) of that allele. In addition, there was unequal expression of the two alleles in three other patients; for example, only 30% expression from one allele. The remaining patients and the controls had equal expression of each allele. Fibrillin protein synthesis by fibroblasts from these heterozygous patients was also examined. After a 30 minute pulse with [{sup 35}S]-cysteine, cell lysates were collected and proteins analyzed by SDS-PAGE. The amount of fibrillin produced relative to a reference protein was determined using a Betagen scanner. Fibrillin protein synthesis was reduced in 2 of the 3 patients with very low RNA production from one of the FBN1 alleles. All other Marfan and control cell strains showed normal amounts of fibrillin synthesized. The low expression levels from one allele may contribute to, but not fully account for, the low detection rate of FBN1 mutations. Interestingly, protein synthesis levels were not affected in 4 of 6 cell strains demonstrating low levels of RNA expression.

  20. Possible involvement of loss of imprinting in immortalization of human fibroblasts.

    Science.gov (United States)

    Okamura, Kotaro; Ohno, Maki; Tsutsui, Takeki

    2011-04-01

    Disruption of the normal pattern of parental origin-specific gene expression is referred to as loss of imprinting (LOI), which is common in various cancers. To investigate a possible role of LOI in the early stage of human cell transformation, we studied LOI in 18 human fibroblast cell lines immortalized spontaneously, by viral oncogenes, by chemical or physical carcinogens, or by infection with a retrovirus vector encoding the human telomerase catalytic subunit, hTERT cDNA. LOI was observed in all the 18 immortal cell lines. The gene most commonly exhibiting LOI was NDN which displayed LOI in 15 of the 18 cell lines (83%). The other genes exhibiting LOI at high frequencies were PEG3 (50%), MAGE-L2 (61%) and ZNF 127 (50%). Expression of NDN that was lost in the immortal cell lines was restored by treatment with 5-aza-2'-deoxycytidine. The ratio of histone H3 lysine 9 methylation to histone H3 lysine 4 methylation of the chromatin containing the NDN promoter in the immortal WI-38VA13 cells was greater than that in the parental cells, suggesting chromatin structure-mediated regulation of NDN expression. We previously demonstrated that inactivation of the p16INK4a/pRb pathway is necessary for immortalization of human cells. Human fibroblasts in the pre-crisis phase and cells with an extended lifespan that eventually senesce, both of which have the normal p16INK4a/pRb pathway, did not show LOI at any imprinted gene examined. Although it is not clear if LOI plays a causal role in immortalization of human cells or is merely coincidental, these findings indicate a possible involvement of LOI in immortalization of human cells or a common mechanism involved in both processes.

  1. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  2. Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

    International Nuclear Information System (INIS)

    Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

    1987-01-01

    In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

  3. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu; Harishankar, M.; Dhinakar Raj, G.

    2011-01-01

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine

  4. FIELD VALIDATION OF A SHEEPSHEAD MINNOW ESTROGEN-RESPONSIVE CDNA MACROARRAY

    Science.gov (United States)

    Hemmer, Michael J., Iris Knoebl, Becky L. Hemmer, Patrick Larkin, Peggy S. Harris and Nancy D. Denslow. In press. Field Validation of a Sheepshead Minnow Estrogen-Responsive cDNA Macroarray (Abstract). To be presented at the SETAC Fourth World Congress, 14-18 November 2004, Portl...

  5. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

    International Nuclear Information System (INIS)

    Chung, T.D.Y.; Drake, F.H.; Tan, K.B.; Per, S.R.; Crooke, S.T.; Mirabelli, C.K.

    1989-01-01

    Several DNA topoisomerase II partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes

  6. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  7. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  8. EXPRESSION PROFILING OF ESTROGENIC COMPOUNDS USING A SHEEPSHEAD MINNOW CDNA MACROARRAY

    Science.gov (United States)

    Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston and Nancy D. Denslow. 2003. Expression Profiling of Estrogenic Compounds Using a Sheepshead Minnow cDNA Macroarray. Environ. Health Perspect. 111(6):839-846. (ERL,GB 1171). A variety of anthropogenic c...

  9. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning

    International Nuclear Information System (INIS)

    Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D.

    1990-01-01

    A λgt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first, screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin CDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA

  10. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  11. APPLICATION OF CDNA MICROARRAY TO THE STUDY OF ARSENIC TOXICOLOGY AND CARCINOGENESIS

    Science.gov (United States)

    Arsenic (As) is a common environmental toxicant and known human carcinogen. Epidemiological studies link As exposure to various disorders and cancers. However, the molecular mechanisms for As toxicity and carcinogenicity are not completely known. The cDNA microarray, a high-th...

  12. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  13. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... cDNA and the genomic sequence of RPS4X were cloned successfully from ... S4 genes plays a role in Turner syndrome; however, this ..... Project of Educational Committee of Sichuan Province ... Molecular biology of the cell.

  14. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    Science.gov (United States)

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

  15. Construction and characterization of a cDNA library from human ...

    African Journals Online (AJOL)

    The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain ...

  16. cDNA, genomic sequence cloning and analysis of the ribosomal ...

    African Journals Online (AJOL)

    Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant ...

  17. Cloning and sequence analysis of cDNA coding for rat nucleolar protein C23

    International Nuclear Information System (INIS)

    Ghaffari, S.H.; Olson, M.O.J.

    1986-01-01

    Using synthetic oligonucleotides as primers and probes, the authors have isolated and sequenced cDNA clones encoding protein C23, a putative nucleolus organizer protein. Poly(A + ) RNA was isolated from rat Novikoff hepatoma cells and enriched in C23 mRNA by sucrose density gradient ultracentrifugation. Two deoxyoligonuleotides, a 48- and a 27-mer, were synthesized on the basis of amino acid sequence from the C-terminal half of protein C23 and cDNA sequence data from CHO cell protein. The 48-mer was used a primer for synthesis of cDNA which was then inserted into plasmid pUC9. Transformed bacterial colonies were screened by hybridization with 32 P labeled 27-mer. Two clones among 5000 gave a strong positive signal. Plasmid DNAs from these clones were purified and characterized by blotting and nucleotide sequence analysis. The length of C23 mRNA was estimated to be 3200 bases in a northern blot analysis. The sequence of a 267 b.p. insert shows high homology with the CHO cDNA with only 9 nucleotide differences and an identical amino acid sequence. These studies indicate that this region of the protein is highly conserved

  18. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  19. Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Ficarelli, A; Tassi, F; Restivo, F M

    1999-03-01

    We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

  20. cDNA sequence and tissue expression analysis of glucokinase from ...

    African Journals Online (AJOL)

    Yomi

    2012-01-10

    Jan 10, 2012 ... distribution of GK mRNA in brain, mesenteric adipose tissue, spleen, white muscle and liver of grass ... expression profile of GK mRNA in liver normalized with β-actin level was 31, 454 and 649-fold compared .... Primers and expected products used for GK gene cDNA RT-PCR, RACE and real-time PCR.

  1. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

    NARCIS (Netherlands)

    Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

    2006-01-01

    The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

  2. Transcription analysis of apple fruit development using cDNA microarrays

    NARCIS (Netherlands)

    Soglio, V.; Costa, F.; Molthoff, J.W.; Weemen-Hendriks, M.; Schouten, H.J.; Gianfranceschi, L.

    2009-01-01

    The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and

  3. cDNA cloning and primary structure analysis of invariant chain in ...

    African Journals Online (AJOL)

    cDNA cloning and primary structure analysis of invariant chain in Chinese Pengze crucian carp. X Liu, W Yu, J Li, F Chen, S Liu, C Wu, J Xu. Abstract. Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been ...

  4. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    OpenAIRE

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; ...

  5. Pros and cons of characterising an optical translocation setup

    CSIR Research Space (South Africa)

    Maphanga, Charles

    2017-02-01

    Full Text Available an optical translocation setup Charles Maphanga 1, 2 , Rudzani Malabi 1, 2 , Saturnin Ombinda-Lemboumba 1 , Malik Maaza 2 , Patience Mthunzi-Kufa 1, 2* 1 Council for Scientific and Industrial Research, National Laser Centre, P O BOX 395, Pretoria...

  6. Centrifugally driven microfluidic disc for detection of chromosomal translocations

    DEFF Research Database (Denmark)

    Brøgger, Anna Line; Kwasny, Dorota; Bosco, Filippo G.

    2012-01-01

    and prognosis of patients. In this work we demonstrate a novel, centrifugally-driven microfluidic system for controlled manipulation of oligonucleotides and subsequent detection of chromosomal translocations. The device is fabricated in the form of a disc with capillary burst microvalves employed to control...

  7. 40 CFR 798.5460 - Rodent heritable translocation assays.

    Science.gov (United States)

    2010-07-01

    ... fertile animals for cytological confirmation as translocation heterozygotes. (3) Animal selection—(i... administration include oral, inhalation, admixture with food or water, and IP or IV injection. (e) Test.... Criteria for determining normal and semisterile males are usually established for each new strain because...

  8. Polymer translocation through a nanopore: a showcase of anomalous diffusion.

    Science.gov (United States)

    Milchev, A; Dubbeldam, Johan L A; Rostiashvili, Vakhtang G; Vilgis, Thomas A

    2009-04-01

    We investigate the translocation dynamics of a polymer chain threaded through a membrane nanopore by a chemical potential gradient that acts on the chain segments inside the pore. By means of diverse methods (scaling theory, fractional calculus, and Monte Carlo and molecular dynamics simulations), we demonstrate that the relevant dynamic variable, the transported number of polymer segments, s(t), displays an anomalous diffusive behavior, both with and without an external driving force being present. We show that in the absence of drag force the time tau, needed for a macromolecule of length N to thread from the cis into the trans side of a cell membrane, scales as tauN(2/alpha) with the chain length. The anomalous dynamics of the translocation process is governed by a universal exponent alpha= 2/(2nu + 2 - gamma(1)), which contains the basic universal exponents of polymer physics, nu (the Flory exponent) and gamma(1) (the surface entropic exponent). A closed analytic expression for the probability to find s translocated segments at time t in terms of chain length N and applied drag force f is derived from the fractional Fokker-Planck equation, and shown to provide analytic results for the time variation of the statistical moments and . It turns out that the average translocation time scales as tau proportional, f(-1)N(2/alpha-1). These results are tested and found to be in perfect agreement with extensive Monte Carlo and molecular dynamics computer simulations.

  9. Selective bowel decontamination results in gram-positive translocation.

    Science.gov (United States)

    Jackson, R J; Smith, S D; Rowe, M I

    1990-05-01

    Colonization by enteric gram-negative bacteria with subsequent translocation is believed to be a major mechanism for infection in the critically ill patient. Selective bowel decontamination (SBD) has been used to control gram-negative infections by eliminating these potentially pathogenic bacteria while preserving anaerobic and other less pathogenic organisms. Infection with gram-positive organisms and anaerobes in two multivisceral transplant patients during SBD led us to investigate the effect of SBD on gut colonization and translocation. Twenty-four rats received enteral polymixin E, tobramycin, amphotericin B, and parenteral cefotaxime for 7 days (PTA + CEF); 23 received parenteral cefotaxime alone (CEF), 19 received the enteral antibiotics alone (PTA), 21 controls received no antibiotics. Cecal homogenates, mesenteric lymph node (MLN), liver, and spleen were cultured. Only 8% of the PTA + CEF group had gram-negative bacteria in cecal culture vs 52% CEF, 84% PTA, and 100% in controls. Log Enterococcal colony counts were higher in the PTA + CEF group (8.0 + 0.9) vs controls (5.4 + 0.4) P less than 0.01. Translocation of Enterococcus to the MLN was significantly increased in the PTA + CEF group (67%) vs controls (0%) P less than 0.01. SBD effectively eliminates gram-negative organisms from the gut in the rat model. Overgrowth and translocation of Enterococcus suggests that infection with gram-positive organisms may be a limitation of SBD.

  10. Toponomics method for the automated quantification of membrane protein translocation.

    Science.gov (United States)

    Domanova, Olga; Borbe, Stefan; Mühlfeld, Stefanie; Becker, Martin; Kubitz, Ralf; Häussinger, Dieter; Berlage, Thomas

    2011-09-19

    Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state. An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results. New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.

  11. Bladder calculus resulting from an intravesical translocation of ...

    African Journals Online (AJOL)

    Although perforation of the uterus by an intrauterine contraceptive device (IUCD) is commonly encountered, intravesical translocation and secondary calculus formation is a very rare complication.We report a case of a 60-year old multiparous woman in whom an intrauterine contraceptive Copper-T device inserted 12 years ...

  12. Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

    International Nuclear Information System (INIS)

    Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

    2005-01-01

    Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids

  13. Resource Control: A Translocation Of The Scramble For Africa ...

    African Journals Online (AJOL)

    Adopting a theoretical framework successfully adapted from the biological and medical sciences, namely; translocation analysis, the paper traces the ancestry of the present resource control problem to the scramble, first, and then, the use of fiscal and revenue allocation commissions during the colonial era, and the ...

  14. Single-Molecule Studies of Bacterial Protein Translocation

    NARCIS (Netherlands)

    Kedrov, Alexej; Kusters, Ilja; Driessen, Arnold J. M.

    2013-01-01

    In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of

  15. Introduction: translocal development, development corridors and development chains.

    NARCIS (Netherlands)

    Zoomers, E.B.; Westen, A.C.M. van

    2011-01-01

    This paper offers an introduction to this Special Issue of International Development Planning Review. It uses the concepts of translocal development, development corridors and development chains to secure a better grasp of what development means in the context of globalisation and how ‘local

  16. Concentration Polarization in Translocation of DNA through Nanopores and Nanochannels

    NARCIS (Netherlands)

    Das, S.; Dubsky, P.; van den Berg, Albert; Eijkel, Jan C.T.

    2012-01-01

    In this Letter we provide a theory to show that high-field electrokinetic translocation of DNA through nanopores or nanochannels causes large transient variations of the ionic concentrations in front and at the back of the DNA due to concentration polarization (CP). The CP causes strong local

  17. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

    Science.gov (United States)

    Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2006-11-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

  18. Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

    Science.gov (United States)

    Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

    2018-06-26

    Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

  19. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

  20. Transcuticular translocation of radionuclides on plant leaf surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Ken-ichi; Watanabe, Tadakazu; Ambe, Shizuko; Yamaguchi, Isamu [Institute of Physical and Chemical Research, Wako, Saitama (Japan)

    1996-12-31

    The cuticle covering all the outermost surfaces of the aerial parts of plants could play a selective role in uptake and translocation of radionuclides from air into plants. In this study, we investigated the transcuticular uptake and translocation behavior via water droplets of various radionuclides in red clover, orchard grass, Japanese radish and mung bean. Ten {mu}l of an aqueous solution of the multitracer generated from Au was applied to the upper surface of the 2nd leaf of the plants at the 5th leaf stage. The plants were then grown for 14 days at 25degC and 70% RH under illumination of artificial solar lights. The transcuticular uptake and translocation throughout the plant were periodically assayed by determining the radioactivity in the surface residue, the cuticle layer beneath the applied site, the leaf area outside the applied site, the other aerial parts and the root of the plant, using an HPGe detector. The applied radionuclides were absorbed into, in turn, the cuticle layer beneath the applied site and then translocated through the cuticle to the inner tissue and eventually to the other aerial parts and finally to the roots, of the plant. The distribution and accumulation in the plant seems to depend upon the characteristics of each radionuclide and plant species. Ca{sup *} and Te{sup *} tended to remain on leaf surfaces without being absorbed into the cuticle. On the other hand, Sc{sup *}, Co{sup *}, Zn{sup *}, Se{sup *}, Rb{sup *}, and Eu{sup *} were easily absorbed and translocated to every part of the plant including the root. The other radionuclides such as Be{sup *}, Mn{sup *}, Sr{sup *}, Y{sup *}, Ba{sup *}, Ce{sup *}, Pm{sup *}, Gd{sup *}, Hf{sup *}, Yb{sup *}, Lu{sup *}, Os{sup *}, Ir{sup *}, and Pt{sup *} remained in the region close to the site of their application. The above results possibly indicate the existence of mechanisms common to these plants for selective transcuticular uptake and translocation of radionuclides within plant

  1. Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Del Valle

    2014-09-01

    Full Text Available Cancer-associated fibroblasts (CAF influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4, lymph node metastasis (N+, n = 3 and bone marrow (BM, n = 4 obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01. Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF. Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

  2. Cold-inhibited phloem translocation in sugar beet

    International Nuclear Information System (INIS)

    Grusak, M.A.

    1985-01-01

    Experimental studies were undertaken on a simplified single source leaf-single sink leaf, or single source leaf-double sink leaf sugar beet system to investigate the responsive nature of the long-distance phloem translocation system to localized cooling perturbations on the source leaf petiole. Experiments were performed by using a steady state [ 14 C]-labelling system for the source leaf, and translocation into the sink leaf (leaves) was monitored with a Geiger-Mueller system. A specially designed Peltier apparatus enabled cooling of the source petiole to 1 0 C (or other desired temperatures) at various positions on the petiole, over different lengths, and at different rates of cooling. Initial experiment were designed to test the predictions of a mathematical recovery model of translocation inhibited by cold. The results did not support the mathematical model, but did suggest that vascular anastomoses may be involved in the recovery response. Selective petiolar incision/excision experiments showed that anastomoses were capable of re-establishing translocation following a disruption of flow. Studies with two monitored sink levels suggested that the inhibition to slow-coolings was not due to reduced translocation through the cooled source petiole region, but rather, was due to a repartitioning of flow among the terminal sinks (sink leaves and hypocotyl/crown region above the heat-girdled root). This repartitioning occurred via a redirection of flow through the vascular connections in the crown region of the plant, and appeared to be promoted by rapid, physical signals originating from the cooled region of the petiole

  3. MiT family translocation renal cell carcinoma.

    Science.gov (United States)

    Argani, Pedram

    2015-03-01

    The MiT subfamily of transcription factors includes TFE3, TFEB, TFC, and MiTF. Gene fusions involving two of these transcription factors have been identified in renal cell carcinoma (RCC). The Xp11 translocation RCCs were first officially recognized in the 2004 WHO renal tumor classification, and harbor gene fusions involving TFE3. The t(6;11) RCCs harbor a specific Alpha-TFEB gene fusion and were first officially recognized in the 2013 International Society of Urologic Pathology (ISUP) Vancouver classification of renal neoplasia. These two subtypes of translocation RCC have many similarities. Both were initially described in and disproportionately involve young patients, though adult translocation RCC may overall outnumber pediatric cases. Both often have unusual and distinctive morphologies; the Xp11 translocation RCCs frequently have clear cells with papillary architecture and abundant psammomatous bodies, while the t(6;11) RCCs frequently have a biphasic appearance with both large and small epithelioid cells and nodules of basement membrane material. However, the morphology of these two neoplasms can overlap, with one mimicking the other. Both of these RCCs underexpress epithelial immunohistochemical markers like cytokeratin and epithelial membrane antigen (EMA) relative to most other RCCs. Unlike other RCCs, both frequently express the cysteine protease cathepsin k and often express melanocytic markers like HMB45 and Melan A. Finally, TFE3 and TFEB have overlapping functional activity as these two transcription factors frequently heterodimerize and bind to the same targets. Therefore, on the basis of clinical, morphologic, immunohistochemical, and genetic similarities, the 2013 ISUP Vancouver classification of renal neoplasia grouped these two neoplasms together under the heading of "MiT family translocation RCC." This review summarizes our current knowledge of these recently described RCCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Immortalization of Werner syndrome and progeria fibroblasts

    International Nuclear Information System (INIS)

    Saito, H.; Moses, R.E.

    1991-01-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

  5. Coupling of cytoskeleton functions for fibroblast locomotion

    DEFF Research Database (Denmark)

    Couchman, J R; Lenn, M; Rees, D A

    1985-01-01

    caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

  6. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...

  7. Genotype/phenotype analysis in a male patient with partial trisomy 4p and monosomy 20q due to maternal reciprocal translocation (4;20): A case report.

    Science.gov (United States)

    Wu, Dong; Zhang, Hui; Hou, Qiaofang; Wang, Hongdan; Wang, Tao; Liao, Shixiu

    2017-11-01

    Translocations are the most frequent structural aberration in the human genome. Carriers of balanced chromosome rearrangement exhibit an increased risk of abortion and/or a chromosomally‑unbalanced child. The present study reported a clinical and cytogenetic analysis of a child who exhibited typical trisomy 4p and monosomy 20q features, including intellectual disability, delayed speech, tall stature, seizures and facial dysmorphism. The karyotype of the proband exhibited 46, XY, add(20) (q13.3). The karyotype of the mother indicated a balanced translocation karyotype: 46, XX, t(4;20) (p15.2;q13.1). The array‑based comparative genomic hybridization (aCGH) analysis identified partial trisomy of the short arm of chromosome 4 and partial monosomy of distal 20q in the proband due to maternal balanced reciprocal translocation 4;20. The analysis of genotype/phenotype correlation demonstrated that fibroblast growth factor receptor 3 and msh homeobox 1 may be the important genes for 4p duplication, and that potassium voltage‑gated channel subfamily Q member 2, myelin transcription factor 1 and cholinergic receptor nicotinic α4 subunit may be the important genes for 20q deletion. To the best of our knowledge, the present study was the first to report an unbalanced translocation involving chromosomes 4p and 20q. The present study additionally demonstrated that aCGH analysis is able to reliably detect unbalanced submicroscopic chromosomal aberrations.

  8. Atlas of alien and translocated indigenous aquatic animals in southern Africa

    CSIR Research Space (South Africa)

    De Moor, IJ

    1988-01-01

    Full Text Available This report serves as an introduction to the problem of alien and translocated aquatic animals in southern Africa is given followed by checklists of the different species which have been introduced into or translocated within the subcontinent...

  9. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  10. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  11. Translocation Study of Some Zooxanthellae Clade to the Survival and Growth of Goniastrea Aspera After Bleaching

    OpenAIRE

    Purnomo, Pujiono W

    2014-01-01

    Inter-host translocation technique of zooxanthellae was attempted to prove Buddemier and Futin's (1993) theory on adaptation. The recent trend of coral products trading must be anticipated by its mass production through artificial techniques, the alternation of natural resources. Translocation bio-technique of zooxanthellae on coral was expected to resolve the problem and the translocation study should provide fundamental answer to coral recovery. The study of zooxanthellae translocation was ...

  12. Microbial Translocation in HIV Infection is Associated with Dyslipidemia, Insulin Resistance, and Risk of Myocardial Infarction

    DEFF Research Database (Denmark)

    Pedersen, Karin Kaereby; Pedersen, Maria; Trøseid, Marius

    2013-01-01

    Microbial translocation has been suggested to be a driver of immune activation and inflammation. We hypothesized that microbial translocation may be related to dyslipidemia, insulin resistance, and the risk of coronary heart disease in HIV-infected individuals.......Microbial translocation has been suggested to be a driver of immune activation and inflammation. We hypothesized that microbial translocation may be related to dyslipidemia, insulin resistance, and the risk of coronary heart disease in HIV-infected individuals....

  13. Toll-like receptor 9 mediated responses in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Ingrid Kristine Ohm

    Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

  14. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  15. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-10-01

    Full Text Available We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1 from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4(q21;q25 that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY. These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  16. 40 CFR 798.5955 - Heritable translocation test in drosophila melanogaster.

    Science.gov (United States)

    2010-07-01

    ... drosophila melanogaster. 798.5955 Section 798.5955 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5955 Heritable translocation test in drosophila melanogaster. (a) Purpose. The heritable translocation test in Drosophila measures the induction of chromosomal translocations in germ cells of insects...

  17. Translocation as a conservation tool for Agassiz's desert tortoises: Survivorship, reproduction, and movements

    Science.gov (United States)

    K. E. Nussear; C. R. Tracy; P. A. Medica; D. S. Wilson; R. W. Marlow; P. S. Corn

    2012-01-01

    We translocated 120 Agassiz's desert tortoises to 5 sites in Nevada and Utah to evaluate the effects of translocation on tortoise survivorship, reproduction, and habitat use. Translocation sites included several elevations, and extended to sites with vegetation assemblages not typically associated with desert tortoises in order to explore the possibility of moving...

  18. Meiotic behaviour and spermatogenesis in male mice heterozygous for translocation types also occurring in man

    NARCIS (Netherlands)

    Nijhoff, J.H.

    1981-01-01

    In this thesis a start was made with meiotic observations of mouse translocation types - a Robertsonian translocation and a translocation between a metacentric and an acrocentric chromosome - which also occur in man. It is generally accepted that, when no chromosomal rearrangements are involved, man

  19. Dek-can rearrangement in translocation (6;9)(p23;q34)

    NARCIS (Netherlands)

    Soekarman, D.; von Lindern, M.; van der Plas, D. C.; Selleri, L.; Bartram, C. R.; Martiat, P.; Culligan, D.; Padua, R. A.; Hasper-Voogt, K. P.; Hagemeijer, A.

    1992-01-01

    The translocation (6;9)(p23;q34) is mainly found in specific subtypes of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The diagnosis of this translocation is not easy since the cytogenetic change is quite subtle. The two genes involved in this translocation were recently isolated

  20. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Salahuddin, E-mail: Salah.Ahmed@utoledo.edu [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V. [Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States); Bhansali, Pravin; Tillekeratne, L.M. Viranga [Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH (United States)

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  1. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

    1987-01-01

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  2. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  3. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    Energy Technology Data Exchange (ETDEWEB)

    Inadomi, Chiaki, E-mail: inadomic@nagasaki-u.ac.jp [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Murata, Hiroaki [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Ihara, Yoshito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509 (Japan); Goto, Shinji; Urata, Yoshishige [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yodoi, Junji [Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Kondo, Takahito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Sumikawa, Koji [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  4. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    International Nuclear Information System (INIS)

    Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

    2012-01-01

    Highlights: ► GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. ► NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. ► Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  5. Myogenic conversion of bladder fibroblasts by construction and ...

    African Journals Online (AJOL)

    The cultured primary bladder fibroblasts were transfected by pEGFP-Myod1 with Lipofection 2000 reagent. The results showed that expression of Myod1 could cause myogenic differentiation of bladder fibroblasts. These findings support the possibility of an alternative approach to exploit the capacity of Myod1 to activate ...

  6. Rac inhibition reverses the phenotype of fibrotic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shi-wen Xu

    Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

  7. Mesenchymal stem cells induce dermal fibroblast responses to injury

    International Nuclear Information System (INIS)

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  8. Collagen expression in fibroblasts with a novel LMNA mutation

    International Nuclear Information System (INIS)

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy

  9. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

    Science.gov (United States)

    Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

    2016-01-01

    Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

  10. RFLP for Duchenne muscular dystrophy cDNA clone 44-1

    Energy Technology Data Exchange (ETDEWEB)

    Laing, N G; Siddique, T; Bartlett, R J; Yamaoka, L H; Chen, J C; Walker, A P; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

    1988-07-25

    Clone 44-1 is one of six cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 0.9kb fragment in the EcoR1 site of Bluescript. Taq1 (TlCGA) identifies two alleles with bands at 6.8 and 5.7kb, as well as four constant bands at 4.8, 3.9, 3.5 and 2.5kb. Its frequency was studied in 62 unrelated individuals. Mendelian inheritance was demonstrated in one three generation and three two generation informative families, 26 individuals. There were no problems on RFLP analysis under normal stringency conditions.

  11. RFLP for Duchenne muscular dystrophy cDNA clone 30-2

    Energy Technology Data Exchange (ETDEWEB)

    Walker, A P; Bartlett, R J; Laing, N G; Siddique, T; Yamaoka, L H; Chen, J C; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

    1988-09-26

    30-2 is one of 6 cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 1.15 kb fragment in the EcoRI site of Bluescribe. TaqI (T{down arrow}CGA) identifies two bands with alleles at 3.7 and 3.5 kb, as well as eight constant bands at 9.0, 7.5, 4.6, 3.6, 3.4, 2.5, 1.7 and 1.4 kb. The allele frequency was studied in 47 unrelated DMD males: 3.7 kb allele 0.45; and 3.5 kb allele 0.55. Co-dominant X-linked segregation was demonstrated in two 2-generation families. 1.1% agarose gels required to resolve the bands. The polymorphism is also recognized by PERT 87-15.

  12. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  13. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

  14. cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

    Science.gov (United States)

    Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

    1990-09-25

    The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

  15. Markers of immunity and bacterial translocation in cirrhosis

    DEFF Research Database (Denmark)

    Mortensen, Christian

    2015-01-01

    to be correlated to portal hypertension, a clinically relevant haemodynamic alteration, and appeared to be associated with increased mortality. To assess the consequences of BT on immunity, we developed an assay for the detection of bacterial DNA (bDNA), a novel marker of BT. Using the assay in the second study......Bacterial translocation (BT), the migration of enteric bacteria to extraintestinal sites, is related to immune stimulation and haemodynamic changes in experimental cirrhosis. These changes may be highly relevant to patients with cirrhosis, where changes in the circulation cause serious......, in 38 patients with ascites, we found no association between bDNA and immunity, in contrast to some previous findings. In the final paper, exploring one possible translocation route, we hypothesized a difference in bDNA levels between the blood from the veins draining the gut on one hand and the liver...

  16. Regulation of Neuronal Protein Trafficking and Translocation by SUMOylation

    Directory of Open Access Journals (Sweden)

    Jeremy M. Henley

    2012-05-01

    Full Text Available Post-translational modifications of proteins are essential for cell function. Covalent modification by SUMO (small ubiquitin-like modifier plays a role in multiple cell processes, including transcriptional regulation, DNA damage repair, protein localization and trafficking. Factors affecting protein localization and trafficking are particularly crucial in neurons because of their polarization, morphological complexity and functional specialization. SUMOylation has emerged as a major mediator of intranuclear and nucleo-cytoplasmic translocations of proteins involved in critical pathways such as circadian rhythm, apoptosis and protein degradation. In addition, SUMO-regulated re-localization of extranuclear proteins is required to sustain neuronal excitability and synaptic transmission. Thus, SUMOylation is a key arbiter of neuronal viability and function. Here, we provide an overview of recent advances in our understanding of regulation of neuronal protein localization and translocation by SUMO and highlight exciting areas of ongoing research.

  17. Sex Chromosome Translocations in the Evolution of Reproductive Isolation

    Science.gov (United States)

    Tracey, Martin L.

    1972-01-01

    Haldane's rule states that in organisms with differentiated sex chromosomes, hybrid sterility or inviability is generally expressed more frequently in the heterogametic sex. This observation has been variously explained as due to either genic or chromosomal imbalance. The fixation probabilities and mean times to fixation of sex-chromosome translocations of the type necessary to explain Haldane's rule on the basis of chromosomal imbalance have been estimated in small populations of Drosophila melanogaster. The fixation probability of an X chromosome carrying the long arm of the Y(X·YL) is approximately 30% greater than expected under the assumption of no selection. No fitness differences associated with the attached YL segment were detected. The fixation probability of a deficient Y chromosome is 300% greater than expected when the X chromosome contains the deleted portion of the Y. It is suggested that sex-chromosome translocations may play a role in the establishment of reproductive isolation. PMID:4630586

  18. Doubling potential of fibroblasts from different species after ionising radiation

    International Nuclear Information System (INIS)

    Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

    1976-01-01

    It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerates acquisition of this potential; human fibroblasts showed an intermediate response, since ionising radiation neither established the cultures as with mouse cells nor reduced the number of cells produced as with chicken fibroblasts. Possible explanations for the different behaviour of the species are offered. (U.K.)

  19. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

    OpenAIRE

    Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

    1988-01-01

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

  20. Analysis of photosynthate translocation velocity and measurement of weighted average velocity in transporting pathway of crops

    International Nuclear Information System (INIS)

    Ge Cailin; Luo Shishi; Gong Jian; Zhang Hao; Ma Fei

    1996-08-01

    The translocation profile pattern of 14 C-photosynthate along the transporting pathway in crops were monitored by pulse-labelling a mature leaf with 14 CO 2 . The progressive spreading of translocation profile pattern along the sheath or stem indicates that the translocation of photosynthate along the sheath or stem proceed with a range of velocities rather than with just a single velocity. The method for measuring the weighted average velocity of photosynthate translocation along the sheath or stem was established in living crops. The weighted average velocity and the maximum velocity of photosynthate translocation along the sheath in rice and maize were measured actually. (4 figs., 3 tabs.)

  1. A strategy for generation and balancing of autosome: Y chromosome translocations.

    Science.gov (United States)

    Joshi, Sonal S; Cheong, Han; Meller, Victoria H

    2014-01-01

    We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced ( 2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.

  2. Polymer translocation in the presence of excluded volume and explicit hydrodynamic interactions

    International Nuclear Information System (INIS)

    Guillouzic, Steve; Slater, Gary W.

    2006-01-01

    Molecular Dynamics simulations of polymer translocation are hereby reported. No external force was applied to the polymer during translocation, and the dynamics was dominated by polymer-pore interactions. It was found that hydrodynamic interactions play an important role in the relaxation of the polymer on each side of the membrane but have a negligible impact on the translocation process itself. Also, the scaling laws obtained for the relaxation and translocation times indicate that long translocating polymers may be considered to be following a quasi-equilibrium anomalous diffusion process in the absence of external forces

  3. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  4. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    Qian, X.; Xu, S.Z.

    2015-01-01

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  5. Human pro. cap alpha. 1(III) collagen: cDNA sequence for the 3' end

    Energy Technology Data Exchange (ETDEWEB)

    Mankoo, B S; Dalgleish, R

    1988-03-25

    The authors have previously isolated two overlapping cDNA clones, pIII-21 and pIII-33, which encode the C-terminal end of human type III procollagen. They now present the sequence of 2520 bases encoded in these cDNAs which overlaps other previously published sequences for the same gene. The sequence presented differs from previously published sequences at five positions.

  6. Cloning, sequencing and expression of a novel xylanase cDNA from ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-03

    Dec 3, 2008 ... First strand cDNA was synthesized by RT-PCR with Oligo(dT)15 using mRNA isolated ... 4°C. Single colonies were picked into 5 mL BMGY medium for preculture, and incubated ... to fold properly into a native conformation. Without the .... polymorphism is often used in taxonomy, but now, it is being well ...

  7. Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

    2010-01-01

    A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

  8. International study of factors affecting human chromosome translocations

    Czech Academy of Sciences Publication Activity Database

    Sigurdson, A.J.; Ha, M.; Hauptmann, M.; Bhatti, P.; Šrám, Radim; Beskid, Olena; Tawn, E.J.; Whitehouse, C.A.; Lindholm, C.; Nakano, M.; Kodama, Y.; Nakamura, N.; Vorobtsova, I.; Oestreicher, U.; Stephan, G.; Yong, L.C.; Bauchinger, M.; Schmid, E.; Chung, H.W.; Darroudi, F.; Roy, L.; Voisin, P.; Barquinero, J.F.; Livingston, G.; Blakey, D.; Hayata, I.; Zhang, W.; Wang, Ch.; Benett, L.M.; Littlefield, L.G.; Edwards, A.A.; Kleinerman, R.A.; Tucker, J.D.

    2008-01-01

    Roč. 652, č. 2 (2008), s. 112-121 ISSN 1383-5718 R&D Projects: GA MŽP SL/5/160/05; GA MŽP SI/340/2/00; GA MŽP SL/740/5/03 Institutional research plan: CEZ:AV0Z50390512 Keywords : Chromosome translocations * FISH * Background frequency Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 2.363, year: 2008

  9. "Translocal Express" juba täna! / Rael Artel

    Index Scriptorium Estoniae

    Artel, Rael, 1980-

    2009-01-01

    27. märtsil algab Kumu Kunstimuuseumis "Public Preparation'i" ("Avalik ettevalmistus") sarja rahvusvaheline seminar "Translocal Express. Golden Age" ("Translokaalne ekspress. Kuldaeg"), kus on kõne all ajalookirjutamise ja kollektiivse mälu roll praegu domineerivas natsionalistlikus diskursuses ja selle käsitlemine kaasaegses kunstis. Seminaril on lähtutud eelkõige kunstnike Martin Krenni (Viin) ja Kristina Normani teoste tutvustamisest

  10. Cloning of the gene encoding the δ subunit of the human T-cell receptor reveals its physical organization within the α-subunit locus and its involvement in chromosome translocations in T-cell malignancy

    International Nuclear Information System (INIS)

    Isobe, M.; Russo, G.; Haluska, F.G.; Croce, C.M.

    1988-01-01

    By taking advantage of chromosomal walking techniques, the authors have obtained clones that encompass the T-cell receptor (TCR) δ-chain gene. They analyzed clones spanning the entire J α region extending 115 kilobases 5' of the TCR α-chain constant region and have shown that the TCR δ-chain gene is located over 80 kilobases 5' of C α . TCR δ-chain gene is rearranged in the γ/δ-expressing T-cell line Peer and is deleted in α/β-expressing T-cell lines. Sequence analysis of portions of this genomic region demonstrates its identity with previously described cDNA clones corresponding to the C δ and J δ segments. Furthermore, they have analyzed a t(8;14)-(q24;q11) chromosome translocation from a T-cell leukemia and have shown that the J δ segment is rearranged in cells deriving from this tumor and probably directly involved in the translocation. Thus, the newly clones TCR δ chain is implicated in the genesis of chromosome translocations in T-cell malignancies carrying cytogenetic abnormalities of band 14q11

  11. Single-strand DNA molecule translocation through nanoelectrode gaps

    International Nuclear Information System (INIS)

    Zhao Xiongce; Payne, Christina M; Cummings, Peter T; Lee, James W

    2007-01-01

    Molecular dynamics simulations were performed to investigate the translocation of single-strand DNA through nanoscale electrode gaps under the action of a constant driving force. The application behind this theoretical study is a proposal to use nanoelectrodes as a screening gap as part of a rapid genomic sequencing device. Preliminary results from a series of simulations using various gap widths and driving forces suggest that the narrowest electrode gap that a single-strand DNA can pass is ∼1.5 nm. The minimum force required to initiate the translocation within nanoseconds is ∼0.3 nN. Simulations using DNA segments of various lengths indicate that the minimum initiation force is insensitive to the length of DNA. However, the average threading velocity of DNA varies appreciably from short to long DNA segments. We attribute such variation to the different nature of drag force experienced by the short and long DNA segments in the environment. It is found that DNA molecules deform significantly to fit in the shape of the nanogap during the translocation

  12. Spatial behaviour and survival of translocated wild brown hares

    Directory of Open Access Journals (Sweden)

    Fischer, C.

    2012-01-01

    Full Text Available The fragility of many populations of brown hares in Western Europe is a concern for managers, hunters and naturalists. We took advantage of a locally high density population to use wild individuals to restock areas where the species had disappeared or was close to disappearing. The aim of the project was to assess the evolution of the spatial behaviour after release using radio–tracking. Over 150 wild brown hares were translocated, one third of which were fitted with radio collars. In addition, fifteen individuals were radio–tagged and released back into the source population as a control. Most individuals settled in less than two months and their seasonal home range, once settled, was similar to that observed in the source population. Mean duration of tracking was not significantly different between the two groups. Moreover, two years after the last translocation, tagged individuals can still be observed, but most hares present are not tagged, which indicates natural reproduction of the released individuals. The translocation of wild individuals thus appears to give encouraging results.

  13. Dominant-lethal mutations and heritable translocations in mice

    International Nuclear Information System (INIS)

    Generoso, W.M.

    1983-01-01

    Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of these two endpoints appears to be determined by the stability of alkylation products with the chromosomes. If the reaction products are intact in the male chromosomes at the time of sperm entry, they may be repaired in fertilized eggs. If repair is not effected and the alkylation products persist to the time of pronuclear chromosome replication, they lead to chromatid-type aberrations and eventually to dominant-lethality. The production of heritable translocations, on the other hand, requires a transformation of unstable alkylation products into suitable intermediate lesions. The process by which these lesions are converted into chromosome exchange within the male genome takes place after sperm enters the egg but prior to the time of pronuclear chromosome replication (i.e., chromosome-type). Thus, dominant-lethal mutations result from both chromatid- and chromosome-type aberrations while heritable translocations result primarily from the latter type. DNA target sites associated with the production of these two endpoints are discussed

  14. Dominant-lethal mutations and heritable translocations in mice

    Energy Technology Data Exchange (ETDEWEB)

    Generoso, W.M.

    1983-01-01

    Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of these two endpoints appears to be determined by the stability of alkylation products with the chromosomes. If the reaction products are intact in the male chromosomes at the time of sperm entry, they may be repaired in fertilized eggs. If repair is not effected and the alkylation products persist to the time of pronuclear chromosome replication, they lead to chromatid-type aberrations and eventually to dominant-lethality. The production of heritable translocations, on the other hand, requires a transformation of unstable alkylation products into suitable intermediate lesions. The process by which these lesions are converted into chromosome exchange within the male genome takes place after sperm enters the egg but prior to the time of pronuclear chromosome replication (i.e., chromosome-type). Thus, dominant-lethal mutations result from both chromatid- and chromosome-type aberrations while heritable translocations result primarily from the latter type. DNA target sites associated with the production of these two endpoints are discussed.

  15. Black bears in Arkansas: Characteristics of a successful translocation

    Science.gov (United States)

    Smith, Kimberly G.; Clark, Joseph D.

    1994-01-01

    In 1958, the Arkansas Game and Fish Commission began translocating black bears (Ursus americanus) from Minnesota to the Interior Highlands (Ozark and Ouachita mountains) of Arkansas where bears had been extirpated early in this century. This project continued for 11 years with little public imput, during which time an estimated 254 bears were released. We estimate there are now >2,500 bears in the Interior Highlands of Arkansas, Missouri, and Oklahoma, making it one of the most successful translocations of a Carnivora. Factors that contributed to the success include use of wild-captured animals, elimination of major factors associated with extirpation, release into prime habitats within the former range, multiple release sites, release of 20–40 animals/year for eight years, and release of mostly males prior to release of mostly females. Studies on two allopatric populations demonstrate that they are now diverging in some demographic characteristics, including litter size, cub survivorship, and adult sex-ratio. Translocation of black bears to the Interior Highlands is successful in terms of numbers of animals, but it will not be truly successful until people accept black bears as part of the regional fauna. To that end, those associated with management and research of bears in Arkansas are now focussing on public education and control of nuisance bears.

  16. Translocation of 11C from leaves of Helianthus: preliminary results

    International Nuclear Information System (INIS)

    Fensom, D.S.; Aikman, D.; Scobie, J.; Drinkwater, A.; Ledingham, K.W.O.

    1977-01-01

    11 C fed to leaves as 11 CO 2 was used to study the dynamics of short-term translocation of photosynthate in Helianthus. As in 14 C studies small amounts of tracer were often detected in the stem close to the fed leaf in th first 5 min, followed by a larger mass flow after 15 min. The speed of mass flow of tracer movement was calculated to be 60 to 400 cm.h -1 depending on the method of calculation. There was no evidence in the premass flow for discrete spots along the stem or petiole where tracer accumulated. Neither was there firm evidence for pulses of tracer moving steadily forward, but there were point fluctuations of greater variability than would be expected by chance alone, which suggest the possibility of aberrations of movement superimposed on the mass flow. Details of these aberrations could not be assessed with certainty from these preliminary experiments owing to the rather low tracer activity. The translocation profiles were sensitive to the prior light conditioning of the plant and above all to chilling. In Helianthus the latter produced temporary restrictions in translocation which lasted for some 10-12 min. (author)

  17. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  18. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    Science.gov (United States)

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  19. Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

    Science.gov (United States)

    Mattsson, J G; Ljunggren, E L; Bergström, K

    2001-05-01

    The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

  20. Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis.

    Science.gov (United States)

    Yang, Liping; Meng, Zining; Liu, Yun; Zhang, Yong; Liu, Xiaochun; Lu, Danqi; Huang, Junhai; Lin, Haoran

    2010-01-15

    The Chinese giant salamander (Andrias davidianus) is one of the largest and 'living fossil' species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.

  1. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

  2. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  3. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    International Nuclear Information System (INIS)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-01-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

  4. cDNA encoding a polypeptide including a hev ein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  5. [Construction of fetal mesenchymal stem cell cDNA subtractive library].

    Science.gov (United States)

    Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

    2002-04-01

    To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

  6. Persistence of X-ray-induced chromosomal rearrangements in long-term cultures of human diploid fibroblasts

    International Nuclear Information System (INIS)

    Kano, Y.; Little, J.B.

    1984-01-01

    As part of a long-term study of mechanisms of human cell neoplastic transformation, the authors have examined the change in the frequencies of X-ray-induced chromosome rearrangements in density-inhibited human foreskin fibroblasts as a function of subculture time. In nonproliferating cells, the frequency of chromosomal aberrations declined within 24 to 48 hr but still remained at a relatively high level up to 43 days after irradiation. Aberrations disappeared rapidly, however, when the cells were allowed to proliferate, indicating that these lesions are lethal to dividing cells. The frequency of induced translocations, as determined by analysis of G-banded karyotypes, was dose dependent and remained stable up to 20 mean population doublings after irradiation. When subculture of density-inhibited cultures was delayed for 4 hr after irradiation (confluent holding), the frequency of chromosomal aberrations in the first mitosis declined, whereas the translocation frequencies at later passage were elevated as compared with cells subcultured immediately. This correlates with the reported increase in the frequency of transformation under similar conditions. These findings support the hypothesis that chromosomal rearrangements induced by DNA damage may be involved in the initiation of cancer

  7. LXA4 actions direct fibroblast function and wound closure

    International Nuclear Information System (INIS)

    Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

    2015-01-01

    Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

  8. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  9. CDNA Microarray Based Comparative Gene Expression Analysis of Primary Breast Tumors Versus In Vitro Transformed Neoplastic Breast Epithelium

    National Research Council Canada - National Science Library

    Szallasi, Zoltan

    2001-01-01

    .... The first group of clones is being sorted by their ability to form tumors. We are currently performing cDNA microarray analysis quantifying the expression level of about 15,000 genes in these cell lines...

  10. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  11. Two human cDNA molecules coding for the Duchenne muscular dystrophy (DMD) locus are highly homologous

    Energy Technology Data Exchange (ETDEWEB)

    Rosenthal, A.; Speer, A.; Billwitz, H. (Zentralinstitut fuer Molekularbiologie, Berlin-Buch (Germany Democratic Republic)); Cross, G.S.; Forrest, S.M.; Davies, K.E. (Univ. of Oxford (England))

    1989-07-11

    Recently the complete sequence of the human fetal cDNA coding for the Duchenne muscular dystrophy (DMD) locus was reported and a 3,685 amino acid long, rod-shaped cytoskeletal protein (dystrophin) was predicted as the protein product. Independently, the authors have isolated and sequenced different DMD cDNA molecules from human adult and fetal muscle. The complete 12.5 kb long sequence of all their cDNA clones has now been determined and they report here the nucleotide (nt) and amino acid (aa) differences between the sequences of both groups. The cDNA sequence comprises the whole coding region but lacks the first 110 nt from the 5{prime}-untranslated region and the last 1,417 nt of the 3{prime}-untranslated region. They have found 11 nt differences (approximately 99.9% homology) from which 7 occurred at the aa level.

  12. Identification of a cryptic prokaryotic promoter within the cDNA encoding the 5' end of dengue virus RNA genome.

    Directory of Open Access Journals (Sweden)

    Dongsheng Li

    Full Text Available Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV 5' UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

  13. Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

  14. Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

    Science.gov (United States)

    Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

    2017-10-01

    Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

  15. Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie A Wallace

    Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

  16. Matrix metalloproteinase inhibition reduces contraction by dupuytren fibroblasts.

    Science.gov (United States)

    Townley, William A; Cambrey, Alison D; Khaw, Peng T; Grobbelaar, Adriaan O

    2008-11-01

    Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

  17. Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

    Science.gov (United States)

    Nakagawa, S; Pawelek, P; Grinnell, F

    1989-06-01

    To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

  18. TFE3-positive renal cell carcinomas are not always Xp11 translocation carcinomas: Report of a case with a TPM3-ALK translocation.

    Science.gov (United States)

    Thorner, Paul Scott; Shago, Mary; Marrano, Paula; Shaikh, Furqan; Somers, Gino R

    2016-10-01

    Translocation-associated renal cell carcinoma (RCC) is a distinct subtype of RCC with gene rearrangements of the TFE3 or TFEB loci. The TFE3 gene is located at Xp11 and can fuse to a number of translocation partners, resulting in high nuclear expression of TFE3 protein. TFE3 immunostaining is often used as a surrogate marker for a TFE3 translocation. We report a case of an RCC that expressed TFE3 but showed only gain of TFE3 rather than a translocation. Moreover, this case had a t(1;2) translocation fusing ALK and TMP3, identical to that seen in inflammatory myofibroblastic tumour. There was resulting overexpression of ALK protein in a cytoplasmic and membranous pattern. The patient was not treated with chemotherapy but following regional nodal recurrence, an ALK inhibitor was added and the patient remains alive one year later. There are only rare reports of RCC with an ALK-TMP3 fusion, and these tumours can express TFE3 on some unknown basis not related to a TFE3 translocation. Any RCC positive for TFE3 and lacking a translocation should be tested for ALK expression and translocation. Recognition of this subtype of RCC will allow ALK inhibitor therapy to be added, in the hope of improving patient outcome. Copyright © 2016 Elsevier GmbH. All rights reserved.

  19. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  20. The Effects of Amphiregulin Induced MMP-13 Production in Human Osteoarthritis Synovial Fibroblast

    Directory of Open Access Journals (Sweden)

    Yi-Te Chen

    2014-01-01

    Full Text Available Osteoarthritis (OA belongs to a group of degenerative diseases. Synovial inflammation, cartilage abrasion, and subchondral sclerosis are characteristics of OA. Researchers do not fully understand the exact etiology of OA. However, matrix metalloproteinases (MMPs, which are responsible for cartilage matrix degradation, play a pivotal role in the progression of OA. Amphiregulin (AREG binds to the EGF receptor (EGFR and activates downstream proteins. AREG is involved in a variety of pathological processes, such as the development of tumors, inflammatory diseases, and rheumatoid arthritis. However, the relationship between AREG and MMP-13 in OA synovial fibroblasts (SFs remains unclear. We investigated the signaling pathway involved in AREG-induced MMP-13 production in SFs. AREG caused MMP-13 production in a concentration- and time-dependent manner. The results of using pharmacological inhibitors and EGFR siRNA to block EGFR revealed that the EGFR receptor was involved in the AREG-mediated upregulation of MMP-13. AREG-mediated MMP-13 production was attenuated by PI3K and Akt inhibitors. The stimulation of cells by using AREG activated p65 phosphorylation and p65 translocation from the cytosol to the nucleus. Our results provide evidence that AREG acts through the EGFR and activates PI3K, Akt, and finally NF-kappaB on the MMP-13 promoter, thus contributing to cartilage destruction during osteoarthritis.

  1. Alzheimer skin fibroblasts show increased susceptibility to free radicals.

    Science.gov (United States)

    Tesco, G; Latorraca, S; Piersanti, P; Piacentini, S; Amaducci, L; Sorbi, S

    1992-11-01

    We have studied the response to toxic oxygen metabolites of fibroblasts derived from skin biopsies of 5 patients with familial (FAD) and 4 with sporadic (AD) Alzheimer's disease compared with those derived from 4 normal controls. Fibroblasts were damaged by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by 50 munits of xanthine-oxidase (Xo). To quantify cell damage we measured lactate dehydrogenase (LDH) activity in the culture medium and cell viability in fibroblast cultures. We found a significant increase in LDH activity in the FAD vs. controls and also in the AD vs. controls.

  2. Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

    International Nuclear Information System (INIS)

    Chan, Waiyee; Liu, Qingrong; Borjigin, J.; Busch, H.; Rennert, O.M.; Tease, L.A.; Chan, Puikwong

    1989-01-01

    A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in δgtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1,311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. When the protein levels were compared with Western blot immunoassays, Navikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver

  3. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1987-01-01

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  4. Construction and identification of subtracted cDNA library in bone marrow cells of radon-exposed mice

    International Nuclear Information System (INIS)

    Li Jianxiang; Nie Jihua; Tong Jian; Fu Chunling; Zhou Jianwei

    2008-01-01

    Objective: To construct and identify subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation. Methods: Adult male BALB/c mice, weighing 18-22 g, were placed in a multi- functional radon chamber. One group of mice was exposed to radon up to the accumulative dose of 105 work level month (WLM). The control group of mice was housed in a room with an accumulative dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and the suppression subtractive hybridization were performed. The obtained forward and reverse cDNA fragments were directly inserted into pMD18-T vector and transformed into E. coli JM109. The inserting cDNA fragments were screened by the blue-and-white blot screening and nested PCR of bacterium liquid. Results: The 244 of 285 white bacteria clones obtained randomly were positive clones contained 100-1100 bp inserted cDNA fragments. Conclusions: The forward and reverse subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation is successfully constructed. (authors)

  5. [Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

    Science.gov (United States)

    Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

    2003-07-01

    Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

  6. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Story, M.T.

    1989-01-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

  7. Efficient induction of Wheat-agropyron cristatum 6P translocation lines and GISH detection.

    Directory of Open Access Journals (Sweden)

    Liqiang Song

    Full Text Available The narrow genetic background restricts wheat yield and quality improvement. The wild relatives of wheat are the huge gene pools for wheat improvement and can broaden its genetic basis. Production of wheat-alien translocation lines can transfer alien genes to wheat. So it is important to develop an efficient method to induce wheat-alien chromosome translocation. Agropyroncristatum (P genome carries many potential genes beneficial to disease resistance, stress tolerance and high yield. Chromosome 6P possesses the desirable genes exhibiting good agronomic traits, such as high grain number per spike, powdery mildew resistance and stress tolerance. In this study, the wheat-A. cristatum disomic addition was used as bridge material to produce wheat-A. cristatum translocation lines induced by (60Co-γirradiation. The results of genomic in situ hybridization showed that 216 plants contained alien chromosome translocation among 571 self-pollinated progenies. The frequency of translocation was 37.83%, much higher than previous reports. Moreover, various alien translocation types were identified. The analysis of M2 showed that 62.5% of intergeneric translocation lines grew normally without losing the translocated chromosomes. The paper reported a high efficient technical method for inducing alien translocation between wheat and Agropyroncristatum. Additionally, these translocation lines will be valuable for not only basic research on genetic balance, interaction and expression of different chromosome segments of wheat and alien species, but also wheat breeding programs to utilize superior agronomic traits and good compensation effect from alien chromosomes.

  8. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

    2011-08-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

  9. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

    2011-01-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

  10. The radiation response of human dermal fibroblasts

    Science.gov (United States)

    Mitchell, Stephen Andrew

    A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: puse of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration

  11. Hyaluronan synthase mediates dye translocation across liposomal membranes

    Directory of Open Access Journals (Sweden)

    Medina Andria P

    2012-01-01

    Full Text Available Abstract Background Hyaluronan (HA is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS, which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS to liposomes preloaded with the fluorophore Cascade Blue (CB. Results CB translocation (efflux was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL. An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL and that, in contrast, tetramyristoyl cardiolipin (TM-CL is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006. Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL. Conclusions The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

  12. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Science.gov (United States)

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant. PMID:25054322

  13. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Directory of Open Access Journals (Sweden)

    Hongbo Wei

    2014-07-01

    Full Text Available Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant.

  14. Relationship among expression of basic-fibroblast growth factor ...

    African Journals Online (AJOL)

    Relationship among expression of basic-fibroblast growth factor, MTDH/Astrocyte elevated gene-1, adenomatous polyposis coli, matrix metalloproteinase 9,and COX-2 markers with prognostic factors in prostate carcinomas.

  15. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  16. Factor XIIIa is expressed by fibroblasts in fibrovascular tumors.

    Science.gov (United States)

    Nemeth, A J; Penneys, N S

    1989-10-01

    Factor XIIIa (FXIIIa), a blood and intracellularly produced coagulation factor, has been found in a variety of cell types including fibroblast-like mesenchymal cells, and has been shown to stimulate the proliferation of fibroblasts and some neoplastic cells in vitro. We have already shown that the dendritic fibroblasts composing the fibrous papule contain this factor. We hypothesized that histopathologically similar fibrovascular tumors may also express FXIIIa and, in this report, show that the large stellate fibroblasts found in acquired digital fibrokeratomas, angiofibromas (adenoma sebaceum of Pringle), and oral fibroma (oral fibrous hyperplasia) also express FXIIIa. We postulate that FXIIIa, possibly acting as a growth factor, may be a common denominator in the pathogenesis of these tumors. Another possibility is that these tumors may be the consequence of a local overproduction of FXIIIa in response to an, as yet, unidentified stimulus.

  17. HGF is released from buccal fibroblasts after smokeless tobacco stimulation

    DEFF Research Database (Denmark)

    Dabelsteen, S; Christensen, S; Gron, B

    2005-01-01

    on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells......To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w....... Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions....

  18. Transient expression of acidic fibroblast growth factor in pea (Pisum ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... a 100 W, long-wave UV lamp (Black Ray model B-100 AP; Ultra- ... frequency) was used to estimate the treatment efficiency during 15 days post .... Crystal structure of fibroblast growth factor receptor ectodomain bound to.

  19. Absorption and translocation of arsenate arsenic by coffee plants

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, J F; Yamaguchi, S

    1964-01-01

    Studies on arsenate arsenic absorption and translocation in coffee plants were carried out using arsenic-77 applied either to leaves or to roots through the nutrient solution. The youngest leaves were unable to export arsenic and only an acropetal movement in the transpiration stream was observed. From the second pair of leaves, it moved into the growing leaves. From the fourth pair it moved downward through the stem and into the roots. Roots absorbed high amounts of arsenate once absorbed it moved via the xylem of the transpiration stream into the leaves. 7 references, 2 figures.

  20. Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

    Science.gov (United States)

    Sasaki, Norihiko; Itakura, Yoko; Toyoda, Masashi

    2017-04-18

    Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast

  1. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  2. A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

    Directory of Open Access Journals (Sweden)

    I. da Silva

    2002-08-01

    Full Text Available In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8 showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries. Plants submitted to stress (wounding, virus infection and ethylene treatment presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

  3. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Christine M Costello

    2005-08-01

    Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

  4. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  5. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    International Nuclear Information System (INIS)

    Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto

    2012-01-01

    Highlights: ► ABA is an endogenous hormone in humans, regulating different cell responses. ► ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. ► UV-B irradiation increases ABA content in SSc cultures. ► SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  6. Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0512 TITLE: Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer PRINCIPAL INVESTIGATOR: Andrew...SUBTITLE 5a. CONTRACT NUMBER Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer 5b. GRANT NUMBER W81XWH-15-1-0512 5c. PROGRAM...blocked by the addition of Pim inhibitors. These results suggest that the Pim protein kinase can regulate stromal cell biology to modulate epithelial

  7. CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Hieu T Nim

    Full Text Available The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP, an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1 relevant to cardiac literature, and (2 differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10 are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

  8. CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

    Science.gov (United States)

    Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

    2015-01-01

    The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

  9. Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

    Science.gov (United States)

    Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

    2017-02-16

    Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

  10. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  11. Sharpening spots: correcting for bleedover in cDNA array images.

    Science.gov (United States)

    Therneau, Terry; Tschumper, Renee C; Jelinek, Diane

    2002-03-01

    For cDNA array methods that depend on imaging of a radiolabel, we show that bleedover of one spot onto another, due to the gap between the array and the imaging media, can be a major problem. The images can be sharpened, however, using a blind convolution method based on the EM algorithm. The sharpened images look like a set of donuts, which concurs with our knowledge of the spotting process. Oversharpened images are actually useful as well, in locating the centers of each spot.

  12. c-DNA of HIV-1 detection on spot of Buffy-Coat of leukocytes (DBCS)

    OpenAIRE

    Marco Rossi de Gasperis; Maria Daniela Caione; Carlo Concato; Ersilia Fiscarelli; Nicola Di Pietro; Vittorio Salotti; Lorenza Putignani; Donato Menichella; Francesco Callea

    2010-01-01

    Introduction:The elective way for the diagnosis of HIV-1-infection in the window period and in children under the age of 16-18 months is to search virus integrated in leukocytes. Aim of the study was to assess the sensitivity and specificity of extraction from Buffy-Dried Coat Spot (DBCS) in leukocyte to detect c-DNA with nested-PCR in HIV-1-infected individuals compared to Dried Blood Spot (DBS) both extracted by automated instrument EZ1 (QIAGEN, Hilden, Germany). Both DBCS and both DBS were...

  13. Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

    Science.gov (United States)

    Pietrowski, D; Förster, M

    2000-01-01

    The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

  14. Characterization of full-length sequenced cDNA inserts (FLIcs from Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

    Full Text Available Abstract Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP, the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91% of the transcripts were annotated using Gene Ontology (GO terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS. The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS. This

  15. Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

    Science.gov (United States)

    Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

    2009-01-01

    Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA

  16. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  17. License - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project License to Use This Database Last updated : 2010/02/15 You may use this databas...ional License described below. The Standard License specifies the license terms regarding the use of this database... and the requirements you must follow in using this database. The Additiona...n the Standard License. Standard License The Standard License for this database is the license specified in ...the Creative Commons Attribution-Share Alike 2.1 Japan . If you use data from this database

  18. Horse cDNA clones encoding two MHC class I genes

    Energy Technology Data Exchange (ETDEWEB)

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  19. Fibroblast Growth Factor Signaling in Metabolic Regulation.

    Science.gov (United States)

    Nies, Vera J M; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T; Atkins, Annette R; Evans, Ronald M; Jonker, Johan W; Downes, Michael Robert

    2015-01-01

    The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.

  20. Use of chromosome translocations for measuring prior environment exposures in humans

    Energy Technology Data Exchange (ETDEWEB)

    Tucker, J. D.

    1997-05-01

    Recent advances in cytogenetic methodology are beginning to have a major impact upon our ability to provide assessments of environmental exposure in humans. The advent of fluorescent-based techniques for `painting` whole chromosomes has made the analysis of chromosome translocations rapid, specific, sensitive and routine. Chromosome painting has been used to address a wide variety of scientific questions, resulting in an increased understanding of the biological consequences of adverse environmental exposure. This paper describes the use of chromosome translocations as a biological marker of exposure and effect in humans. The relevance of translocations is discussed, as are the advantages and disadvantages of painting compared to classical cytogenetic methods for translocation evaluation. The factors to consider in the use of translocations as a retrospective indicator of exposure are then described. Several theoretical parameters that are important to the use of translocations are provided, and the paper concludes with a vision for the future of cytogenetic methodology.

  1. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    International Nuclear Information System (INIS)

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin

    2006-01-01

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions

  2. Polymer translocation under a pulling force: Scaling arguments and threshold forces

    Science.gov (United States)

    Menais, Timothée

    2018-02-01

    DNA translocation through nanopores is one of the most promising strategies for next-generation sequencing technologies. Most experimental and numerical works have focused on polymer translocation biased by electrophoresis, where a pulling force acts on the polymer within the nanopore. An alternative strategy, however, is emerging, which uses optical or magnetic tweezers. In this case, the pulling force is exerted directly at one end of the polymer, which strongly modifies the translocation process. In this paper, we report numerical simulations of both linear and structured (mimicking DNA) polymer models, simple enough to allow for a statistical treatment of the pore structure effects on the translocation time probability distributions. Based on extremely extended computer simulation data, we (i) propose scaling arguments for an extension of the predicted translocation times τ ˜N2F-1 over the moderate forces range and (ii) analyze the effect of pore size and polymer structuration on translocation times τ .

  3. Senescent phenotypes of skin fibroblasts from patients with Tangier disease

    International Nuclear Information System (INIS)

    Matsuura, Fumihiko; Hirano, Ken-ichi; Ikegami, Chiaki; Sandoval, Jose C.; Oku, Hiroyuki; Yuasa-Kawase, Miyako; Tsubakio-Yamamoto, Kazumi; Koseki, Masahiro; Masuda, Daisaku; Tsujii, Ken-ichi; Ishigami, Masato; Nishida, Makoto; Shimomura, Iichiro; Hori, Masatsugu; Yamashita, Shizuya

    2007-01-01

    Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) ∼10 compared with control cells. TD cells practically ceased proliferation at PDL ∼30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated β-galactosidase (SA-β-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients

  4. [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

    Science.gov (United States)

    Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

    2017-01-01

    Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

  5. Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

    Science.gov (United States)

    Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

    2018-04-01

    Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Balanced translocation linked to psychiatric disorder, glutamate, and cortical structure/function

    OpenAIRE

    Thomson, Pippa A; Duff, Barbara; Blackwood, Douglas H R; Romaniuk, Liana; Watson, Andrew; Whalley, Heather C; Li, Xiang; Dauvermann, Maria R; Moorhead, T William J; Bois, Catherine; Ryan, Niamh M; Redpath, Holly; Hall, Lynsey; Morris, Stewart W; van Beek, Edwin J R

    2016-01-01

    Rare genetic variants of large effect can help elucidate the pathophysiology of brain disorders. Here we expand the clinical and genetic analyses of a family with a (1;11)(q42;q14.3) translocation multiply affected by major psychiatric illness and test the effect of the translocation on the structure and function of prefrontal, and temporal brain regions. The translocation showed significant linkage (LOD score 6.1) with a clinical phenotype that included schizophrenia, schizoaffective disorde...

  7. Survival of translocated sharp-tailed grouse: Temporal threshold and age effects

    Science.gov (United States)

    Mathews, Steven; Coates, Peter S.; Delehanty, David J.

    2016-01-01

    Context: The Columbian sharp-tailed grouse (Tympanuchus phasianellus columbianus) is a subspecies of conservation concern in the western United States, currently occupying ≤10% of its historic range. Land and management agencies are employing translocation techniques to restore Columbian sharp-tailed grouse (CSTG) populations. However, establishing self-sustaining populations by translocating grouse often is unsuccessful, owing, in part, to low survivorship of translocated grouse following release.Aims: We measured and modelled patterns of CSTG mortality for 150 days following translocation into historic range, to better understand patterns and causes of success or failure in conservation efforts to re-establish grouse populations.Methods: We conducted two independent multi-year translocations and evaluated individual and temporal factors associated with CSTG survival up to 150 days following their release. Both translocations were reintroduction attempts in Nevada, USA, to establish viable populations of CSTG into their historic range.Key results: We observed a clear temporal threshold in survival probability, with CSTG mortality substantially higher during the first 50 days following release than during the subsequent 100 days. Additionally, translocated yearling grouse exhibited higher overall survival (0.669 ± 0.062) than did adults (0.420 ± 0.052) across the 150-day period and higher survival than adults both before and after the 50-day temporal threshold.Conclusions: Translocated CSTG are especially vulnerable to mortality for 50 days following release, whereas translocated yearling grouse are more resistant to mortality than are adult grouse. On the basis of the likelihood of survival, yearling CSTG are better candidates for population restoration through translocation than are adult grouse.Implications: Management actions that ameliorate mortality factors for 50 days following translocation and translocations that employ yearling grouse will

  8. Meiotic behaviour and spermatogenesis in male mice heterozygous for translocation types also occurring in man

    International Nuclear Information System (INIS)

    Nijhoff, J.H.

    1981-01-01

    In this thesis a start was made with meiotic observations of mouse translocation types - a Robertsonian translocation and a translocation between a metacentric and an acrocentric chromosome - which also occur in man. As an exogeneous factor of possible influence, the meiotic effects of two types of radiation (fission neutrons and X-rays) administered at relatively low doses 2 and 3 hours before prometaphase-metaphase II (probably during metaphase-anaphase I), were determined in Rb4Bnr/+-males. (Auth.)

  9. Occurence of translocations between irradiated and intact chromosomes of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Myasnyankina, E.N.; Abeleva, Eh.A.; Generalova, M.V.

    1980-01-01

    Two translocations between irradiated father and intact mother autosomes are obtained in Drosophila melanogaster. Five out of 283 regular translocations (between the second and the third chromosomes of an irradiated male) are accompanied by a recombination over the second or the third chromosomes. Nine flies out of twenty considered to be recombinants, could originate due to mutations. The data obtained prove that intact female autosomes can take part in the exchange with homologic (recombinations) and heterologic (translocations) irradiated male autosomes

  10. Translocation of photoassimilates in γ-irradiated soybean plants

    International Nuclear Information System (INIS)

    Ursino, D.J.; Schefski, H.; Latour, P.W.

    1977-01-01

    Young soybean plants received on a single occasion a dose of 3.75 krads of γ-radiation after which they were allowed to photoassimilate 14 CO 2 so that the magnitude of translocation and distribution pattern of exported 14 C could be determined. In comparison to the non-irradiated controls, plants exposed to ionizing radiation showed a large reduction, almost 70%, in the magnitude of 14 C exported from the source leaf in a one hour period. Furthermore, the pattern of distribution of exported 14 C was altered such that less was exported to the shoot apex region and more to the region below the node of the source leaf. Replacement of the shoot apex with 20 ppm IAA immediately following irradiation completely restored the pattern of distribution but was able to only partially increase the magnitude of translocation. These results are interpreted with reference to the results from the author's previous study with soybean showing radiation effects on photosynthesis and shoot development. (author)

  11. Myeloid translocation genes differentially regulate colorectal cancer programs

    Science.gov (United States)

    Parang, Bobak; Bradley, Amber M.; Mittal, Mukul K.; Short, Sarah P.; Thompson, Joshua J.; Barrett, Caitlyn W.; Naik, Rishi D.; Bilotta, Anthony J.; Washington, Mary K.; Revetta, Frank L.; Smith, Jesse J.; Chen, Xi; Wilson, Keith T.; Hiebert, Scott W.; Williams, Christopher S.

    2016-01-01

    Myeloid translocation genes (MTGs), originally identified as chromosomal translocations in acute myelogenous leukemia, are transcriptional corepressors that regulate hematopoietic stem cell programs. Analysis of The Cancer Genome Atlas (TCGA) database revealed that MTGs were mutated in epithelial malignancy and suggested that loss of function might promote tumorigenesis. Genetic deletion of MTGR1 and MTG16 in the mouse has revealed unexpected and unique roles within the intestinal epithelium. Mtgr1−/− mice have progressive depletion of all intestinal secretory cells, and Mtg16−/− mice have a decrease in goblet cells. Furthermore, both Mtgr1−/− and Mtg16−/− mice have increased intestinal epithelial cell proliferation. We thus hypothesized that loss of MTGR1 or MTG16 would modify Apc1638/+-dependent intestinal tumorigenesis. Mtgr1−/− mice, but not Mtg16−/− mice, had a 10-fold increase in tumor multiplicity. This was associated with more advanced dysplasia, including progression to invasive adenocarcinoma, and augmented intratumoral proliferation. Analysis of ChIP-seq datasets for MTGR1 and MTG16 targets indicated that MTGR1 can regulate Wnt and Notch signaling. In support of this, immunohistochemistry and gene expression analysis revealed that both Wnt and Notch signaling pathways were hyperactive in Mtgr1−/− tumors. Furthermore, in human colorectal cancer (CRC) samples MTGR1 was downregulated at both the transcript and protein level. Overall our data indicates that MTGR1 has a context dependent effect on intestinal tumorigenesis. PMID:27270437

  12. [Bacterial Translocation from Intestine: Microbiological, Immunological and Pathophysiological Aspects].

    Science.gov (United States)

    Podoprigora, G I; Kafarskaya, L I; Bainov, N A; Shkoporov, A N

    2015-01-01

    Bacterial translocation (BT) is both pathology and physiology phenomenon. In healthy newborns it accompanies the process of establishing the autochthonous intestinal microbiota and the host microbiome. In immunodeficiency it can be an aethio-pathogenetic link and a manifestation of infection or septic complications. The host colonization resistance to exogenous microbic colonizers is provided by gastrointestinal microbiota in concert with complex constitutional and adaptive defense mechanisms. BT may be result of barrier dysfunction and self-purification mechanisms involving the host myeloid cell phagocytic system and opsonins. Dynamic cell humoral response to microbial molecular patterns that occurs on the mucous membranes initiates receptorsignalingpathways and cascade ofreactions. Their vector and results are largely determined by cross-reactivity between microbiome and the host genome. Enterocyte barriers interacting with microbiota play leading role in providing adaptive, homeostatic and stress host reactivity. Microcirculatory ischemic tissue alterations and inflammatory reactions increase the intestinal barrier permeability and BT These processes a well as mechanisms for apoptotic cells and bacteria clearance are justified to be of prospective research interest. The inflammatory and related diseases caused by alteration and dysfunction of the intestinal barrier are reasonably considered as diseases of single origin. Maternal microbiota affects theformation of the innate immune system and the microbiota of the newborn, including intestinal commensal translocation during lactation. Deeper understanding of intestinal barrier mechanisms needs complex microbiological, immunological, pathophysiological, etc. investigations using adequate biomodels, including gnotobiotic animals.

  13. Translocation of the neonicotinoid seed treatment clothianidin in maize.

    Science.gov (United States)

    Alford, Adam; Krupke, Christian H

    2017-01-01

    Neonicotinoid seed treatments, typically clothianidin or thiamethoxam, are routinely applied to >80% of maize (corn) seed grown in North America where they are marketed as a targeted pesticide delivery system. Despite this widespread use, the amount of compound translocated into plant tissue from the initial seed treatment to provide protection has not been reported. Our two year field study compared concentrations of clothianidin seed treatments in maize to that of maize without neonicotinoid seed treatments and found neonicotinoids present in root tissues up to 34 days post planting. Plant-bound clothianidin concentrations followed an exponential decay pattern with initially high values followed by a rapid decrease within the first ~20 days post planting. A maximum of 1.34% of the initial seed treatment was successfully recovered from plant tissues in both study years and a maximum of 0.26% was recovered from root tissue. Our findings show neonicotinoid seed treatments may provide protection from some early season secondary maize pests. However, the proportion of the neonicotinoid seed treatment clothianidin translocated into plant tissues throughout the growing season is low overall and this observation may provide a mechanism to explain reports of inconsistent efficacy of this pest management approach and increasing detections of environmental neonicotinoids.

  14. Translocation of the neonicotinoid seed treatment clothianidin in maize.

    Directory of Open Access Journals (Sweden)

    Adam Alford

    Full Text Available Neonicotinoid seed treatments, typically clothianidin or thiamethoxam, are routinely applied to >80% of maize (corn seed grown in North America where they are marketed as a targeted pesticide delivery system. Despite this widespread use, the amount of compound translocated into plant tissue from the initial seed treatment to provide protection has not been reported. Our two year field study compared concentrations of clothianidin seed treatments in maize to that of maize without neonicotinoid seed treatments and found neonicotinoids present in root tissues up to 34 days post planting. Plant-bound clothianidin concentrations followed an exponential decay pattern with initially high values followed by a rapid decrease within the first ~20 days post planting. A maximum of 1.34% of the initial seed treatment was successfully recovered from plant tissues in both study years and a maximum of 0.26% was recovered from root tissue. Our findings show neonicotinoid seed treatments may provide protection from some early season secondary maize pests. However, the proportion of the neonicotinoid seed treatment clothianidin translocated into plant tissues throughout the growing season is low overall and this observation may provide a mechanism to explain reports of inconsistent efficacy of this pest management approach and increasing detections of environmental neonicotinoids.

  15. Novel Class of Potential Therapeutics that Target Ricin Retrograde Translocation

    Directory of Open Access Journals (Sweden)

    Veronika Redmann

    2013-12-01

    Full Text Available Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTAE177Qegfp to identify compounds that target RTA retrograde translocation. Stabilizing RTAE177Qegfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.

  16. Molecular determinants of nucleolar translocation of RNA helicase A

    International Nuclear Information System (INIS)

    Liu Zhe; Kenworthy, Rachael; Green, Christopher; Tang, Hengli

    2007-01-01

    RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role

  17. Adenine nucleotide translocator transports haem precursors into mitochondria.

    Directory of Open Access Journals (Sweden)

    Motoki Azuma

    2008-08-01

    Full Text Available Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT, the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.

  18. Zinc translocation accelerates infarction after mild transient focal ischemia.

    Science.gov (United States)

    Lee, J-M; Zipfel, G J; Park, K H; He, Y Y; Hsu, C Y; Choi, D W

    2002-01-01

    Excess release of chelatable zinc (Zn(2+)) from central synaptic vesicles may contribute to the pathogenesis of selective neuronal cell death following transient forebrain ischemia, but a role in neurodegeneration after focal ischemia has not been defined. Adult male Long-Evans rats subjected to middle cerebral artery occlusion (MCAO) for 30 min followed by reperfusion developed delayed cerebral infarction reaching completion 3 days after the insult. One day after the insult, many degenerating cerebral neurons exhibited increased intracellular Zn(2+), and some labeled with the antibody against activated caspase-3. I.c.v. administration of the Zn(2+) chelator, EDTA saturated with equimolar Ca(2+) (CaEDTA), 15 min prior to ischemia attenuated subsequent Zn(2+) translocation into cortical neurons, and reduced infarct volume measured 3 days after ischemia. Although the protective effect of CaEDTA at this endpoint was substantial (about 70% infarct reduction), it was lost when insult severity was increased (from 30 to 60 min MCAO), or when infarct volume was measured at a much later time point (14 days instead of 3 days after ischemia). These data suggest that toxic Zn(2+) translocation, from presynaptic terminals to post-synaptic cell bodies, may accelerate the development of cerebral infarction following mild transient focal ischemia.

  19. Translocation of cesium in plants after foliar deposition - Experiments and models

    International Nuclear Information System (INIS)

    Proehl, G.; Voigt, G.; Mueller, H.

    1991-01-01

    The translocation of cesium from the foliage to the edible parts as function of the time period between deposition and harvest has been determined for cereals, potatoes, green beans and carrots. From the results the following conclusions can be drawn: 1. The maximum of the cesium translocation is 40 to 50 and 70 to 90 days before harvest for cereals and potatoes respectively. For green beans a maximum was observed after deposition 15 days before harvest; 2. The variations of the translocation factors are less if the translocation is normalized to the yield; 3. The translocation factors are in good agreement with those of other investigators. The agreement between the experimental series is better for a normalization of the translocation factor on the yield; 4. For cereals and potatoes the translocation can be described with gaussian functions which are consistent with the physiological development of cereals and potatoes. Although the approach in ECOSYS tends to over predict slightly the translocation for barley and potatoes there is a good overall agreement between the experiments and this model; 5. According to the investigations available the translocation of cesium can be predicted within a factor of 3 for cereals and a factor of 4 for potatoes. Sources of the uncertainties besides the biological variability and the inherent experimental error are differences in the development of the plants due to weather conditions, farm management and plant diseases. (9 refs., 5 figs.)

  20. Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

    Science.gov (United States)

    Lakkaraju, Asvin K. K.; Thankappan, Ratheeshkumar; Mary, Camille; Garrison, Jennifer L.; Taunton, Jack; Strub, Katharina

    2012-01-01

    Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120–160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway. PMID:22648169

  1. Use of wild–caught individuals as a key factor for success in vertebrate translocations

    Energy Technology Data Exchange (ETDEWEB)

    Rummel, L.; MartInez-AbraIn, A.; Mayol, J.; Ruiz-Olmo, J.; Mañas, F.; Jimenez, J.; Gomez, J.A.; Oro, D.

    2016-07-01

    Success of vertebrate translocations is crucial to improve efficacy and efficiency of conservation actions but it is often difficult to assess because negative results (failed translocations) are seldom published. We developed surveys and sent them to heads of conservation services in three major Spanish Mediterranean regions. The purpose of our surveys was to determine which methodological factor, that could easily be implemented in practice, was more influential for translocation success. These factors included the origin of translocated individuals (captive or wild) and translocation effort (propagule size and program duration). After analyzing 83 programs, corresponding to 34 different vertebrate species, by means of generalized linear mixed modelling, we found that ‘origin’ was more relevant for translocation success than ‘effort’, although we could not rule out some role of translocation effort. Variance in success of translocation programs involving individuals from wild sources was smaller and consequently results more predictable. Origin interacted with taxa so that success was higher when using wild birds and especially wild fish and mammals, but not when releasing reptiles. Hence, we suggest that, for any given effort, translocation results will be better for most vertebrate taxa if individuals from wild sources are used. When this is not feasible, managers should release captive–reared individuals for a long number of years rather than a short number of years. (Author)

  2. Can Characteristics of Reciprocal Translocations Predict the Chance of Transferable Embryos in PGD Cycles?

    Directory of Open Access Journals (Sweden)

    Elsbeth Dul

    2014-04-01

    Full Text Available Translocation carriers have an increased risk of miscarriage or the birth of a child with congenital anomalies. Preimplantation genetic diagnosis (PGD is performed in translocation carriers to select for balanced embryos and, thus, increase the chance of an ongoing pregnancy. However, a common experience is that reciprocal translocation carriers produce a high percentage of unbalanced embryos, which cannot be transferred. Therefore, the pregnancy rates in PGD in this patient group are low. In a cohort of 85 reciprocal translocation carriers undergoing PGD we have searched for cytogenetic characteristics of the translocations that can predict the percentage of balanced embryos. Using shape algorithms, the most likely segregation mode per translocation was determined. Shape algorithm, breakpoint location, and relative chromosome segment sizes proved not to be independent predictors of the percentage of balanced embryos. The ratio of the relative sizes of the translocated segments of both translocation chromosomes can give some insight into the chance of transferable embryos: Very asymmetrical translocations have a higher risk of unbalanced products (p = 0.048. Counseling of the couples on the pros and cons of all their reproductive options remains very important.

  3. Use of wild–caught individuals as a key factor for success in vertebrate translocations

    Directory of Open Access Journals (Sweden)

    Rummel, L.

    2016-06-01

    Full Text Available Success of vertebrate translocations is crucial to improve efficacy and efficiency of conservation actions but it is often difficult to assess because negative results (failed translocations are seldom published. We developed surveys and sent them to heads of conservation services in three major Spanish Mediterranean regions. The purpose of our surveys was to determine which methodological factor, that could easily be implemented in practice, was more influential for translocation success. These factors included the origin of translocated individuals (captive or wild and translocation effort (propagule size and program duration. After analyzing 83 programs, corresponding to 34 different vertebrate species, by means of generalized linear mixed modelling, we found that ‘origin’ was more relevant for translocation success than ‘effort’, although we could not rule out some role of translocation effort. Variance in success of translocation programs involving individuals from wild sources was smaller and consequently results more predictable. Origin interacted with taxa so that success was higher when using wild birds and especially wild fish and mammals, but not when releasing reptiles. Hence, we suggest that, for any given effort, translocation results will be better for most vertebrate taxa if individuals from wild sources are used. When this is not feasible, managers should release captive–reared individuals for a long number of years rather than a short number of years.

  4. Translocation of reindeer from South Georgia to the Falkland Islands

    Directory of Open Access Journals (Sweden)

    Cameron M. Bell

    2010-03-01

    Full Text Available This report describes the first translocation of reindeer Rangifer tarandus from South Georgia to the Falkland Islands, in the South Atlantic Ocean. Reindeer were introduced from Norway to the subantarctic island of South Georgia on three occasions in the early 1900s by Norwegian whalers, and today they exist as two discrete herds, numbering approximately 2600 individuals in total. Because of concerns over the impact on native vegetation, the long-term eradication of reindeer from South Georgia has recently been proposed. A translocation of reindeer to the Falkland Islands was undertaken in 2001 by the Falkland Island Government with two objectives: (1 to preserve the genetic resources of at least one of the South Georgia herds; and (2 to facilitate the diversification of the agricultural sector of the Falkland Islands by establishing a commercial reindeer herd. Techniques developed and used in North America for the successful relocation of large numbers of calves were adopted for the translocation. A total of 59 calves (26 females and 33 males were successfully translocated from South Georgia to the Falklands Islands in 2001, and subsequently produced their first offspring in 2003. Good husbandry practices and an understanding of biology and behaviour are essential for the successful translocation of reindeer.Flytting av rein fra Sør-Georgia til FalklandsøyeneAbstract in Norwegian / Sammendrag: Artikkelen beskriver den første overføring av rein Rangifer tarandus fra Sør-Georgia til Falklandsøyene i søratlanteren. Tamrein fra Norge ble flyttet til den subarktiske øya Sør-Georgia ved tre anledninger i perioden 1911 til 1925 i forbindelse med den norske hvalfangsten der. I dag består bestanden av rundt regnet 2600 dyr fordelt på to geografisk atskilte flokker. Av hensyn til den naturlige vegetasjonen på øya er det forslag om å på sikt utrydde reinbestanden på øya. Regjeringen på Falklandsøyene foretok en første overføring av

  5. Fibroblast growth factor 21 attenuates hepatic fibrogenesis through TGF-β/smad2/3 and NF-κB signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Pengfei; Zhang, Yingjie; Liu, Yunye; Yuan, Qingyan; Song, Liying; Liu, Mingyao; Liu, Zhihang; Yang, Yongbi; Li, Junyan; Li, Deshan, E-mail: deshanli@163.com; Ren, Guiping, E-mail: renguiping@126.com

    2016-01-01

    Fibroblast growth factor 21 (FGF-21) is a secreted protein, which has anti-diabetic and lipocaic effects, but its ability to protect against hepatic fibrosis has not been studied. In this study, we investigated the ability of FGF-21 to attenuate dimethylnitrosamine (DMN)-induced hepatic fibrogenesis in mice and the mechanism of its action. Hepatic fibrosis was induced by injection of DMN, FGF-21 was administered to the mice once daily in association with DMN injection till the end of the experiment. Histopathological examination, tissue 4-hydroxyproline content and expressions of smooth muscle α-actin (α-SMA) and collagen I were measured to assess hepatic fibrosis. Ethanol/PDGF-BB-activated hepatic stellate cells (HSCs) were used to understand the mechanisms of FGF-21 inhibited hepatic fibrogenesis. Results showed that FGF-21 treatment attenuated hepatic fibrogenesis and was associated with a significant decrease in intrahepatic fibrogenesis, 4-hydroxyproline accumulation, α-SMA expression and collagen I deposition. FGF-21 treatment inhibited the activation of HSCs via down-regulating the expression of TGF-β, NF-κB nuclear translocation, phosphorylation levels of smad2/3 and IκBα. Besides, FGF-21 treatment caused activated HSC apoptosis with increasing expression of Caspase-3, and decreased the ratio of Bcl-2 to Bax. In conclusion, FGF-21 attenuates hepatic fibrogenesis and inhibits the activation of HSC warranting the use of FGF-21 as a potential therapeutic agent in the treatment of hepatic fibrosis. - Highlights: • Fibroblast growth factor 21 attenuates hepatic fibrogenesis. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via TGF-β/smad2/3 signaling pathways. • Fibroblast growth factor 21 attenuates hepatic fibrogenesis via NF-κB signaling pathways.

  6. Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

    Science.gov (United States)

    Chen, Jung-Tsu; Wang, Chen-Ying; Chen, Min-Huey

    2018-01-13

    Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. Copyright © 2018. Published by Elsevier B.V.

  7. Anti-Inflammatory Effect of ETAS®50 by Inhibiting Nuclear Factor-κB p65 Nuclear Import in Ultraviolet-B-Irradiated Normal Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Ken Shirato

    2018-01-01

    Full Text Available Ultraviolet (UV irradiation induces proinflammatory responses in skin cells, including dermal fibroblasts, accelerating premature skin aging (photoaging. ETAS 50, a standardized extract from the Asparagus officinalis stem, is a novel and unique functional food that suppresses proinflammatory responses of hydrogen peroxide-stimulated skin fibroblasts and interleukin- (IL- 1β-stimulated hepatocytes. To elucidate its antiphotoaging potencies, we examined whether ETAS 50 treatment after UV-B irradiation attenuates proinflammatory responses of normal human dermal fibroblasts (NHDFs. UV-B-irradiated NHDFs showed reduced levels of the cytosolic inhibitor of nuclear factor-κB α (IκBα protein and increased levels of nuclear p65 protein. The nuclear factor-κB nuclear translocation inhibitor JSH-23 abolished UV-B irradiation-induced IL-1β mRNA expression, indicating that p65 regulates transcriptional induction. ETAS 50 also markedly suppressed UV-B irradiation-induced increases in IL-1β mRNA levels. Immunofluorescence analysis revealed that ETAS 50 retained p65 in the cytosol after UV-B irradiation. Western blotting also showed that ETAS 50 suppressed the UV-B irradiation-induced increases in nuclear p65 protein. Moreover, ETAS 50 clearly suppressed UV-B irradiation-induced distribution of importin-α protein levels in the nucleus without recovering cytosolic IκBα protein levels. These results suggest that ETAS 50 exerts anti-inflammatory effects on UV-B-irradiated NHDFs by suppressing the nuclear import machinery of p65. Therefore, ETAS 50 may prevent photoaging by suppressing UV irradiation-induced proinflammatory responses of dermal fibroblasts.

  8. Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

    International Nuclear Information System (INIS)

    Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

    2003-01-01

    Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

  9. Monoterpene engineering in a woody plant Eucalyptus camaldulensis using a limonene synthase cDNA.

    Science.gov (United States)

    Ohara, Kazuaki; Matsunaga, Etsuko; Nanto, Kazuya; Yamamoto, Kyoko; Sasaki, Kanako; Ebinuma, Hiroyasu; Yazaki, Kazufumi

    2010-01-01

    Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and alpha-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.

  10. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  11. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  12. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    Science.gov (United States)

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. cDNA sequences of two inducible T-cell genes

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

    1989-03-01

    The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

  14. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

    International Nuclear Information System (INIS)

    Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K.

    2002-01-01

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  15. Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

    Science.gov (United States)

    Ginsberg, Stephen D; Che, Shaoli

    2004-08-01

    The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

  16. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  17. Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions.

    Directory of Open Access Journals (Sweden)

    Valentina Bizzarro

    Full Text Available Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG or high glucose (HG we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

  18. Translocator protein as an imaging marker of macrophage and stromal activation in RA pannus.

    Science.gov (United States)

    Narayan, Nehal; Owen, David; Mandhair, Harpreet; Smyth, Erica; Carlucci, Francesco; Saleem, Azeem; Gunn, Roger; Rabiner, Eugenii Ilan A; Wells, Lisa; Dakin, Stephanie; Sabokbar, Afsie; Taylor, Peter

    2018-01-04

    Positron Emission Tomography (PET) radioligands targeted to Translocator protein (TSPO), offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus; monocytes, macrophages, fibroblast-like synoviocytes (FLS) and CD4+ T lymphocytes, to more accurately interpret TSPO PET signal from RA synovium. Methods: 3 RA patients and 3 healthy volunteers underwent PET both knees using the TSPO radioligand 11 C-PBR28. Through synovial tissue 3H-PBR28 autoradiography and immunostaining of 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO mRNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS and CD4+ T-lymphocytes. Results: 11 C-PBR28 PET signal was significantly higher in RA compared to healthy joints (average SUV 0.82± 0.12 compared to 0.03± 0.004 respectively, p<0.01). Further, 3H-PBR28 specific binding in synovial tissue was approximately 10-fold higher in RA compared to healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS and CD4+ T cells. In vitro study demonstrated highest TSPO mRNA expression and 3H-PBR28 specific binding, in activated FLS, non-activated and activated 'M2' reparative macrophages, with least TSPO expression in activated and non-activated CD4+ T lymphocytes. Conclusion: This study is the first evaluation of cellular TSPO expression in synovium, finding highest TSPO expression and PBR28 binding on activated synovial FLS and M2 phenotype macrophages. TSPO targeted PET may therefore have unique sensitivity to detect FLS and macrophage

  19. An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

    Science.gov (United States)

    Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2009-01-01

    The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

  20. PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus.

    Science.gov (United States)

    Lan, Jiang-Feng; Zhao, Li-Juan; Wei, Shun; Wang, Yuan; Lin, Li; Li, Xin-Cang

    2016-11-01

    Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine

  1. Fibroblast growth factor signaling in metabolic regulation

    Directory of Open Access Journals (Sweden)

    Vera eNies

    2016-01-01

    Full Text Available The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases, and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed.In this review we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease, and to provide starting points for the development of FGF-based therapies against metabolic conditions.

  2. Physiological Actions of Fibroblast Growth Factor-23

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    Reinhold G. Erben

    2018-05-01

    Full Text Available Fibroblast growth factor-23 (FGF23 is a bone-derived hormone suppressing phosphate reabsorption and vitamin D hormone synthesis in the kidney. At physiological concentrations of the hormone, the endocrine actions of FGF23 in the kidney are αKlotho-dependent, because high-affinity binding of FGF23 to FGF receptors requires the presence of the co-receptor αKlotho on target cells. It is well established that excessive concentrations of intact FGF23 in the blood lead to phosphate wasting in patients with normal kidney function. Based on the importance of diseases associated with gain of FGF23 function such as phosphate-wasting diseases and chronic kidney disease, a large body of literature has focused on the pathophysiological consequences of FGF23 excess. Less emphasis has been put on the role of FGF23 in normal physiology. Nevertheless, during recent years, lessons we have learned from loss-of-function models have shown that besides the paramount physiological roles of FGF23 in the control of 1α-hydroxylase expression and of apical membrane expression of sodium-phosphate co-transporters in proximal renal tubules, FGF23 also is an important stimulator of calcium and sodium reabsorption in distal renal tubules. In addition, there is an emerging role of FGF23 as an auto-/paracrine regulator of alkaline phosphatase expression and mineralization in bone. In contrast to the renal actions of FGF23, the FGF23-mediated suppression of alkaline phosphatase in bone is αKlotho-independent. Moreover, FGF23 may be a physiological suppressor of differentiation of hematopoietic stem cells into the erythroid lineage in the bone microenvironment. At present, there is little evidence for a physiological role of FGF23 in organs other than kidney and bone. The purpose of this mini-review is to highlight the current knowledge about the complex physiological functions of FGF23.

  3. Fatty acid effects on fibroblast cholesterol synthesis

    International Nuclear Information System (INIS)

    Shireman, R.B.; Muth, J.; Lopez, C.

    1987-01-01

    Two cell lines of normal (CRL 1475, GM5565) and of familial hypercholesterolemia (FH) (CM 486,488) fibroblasts were preincubated with medium containing the growth factor ITS, 2.5 mg/ml fatty acid-free BSA, or 35.2 μmol/ml of these fatty acids complexed with 2.5 mg BSA/ml: stearic (18:0), caprylic (8:0), oleic (18:1;9), linoleic (18:2;9,12), linolenic (18:3;9,12,15), docosahexaenoic (22:6;4,7,10,13,16,19)(DHA) or eicosapentaenoic (20:5;5,8,11,14,17)(EPA). After 20 h, cells were incubated for 2 h with 0.2 μCi [ 14 C]acetate/ml. Cells were hydrolyzed; an aliquot was quantitated for radioactivity and protein. After saponification and extraction with hexane, radioactivity in the aqueous and organic phases was determined. The FH cells always incorporated 30-90% more acetate/mg protein than normal cells but the pattern of the fatty acid effects was similar in both types. When the values were normalized to 1 for the BSA-only group, cells with ITS had the greatest [ 14 C]acetate incorporation (1.45) followed by the caprylic group (1.14). Cells incubated with 18:3, 20:6 or 22:6 incorporated about the same amount as BSA-only. Those preincubated with 18:2, 18:1, 18:0 showed the least acetate incorporation (0.87, 0.59 and 0.52, respectively). The percentage of total 14 C counts which extracted into hexane was much greater in FH cells; however, these values varied with the fatty acid, e.g., 1.31(18:0) and 0.84(8:0) relative to 1

  4. Fibroblasts and myofibroblasts in wound healing

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    Darby IA

    2014-11-01

    Full Text Available Ian A Darby,1 Betty Laverdet,2 Frédéric Bonté3, Alexis Desmoulière2 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia; 2Department of Physiology and EA 6309, FR 3503, Faculties of Medicine and Pharmacy, University of Limoges, Limoges, France; 3LVMH Recherche, Saint Jean de Braye, France Abstract: (Myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myofibroblasts are embedded in a sophisticated extracellular matrix (ECM that they secrete, and a complex and interactive dialogue exists between (myofibroblasts and their microenvironment. In addition to the secretion of the ECM, (myofibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myofibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myofibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis, or during aging, this dialogue between the (myofibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myofibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment. Keywords: myofibroblast, fibroblast, α-smooth muscle actin

  5. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  6. [The influence of combinations of alien translocations on in vitro androgenesis in near-isogenic lines of spring bread wheat].

    Science.gov (United States)

    Sibikeeva, Yu E; Sibikeev, S N

    2014-07-01

    The features of in vitro androgenesis were studied in Cultured anthers of spring bread wheats L503 and Dobrynya, having 7DS-7DL-7Ae#1 L translocation with genes Lrl9/Sr25 (Lrl9 translocation) from Agropyron elongatum (Host.) P.B. and their near-isogenic lines carrying combinations of Lrl9 translocation with translocations: 1BL-IR#1S with genes Pm8/Sr31/Lr26/Yr9 (Lr26translocation) from Secale cereal L., 4BS-4BL-2R#1L with genes Lr25/Pm7 (Lr25 translocation) from Secale cereal, 3DS-3DL-3Ae#1L with genes Lr24/Sr24 (Lr24 translocation) from Agropyron elongatum and 6BS-6BL-6U#1L with gene Lr9 (Lr9 translocation) from Aegilops umbellulata Zhuk. In comparison with those varieties having received the Lrl9 translocation, the following was established: (1) the combination of translocations Lr19+26 increased embryo frequency and green plant regeneration; (2) the combination of translocations Lr19+9 decreased embryo frequency but increased green plant regeneration; (3) the combination of translocations Lr19+24 decreased embryo frequency but increased green and albino plant regeneration; (4) the combination of translocations Lr19+25 increased embryo frequency and green plant regeneration but decreased albino plant regeneration. Thus, on near-isogenic lines of spring bread wheat, the influences of genotypes of four alien translocation combinations on in vitro androgenesis were determined.

  7. Construction of cDNA libraries from Pseudocercospora fijiensis Morelet infected leaves of the cultivars Calcutta 4 and Niyarma Yik

    Directory of Open Access Journals (Sweden)

    Milady Mendoza-Rodríguez

    2004-01-01

    Full Text Available Molecular studies of plant-pathogen interaction are very important for the identification of gene (s related with the pathogenic process, as well as with the plant resistance. These gene (s could be use for the genetic improvement programs in order to obtain resistant cultivars. The aim of this work was to construct complementary DNA (cDNA libraries from infected leaves with Pseudocercospora fijiensis CCIBP-Pf1 isolated of two banana cultivars (a resistant one Calcutta4 and another one susceptible Niyarma Yik. First-strand cDNA synthesis, was made beginning with one microgram of total RNA by using oligo dT primer and cDNA quality was checked by Polimerase chain reaction (PCR with cytochrome b specific primers. Second-strand cDNA synthesis was performed by using the homopolymeric tailing with dC-BamH I + dT-Not I primer combination. Four cDNA libraries of infected plants at different times of infection with the pathogen were obtained. Forty one clones of one of the libraries of Niyarma Yik were sequenced and the obtained sequences correspond with genes related to fungi. Key words: Banana-Mycosphaerella fijiensis interaction,Black Sigatoka, Musa spp.

  8. Cloning and characterization of transferrin cDNA and rapid detection of transferrin gene polymorphism in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Tange, N; Jong-Young, L; Mikawa, N; Hirono, I; Aoki, T

    1997-12-01

    A cDNA clone of rainbow trout (Oncorhynchus mykiss) transferrin was obtained from a liver cDNA library. The 2537-bp cDNA sequence contained an open reading frame encoding 691 amino acids and the 5' and 3' noncoding regions. The amino acid sequences at the iron-binding sites and the two N-linked glycosylation sites, and the cysteine residues were consistent with known, conserved vertebrate transferrin cDNA sequences. Single N-linked glycosylation sites existed on the N- and C-lobe. The deduced amino acid sequence of the rainbow trout transferrin cDNA had 92.9% identities with transferrin of coho salmon (Oncorhynchus kisutch); 85%, Atlantic salmon (Salmo salar); 67.3%, medaka (Oryzias latipes); 61.3% Atlantic cod (Gadus morhua); and 59.7%, Japanese flounder (Paralichthys olivaceus). The long and accurate polymerase chain reaction (LA-PCR) was used to amplify approximately 6.5 kb of the transferrin gene from rainbow trout genomic DNA. Restriction fragment length polymorphisms (RFLPs) of the LA-PCR products revealed three digestion patterns in 22 samples.

  9. Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

    Science.gov (United States)

    Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

    2016-08-24

    To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

  10. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  11. Genetic factors affecting radiosensitivity and cancer predisposition: application of a continuous low dose-rate irradiation colony formation assay to select radiosensitive retinoblastoma family members for correction with a cDNA library

    International Nuclear Information System (INIS)

    Wilson, P.F.; Nagasawa, H.; Bedford, J.S.; Little, J.B.

    2003-01-01

    Full text: The aim of this study is to identify new or undescribed functions of radiosensitivity and genomic instability genes using a continuous low dose-rate colony formation assay. This assay expands on the standard colony formation assay, whereby colony formation ability (retention of proliferative capacity) is measured during continuous low dose-rate irradiation rather than 10-14 days following the completion of such exposures. This approach has previously employed by the Bedford laboratory to identify a Prkdc (DNA-PKcs) mutant of CHO cells, irs-20. In this study we examine the growth response of fibroblasts derived from recently identified radiosensitive retinoblastoma family members, both affected probands and their unaffected parents, and various apparently normal fibroblast lines obtained from the NIGMS Human Genetic Cell Repository (Coriell Medical Institute, Camden, NJ). Colony formation was assayed by plating single cells, exposing them at 37 deg C to continuous Cs-137 gamma irradiation at dose rates of 0.5-8.5 cGy/h, and scoring survivors as colonies with >100 viable cells. The retinoblastoma family members display severely limited growth (survival less than 10E-3) at dose rates greater than 2-2.5 cGy/h, while the apparently normal cell lines do not display such inhibited growth until 6-7 cGy/h. Two of the retinoblastoma family cell lines, MF-6F and MF-15F (both unaffected but radiosensitive parents), were selected as targets of transfection with a viral cDNA library (ViraPort human cDNA library, Stratagene Cloning Systems, La Jolla, CA) and subjected to a ∼3 cGy/h selection dose rate, where uncorrected survival relative to normal cells is lower by a factor of 50-150. Colonies recovered will provide valuable information regarding the genetic nature of their radiosensitivity (possibly involving chromosome stability, DNA repair, and/or cell cycle regulatory pathways), that may influence risks for cancer and heritable effects for a previously

  12. Increased fibroblast functionality on CNN2-loaded titania nanotubes

    Directory of Open Access Journals (Sweden)

    Wei HB

    2012-02-01

    Full Text Available Hongbo Wei*, Shuyi Wu*, Zhihong Feng, Wei Zhou, Yan Dong, Guofeng Wu, Shizhu Bai, Yimin Zhao Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China *These authors contributed equally to this workAbstract: Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.Keywords: anodization, titania nanotubes, adhesion, connective

  13. Binding, uptake, and release of nicotine by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Hanes, P.J.; Schuster, G.S.; Lubas, S.

    1991-01-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4 degree C using a mixture of 3 H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3 H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37 degree C after treating cells with 3 H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours

  14. The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

    Science.gov (United States)

    Clipson, Alexandra; Barrans, Sharon; Zeng, Naiyan; Crouch, Simon; Grigoropoulos, Nicholas F; Liu, Hongxiang; Kocialkowski, Sylvia; Wang, Ming; Huang, Yuanxue; Worrillow, Lisa; Goodlad, John; Buxton, Jenny; Neat, Michael; Fields, Paul; Wilkins, Bridget; Grant, John W; Wright, Penny; Ei-Daly, Hesham; Follows, George A; Roman, Eve; Watkins, A James; Johnson, Peter W M; Jack, Andrew; Du, Ming-Qing

    2015-07-01

    A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP ( n  = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP ( n  = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.

  15. Experience with FISH-detected translocations as an indicator in retrospective dose reconstructions

    International Nuclear Information System (INIS)

    Pressl, S.; Romm, H.; Ganguly, B.B.; Stephan, G.

    2000-01-01

    The prerequisite for the use of translocations as an indicator in retrospective dose reconstructions, is knowledge of the background level, persistence, and the availability of dose response curves for the conversion of translocation frequencies into doses. The results obtained in these areas are summarised. Cells with complete painted chromosome material are evaluated. Those showing any aberrations which involve painted material are stored in a computerised system, and described in detail. The simultaneous painting of whole chromosomes and centromeres has proved to provide a better level of discrimination between translocations and dicentrics. Following irradiation, direct proportionality was observed between DNA content covered by the painted chromosomes (11-19%) and the translocation frequency. The background level of translocations was determined in 42 healthy subjects, aged between 21 and 73 years of age. The statistical analyses of the data revealed no influence from sex and smoking habits on the translocation frequency. A clear increase in translocation yield was, however, observed for age. For the whole genome the frequency is at a level of 3 to 11 per 1000 cells, for all types of translocations. In a radiation accident victim (Estonia) the frequency of translocations was determined over a post-exposure time of four years. For two-way translocations, the half-time was calculated to be 7.0 years, and that for one-way translocations 5.2 years. On the basis of our control data and our dose response curve, the lowest detectable radiation dose is about 0.3 Gy in subjects under 40 years of age, and about 0.5 Gy for those older than 40 years of age. (author)

  16. Translocation of iodine-129 in soil and transfer to vegetation

    International Nuclear Information System (INIS)

    Handl, J.

    1989-01-01

    The translocation of I-129 in the soil and its long-term transfer from soil to plant have been investigated using an undisturbed soil column and a pasture on a river bank. The I-129 profiles in the soils of the monolith and the pasture exhibit clear differences in the distribution of radioiodine after 52 and 47 months, respectively. At those times after surface contamination of both soil types 88% (monolith) and 66% (pasture) of the total I-129 activity were found in the top soil layer of 10 cm, indicating that translocation in the pasture soil is faster than in the monolith. Apart from soil-specific parameters this behaviour may be explained by anaerobic conditions created in the soil due to high water levels in the nearby river attecting the chemical form of iodine. The distribution of stable iodine was found rather homogeneous in both soils with mean contents of 3 mg I-127 kg -1 d.w. in the upper 15 cm and 4 mg I-127 kg -1 d.w. in deeper layers in case of the monolith. For the pasture soil the corresponding values are about 4 and 5 mg I-127 kg -1 d.w. Transfer factors plant/soil (dry weight basis) of I-129 obtained from the monolith 39 g and 52 months after contamination are 1.5x10 -3 and 2.1x10 -3 , respectively. Data from the pasture 35 and 47 months after contamination show values of 9.0x10 -3 and 8.4x10 -3 , respectively. Transfer factors of stable iodine are significantly higher in both soils yielding an average value of 1.0 for the monolith and 0.3 for the pasture. Delay constants (related to a top soil layer of 10 cm) calculated for I-129 using data obtained from the long-term translocation processes to deeper soil layers in the monolith and in the pasture show values of 1x10 -9 s -1 and 4x10 -9 s -1 , respectively. (orig.) [de

  17. Cd translocation into generative organs of linseed (Linum usitatissimum L.)

    Energy Technology Data Exchange (ETDEWEB)

    Becher, M.; Woerner, A.; Schubert, S. [Giessen Univ. (Germany). Inst. fuer Pflanzenernaehrung

    1997-12-31

    Linseed is able to accumulate considerably high concentrations of Cd in generative organs, the dietary critical value of 0.3 {mu}g Cd/g seed often being exceeded. Differences between genotypes of linseed in this respect, however, have been observed but the underlying mechanisms responsible for these differences are unknown. The aim of the present work was to identify these mechanisms by studying the Cd accumulation of the linseed genotypes Antares and McGregor which differ in their ability to accumulate Cd in the seeds. Cultivar Antares is a high and cv. McGregor is low Cd accumulator which was confirmed in a pot experiment. It was found that the differences between these genotypes were Cd-specific and were caused neither by single seed weight nor by Cd translocation into the shoot. The distribution pattern of Cd within mature capsules between the pericarp and the seeds differed from that of Ca which was used as a phloem-immobile reference ion. From these results we conclude that Cd was translocated from the pericarp into the seeds via the phloem. This conclusion was supported by direct Cd determination in collected phloem sap from linseed stems. As sources of seed-Cd we identified the pericarps of capsules and the leaves. The genotype differences concerning the Cd concentrations in the seeds may be explained in terms of differences in phloem translocation of Cd. (orig.) [Deutsch] Oellein vermag Cd in betraechtlichen Konzentrationen in den Samen zu akkumulieren. Aus diesem Grund wird der Grenzwert fuer Diaetlein von 0,3 {mu}g/g Samen im Anbau haeufig ueberschritten. Es gibt jedoch genotypische Unterschiede, deren zugrundeliegende Mechanismen nicht bekannt sind. Das Ziel der vorliegenden Arbeit bestand darin, diese Mechanismen an den Oelleinsorten Antares und McGregor zu untersuchen. Antares akkumuliert grosse, McGregor hingegen geringe Mengen Cd in den Samen. Diese Beobachtungen konnten in einem Gefaessversuch bestaetigt werden. Es wurde gezeigt, dass die

  18. Characterization of the growth of murine fibroblasts that express human insulin receptors. I. The effect of insulin in the absence of other growth factors

    International Nuclear Information System (INIS)

    Randazzo, P.A.; Morey, V.A.; Polishook, A.K.; Jarett, L.

    1990-01-01

    The effect of insulin on the growth of murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental cells (NIH/3T3) was characterized. Insulin in the absence of other mitogens increased the rate of incorporation of thymidine into NIH 3T3/HIR cells with a half-maximal response occurring at an insulin concentration of 35 ng/ml and a maximal response that was equivalent to that elicited by 10% fetal calf serum. The thymidine incorporation rate was increased by 12 h, was maximal at approximately 16 h, and returned to basal rates at 24 h after the addition of insulin. Insulin induced a maximum of 65% of cells to incorporate thymidine. The increased DNA synthesis was accompanied by net growth. Addition of insulin to the NIH 3T3/HIR cells resulted in increased DNA content with a half-maximal response occurring at approximately 30 ng/ml insulin and a maximal response equivalent to that elicited by serum. An increase in cell number detected after the addition of insulin to the NIH 3T3/HIR suggests that the cells had progressed through mitosis. Insulin did not increase the rate of thymidine incorporation, DNA content, or number of the parental NIH 3T3 cells. These data show that insulin, in the absence of a second mitogen, is able to induce NIH 3T3/HIR fibroblasts to traverse the cell cycle

  19. Role of insulin-like growth factor-I receptor (IGF-IR) in survival kinetics and radioresistance of mouse embryo fibroblasts in a hypoxic environment

    International Nuclear Information System (INIS)

    Okochi, Kiyoshi

    2002-01-01

    The role of insulin-like growth factor-I receptor (IGF-IR) in survival kinetics and radioresistance of fibroblasts in a severely hypoxic environment (partial oxygen pressure of less than 3 mmHg) was analyzed, in both low and high cell-density conditions. Mouse embryonic fibroblasts R(-), with a targeted disruption of the IGF-IR gene, and R(+) cells, derived from R(-) cells stably transfected with a plasmid containing a human IGF-IR cDNA, were used for this purpose. Survival time in hypoxia was longer in R(+) cells than R(-) cells, which correlated with highly elevated expression of caspase 3-like activity in R(-) cells, but not with HIF-Iα expression. Under euoxia, R(+) cells were more radioresistant, by a factor of 1.9, than R(-) cells. Under hypoxia, R(+) cells became more radioresistant, with an oxygen-enhancement ratio (OER) of 2.7, than R(-) cells, with an OER of 1.5, in a low cell density. However, unexpected hyper-radiosensitivity in hypoxia was observed for both R(+) and R(-) cells in a high cell density, which further increased with incubation time in hypoxia following X-irradiation. The hyper-radiosensitivity was more pronounced for R(-) cells. The result thus implies that IGF-IR may be an important target molecule for radioresistant tumors in radiotherapy. (author)

  20. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  1. A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

    Science.gov (United States)

    Cui, Y H; Zhu, G Q; Chen, Q J; Wang, Y F; Yang, M M; Song, Y X; Wang, J G; Cao, B Y

    2011-11-25

    Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

  2. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  3. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H.

    1988-01-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10 6 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  4. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell.

    Science.gov (United States)

    Chuang, Pan; Shoichiro, Ishizaki; Yuji, Nagashima; Jialong, Gao; Shugo, Watabe

    2018-02-01

    In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C) on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article "Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei " (Chuang et al., 2017) [1].

  5. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell

    Directory of Open Access Journals (Sweden)

    Pan Chuang

    2018-02-01

    Full Text Available In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article “Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei” (Chuang et al., 2017 [1].

  6. Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

    Science.gov (United States)

    Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

    1987-01-01

    Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

  7. Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

    International Nuclear Information System (INIS)

    Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

    2001-01-01

    Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

  8. Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15(q27;q11.2 associated with Prader-Willi syndrome

    Directory of Open Access Journals (Sweden)

    Slater Howard R

    2005-05-01

    Full Text Available Abstract Background Prader-Willi syndrome (MIM #176270; PWS is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15(q27;q11.2. Methods We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Results Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. Conclusion As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype.

  9. Tailoring particle translocation via dielectrophoresis in pore channels

    Science.gov (United States)

    Tanaka, Shoji; Tsutsui, Makusu; Theodore, Hu; Yuhui, He; Arima, Akihide; Tsuji, Tetsuro; Doi, Kentaro; Kawano, Satoyuki; Taniguchi, Masateru; Kawai, Tomoji

    2016-01-01

    Understanding and controlling electrophoretic motions of nanoscopic objects in fluidic channels are a central challenge in developing nanopore technology for molecular analyses. Although progress has been made in slowing the translocation velocity to meet the requirement for electrical detections of analytes via picoampere current measurements, there exists no method useful for regulating particle flows in the transverse directions. Here, we report the use of dielectrophoresis to manipulate the single-particle passage through a solid-state pore. We created a trap field by applying AC voltage between electrodes embedded in a low-aspect-ratio micropore. We demonstrated a traffic control of particles to go through center or near side surface via the voltage frequency. We also found enhanced capture efficiency along with faster escaping speed of particles by virtue of the AC-mediated electroosmosis. This method is compatible with nanopore sensing and would be widely applied for reducing off-axis effects to achieve single-molecule identification. PMID:27527126

  10. Mitochondrial tRNA gene translocations in highly eusocial bees

    Directory of Open Access Journals (Sweden)

    Daniela Silvestre

    2006-01-01

    Full Text Available Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Considering the high number of rearrangements observed, the data presented here contradict the general rule of high gene order conservation among closely related organisms, and also represent a powerful molecular tool to help solve questions about phylogeny and evolution in bees.

  11. Physical insights into the blood-brain barrier translocation mechanisms

    Science.gov (United States)

    Theodorakis, Panagiotis E.; Müller, Erich A.; Craster, Richard V.; Matar, Omar K.

    2017-08-01

    The number of individuals suffering from diseases of the central nervous system (CNS) is growing with an aging population. While candidate drugs for many of these diseases are available, most of these pharmaceutical agents cannot reach the brain rendering most of the drug therapies that target the CNS inefficient. The reason is the blood-brain barrier (BBB), a complex and dynamic interface that controls the influx and efflux of substances through a number of different translocation mechanisms. Here, we present these mechanisms providing, also, the necessary background related to the morphology and various characteristics of the BBB. Moreover, we discuss various numerical and simulation approaches used to study the BBB, and possible future directions based on multi-scale methods. We anticipate that this review will motivate multi-disciplinary research on the BBB aiming at the design of effective drug therapies.

  12. Translocation of labelled fertilizer nitrogen in soil columns

    International Nuclear Information System (INIS)

    Haunold, E.; Zvara, J.

    1975-01-01

    The translocation of 15 labeled ammonium and nitrate fertilizer was studied under normal weather conditions for two years in columns filled with different soils. At the end of the experimental period, which usually lasted for 9 months, between 5.9-10.3% of the ammonium fertilizer was leached out, 33.7-50.1% remained in the soil and 39.5-59.7% was lost as gas. For nitrate nitrogen the figures were: 22.6-47.3% leached out, 16.7-40% remaining in the soil, 12.7-60.0% lost as gas. The ammonium fertilizer moving through the soil interchanged with 1-13% of the soil nitrogen, the nitrate fertilizer with only 0.5-2%

  13. Real-time analysis of nitrogen translocation in plants

    International Nuclear Information System (INIS)

    Hayashi, Hiroaki

    2000-01-01

    Nitrogen absorbed by roots is transported to the leaves through xylem vessels and then retranslocated to the new leaves, such as root and storage organs through sieve tubes. It is very important to know how this nitrogen movement occurs in the plants and what mechanisms are involved in controlling this movement in order to increase the efficiency of fertilizer. In this experiments, 13 N and 15 N was used to detect the nitrogen circulation in plants, in combination with the technique for positron detection in real time and for collection of sap in sieve tubes and analysis of 15 N in it. By using 13 N, nitrogen movement from root to shoot was analyzed within 10 min after 13 N was applied to the roots. On the other hand, nitrogen retranslocation through sieve tubes was detected by the analysis of 15 N in the phloem sap over 6 hrs. All data suggest the dynamic translocation of nitrogen in rice plants. (author)

  14. Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    François Huaux

    Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

  15. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

    Science.gov (United States)

    Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

    2017-01-01

    Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing. PMID:28773152

  16. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  17. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

    Directory of Open Access Journals (Sweden)

    Rebeca Illescas-Montes

    2017-07-01

    Full Text Available Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2 using different transmission modes (continuous or pulsed. The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing.

  18. Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

    OpenAIRE

    Liew, C C; Jandreski, M A

    1986-01-01

    A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequen...

  19. Translocation of Cell Penetrating Peptide Engrafted Nanoparticles Across Skin Layers

    Science.gov (United States)

    Patlolla, Ram R; Desai, Pinaki; Belay, Kalayu; Singh, Mandip

    2010-01-01

    The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24 h, fluorescence was observed upto a depth of 120 µm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 µm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24 h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers. PMID:20413152

  20. Distribution and Translocation of 141Ce (III) in Horseradish

    Science.gov (United States)

    Guo, Xiaoshan; Zhou, Qing; Lu, Tianhong; Fang, Min; Huang, Xiaohua

    2007-01-01

    Background and Aims Rare earth elements (REEs) are used in agriculture and a large amount of them contaminate the environment and enter foods. The distribution and translocation of 141Ce (III) in horseradish was investigated in order to help understand the biochemical behaviour and toxic mechanism of REEs in plants. Method The distribution and translocation of 141Ce (III) in horseradish were investigated using autoradiography, liquid scintillation counting (LSC) and electron microscopic autoradiography (EMARG) techniques. The contents of 141Ce (III) and nutrient elements were analysed using an inductively coupled plasma-atomic emission spectrometer (ICP-AES). Results The results from autoradiography and LSC indicated that 141Ce (III) could be absorbed by horseradish and transferred from the leaf to the leaf-stalk and then to the root. The content of 141Ce (III) in different parts of horseradish was as follows: root > leaf-stalk > leaf. The uptake rates of 141Ce (III) in horseradish changed with the different organs and time. The content of 141Ce (III) in developing leaves was greater than that in mature leaves. The results from EMARG indicated that 141Ce (III) could penetrate through the cell membrane and enter the mesophyll cells, being present in both extra- and intra-cellular deposits. The contents of macronutrients in horseradish were decreased by 141Ce (III) treatment. Conclusions 141Ce (III) can be absorbed and transferred between organs of horseradish with time, and the distribution was found to be different at different growth stages. 141Ce (III) can enter the mesophyll cells via apoplast and symplast channels or via plasmodesmata. 141Ce (III) can disturb the metabolism of macronutrients in horseradish. PMID:17921527