Science.gov

Sample records for mammalian wnt4 promoter

  1. Follistatin Operates Downstream of Wnt4 in Mammalian Ovary Organogenesis

    PubMed Central

    Yao, Humphrey H.C.; Matzuk, Martin M.; Jorgez, Carolina J.; Menke, Douglas B.; Page, David C.; Swain, Amanda; Capel, Blanche

    2014-01-01

    Wnt4−/− XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFβ superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary. Developmental Dynamics 230:210–215, 2004. PMID:15162500

  2. Combination of melatonin and Wnt-4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury.

    PubMed

    Gao, Yuhua; Bai, Chunyu; Zheng, Dong; Li, Changli; Zhang, Wenxiu; Li, Mei; Guan, Weijun; Ma, Yuehui

    2016-04-01

    Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promoting differentiation of neural cells remain unknown. Wnt signaling mediates major developmental processes during embryogenesis and regulates maintenance, self-renewal, and differentiation of adult mammalian stem cells. However, the role of the noncanonical Wnt pathway during neurogenesis remains poorly understood. In this study, the amniotic epithelial cells ( AECs) were isolated from bovine amnion and incubated with various melatonin concentrations (0.01, 0.1, 1, 10, or 100 μm) and 5 × 10(-5) m all-trans retinoic acid (RA) for screening optimum culture medium of neural differentiation, compared with each groups, 1 μm melatonin and 5 × 10(-5) m RA were selected to induce neural differentiation of AECs, and then siMT1, siMT2, oWnt-4, and siWnt-4 were expressed in AECs to research role of these genes in neural differentiation. Efficiency of neural differentiation was evaluated after expressed above genes using flow cytometry. Cell function of neural cells was demonstrated in vivo using spinal cord injury model after cell transplantation, and damage repair of spinal cord was assessed using cell tracking and Basso, Beattie, Bresnahan Locomotor Rating Scale scores. Results demonstrated that melatonin stimulated melatonin receptor 1, which subsequently increased bovine amniotic epithelial cell vitality and promoted differentiation into neural cells. This took place through cooperation with Wnt-4. Additionally, following cotreatment with melatonin and Wnt-4, neurogenesis gene expression was significantly altered. Furthermore, single inhibition of melatonin receptor 1 or Wnt-4 expression decreased expression of neurogenesis-related genes, and bovine amniotic epithelial cell-derived neural cells were successfully colonized into injured spinal cord, which suggested participation in tissue repair.

  3. Wnt4 Participates in the Formation of Vertebrate Neuromuscular Junction

    PubMed Central

    Strochlic, Laure; Falk, Julien; Goillot, Evelyne; Sigoillot, Séverine; Bourgeois, Francine; Delers, Perrine; Rouvière, Jérôme; Swain, Amanda; Castellani, Valérie; Schaeffer, Laurent; Legay, Claire

    2012-01-01

    Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4−/− NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis. PMID:22253844

  4. Identification of the genes regulated by Wnt-4, a critical signal for commitment of the ovary.

    PubMed

    Naillat, Florence; Yan, Wenying; Karjalainen, Riikka; Liakhovitskaia, Anna; Samoylenko, Anatoly; Xu, Qi; Sun, Zhandong; Shen, Bairong; Medvinsky, Alexander; Quaggin, Susan; Vainio, Seppo J

    2015-03-15

    The indifferent mammalian embryonic gonad generates an ovary or testis, but the factors involved are still poorly known. The Wnt-4 signal represents one critical female determinant, since its absence leads to partial female-to-male sex reversal in mouse, but its signalling is as well implicated in the testis development. We used the Wnt-4 deficient mouse as a model to identify candidate gonadogenesis genes, and found that the Notum, Phlda2, Runx-1 and Msx1 genes are typical of the wild-type ovary and the Osr2, Dach2, Pitx2 and Tacr3 genes of the testis. Strikingly, the expression of these latter genes becomes reversed in the Wnt-4 knock-out ovary, suggesting a role in ovarian development. We identified the transcription factor Runx-1 as a Wnt-4 signalling target gene, since it is expressed in the ovary and is reduced upon Wnt-4 knock-out. Consistent with this, introduction of the Wnt-4 signal into early ovary cells ex vivo induces Runx-1 expression, while conversely Wnt-4 expression is down-regulated in the absence of Runx-1. We conclude that the Runx-1 gene can be a Wnt-4 signalling target, and that Runx-1 and Wnt-4 are mutually interdependent in their expression. The changes in gene expression due to the absence of Wnt-4 in gonads reflect the sexually dimorphic role of this signal and its complex gene network in mammalian gonad development.

  5. Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

    PubMed

    Weng, Shenda; You, Feng; Fan, Zhaofei; Wang, Lijuan; Wu, Zhihao; Zou, Yuxia

    2016-08-01

    WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.

  6. Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in Mice

    PubMed Central

    Lee, Hu-Hui; Behringer, Richard R.

    2007-01-01

    Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype. PMID:17505543

  7. Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary.

    PubMed

    Maatouk, Danielle M; Mork, Lindsey; Chassot, Anne-Amandine; Chaboissier, Marie-Christine; Capel, Blanche

    2013-11-15

    Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage. © 2013 Elsevier Inc. All rights reserved.

  8. Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary

    PubMed Central

    Maatouk, Danielle M.; Mork, Lindsey; Chassot, Anne-Amandine; Marie-Christine, Chaboissier; Capel, Blanche

    2014-01-01

    Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage. PMID:24036309

  9. A promoter-level mammalian expression atlas

    PubMed Central

    2015-01-01

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. PMID:24670764

  10. A promoter-level mammalian expression atlas.

    PubMed

    Forrest, Alistair R R; Kawaji, Hideya; Rehli, Michael; Baillie, J Kenneth; de Hoon, Michiel J L; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V; Lizio, Marina; Itoh, Masayoshi; Andersson, Robin; Mungall, Christopher J; Meehan, Terrence F; Schmeier, Sebastian; Bertin, Nicolas; Jørgensen, Mette; Dimont, Emmanuel; Arner, Erik; Schmidl, Christian; Schaefer, Ulf; Medvedeva, Yulia A; Plessy, Charles; Vitezic, Morana; Severin, Jessica; Semple, Colin A; Ishizu, Yuri; Young, Robert S; Francescatto, Margherita; Alam, Intikhab; Albanese, Davide; Altschuler, Gabriel M; Arakawa, Takahiro; Archer, John A C; Arner, Peter; Babina, Magda; Rennie, Sarah; Balwierz, Piotr J; Beckhouse, Anthony G; Pradhan-Bhatt, Swati; Blake, Judith A; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Burroughs, A Maxwell; Califano, Andrea; Cannistraci, Carlo V; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C; Dalla, Emiliano; Davis, Carrie A; Detmar, Michael; Diehl, Alexander D; Dohi, Taeko; Drabløs, Finn; Edge, Albert S B; Edinger, Matthias; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey; Fang, Hai; Farach-Carson, Mary C; Faulkner, Geoffrey J; Favorov, Alexander V; Fisher, Malcolm E; Frith, Martin C; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furino, Masaaki; Furusawa, Jun-ichi; Geijtenbeek, Teunis B; Gibson, Andrew P; Gingeras, Thomas; Goldowitz, Daniel; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Takehiro; Herlyn, Meenhard; Hitchens, Kelly J; Ho Sui, Shannan J; Hofmann, Oliver M; Hoof, Ilka; Hori, Furni; Huminiecki, Lukasz; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R; Jia, Hui; Joshi, Anagha; Jurman, Giuseppe; Kaczkowski, Bogumil; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I; Kawashima, Tsugumi; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klinken, S Peter; Knox, Alan J; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T; Laros, Jeroen F J; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Lipovich, Leonard; Mackay-Sim, Alan; Manabe, Ri-ichiroh; Mar, Jessica C; Marchand, Benoit; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison; Mizuno, Yosuke; de Lima Morais, David A; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Noma, Shohei; Noazaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohimiya, Hiroko; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Pain, Arnab; Passier, Robert; Patrikakis, Margaret; Persson, Helena; Piazza, Silvano; Prendergast, James G D; Rackham, Owen J L; Ramilowski, Jordan A; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Rye, Morten B; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Savvi, Suzana; Saxena, Alka; Schneider, Claudio; Schultes, Erik A; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W; Simon, Christophe; Sugiyama, Daisuke; Sugiyama, Takaai; Suzuki, Masanori; Suzuki, Naoko; Swoboda, Rolf K; 't Hoen, Peter A C; Tagami, Michihira; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyodo, Hiroo; Toyoda, Tetsuro; Valen, Elvind; van de Wetering, Marc; van den Berg, Linda M; Verado, Roberto; Vijayan, Dipti; Vorontsov, Ilya E; Wasserman, Wyeth W; Watanabe, Shoko; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Wood, Emily J; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Susan E; Zhang, Peter G; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M; Suzuki, Harukazu; Daub, Carsten O; Kawai, Jun; Heutink, Peter; Hide, Winston; Freeman, Tom C; Lenhard, Boris; Bajic, Vladimir B; Taylor, Martin S; Makeev, Vsevolod J; Sandelin, Albin; Hume, David A; Carninci, Piero; Hayashizaki, Yoshihide

    2014-03-27

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  11. WNT4-like protein is a cortical granule component in mouse oocytes and functions in regulating preimplantation embryogenesis.

    PubMed

    Liu, Min; Yang, Huei-Ting

    2016-01-01

    Mammalian cortical granules (CG) are membrane-bound organelles located in the cortex of the unfertilized oocytes. Upon fertilization, CG undergo exocytosis to function in blocking polyspermy. While cortical granules are important in fertilization, their exact biochemical composition and reproductive function have not been fully defined. In the present study, a 66 kDa wingless-type MMTV integration site family, member 4 (WNT4)-like protein, with mouse CG origin was identified. Oocytes that were double labeled with lectin Lens culinaris agglutinin (LCA) and WNT4 antibody showed colocalization of the WNT4 molecules and cortical granules. The disappearance of WNT4 molecules in the artificially activated oocytes that were devoid of cortical granules confirmed their granule origin. Following fertilization, WNT4 remained associated with zygotes and blastomeres of 2-cell and 8-cell embryos; however the amount of protein present was reduced more than 2-fold as embryos developed. Prior to implantation, WNT4 appeared to be detectable only in the trophoblast cells. Our functional study revealed that WNT4 molecules were involved in regulating zygotic cleavage and early embryogenesis. To our knowledge, this is the first study demonstrating mammalian cortical granules contain signaling molecules that are involved in the regulation of the first phase of embryonic development.

  12. Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity, and antimüllerian hormone expression during oocyte maturation in the female follicle.

    PubMed

    Prunskaite-Hyyryläinen, Renata; Shan, Jingdong; Railo, Antti; Heinonen, Krista M; Miinalainen, Ilkka; Yan, Wenying; Shen, Bairong; Perreault, Claude; Vainio, Seppo J

    2014-04-01

    Wnt4 is a key signal that channels the developmental fate of the indifferent mammalian gonad toward the ovary, but whether Wnt4 has later roles during ovary development remains unknown. To investigate this, we inactivated the Wnt4 gene by crossing Amhr2Cre and doxycycline-inducible Rosa(rtTA)-knock-in Cre mice with mice carrying a floxed Wnt4 allele and used a novel Wnt4(mCherry)-knock-in mouse. In these models, ovarian folliculogenesis was compromised, and female fertility was severely reduced, and Wnt4 deficiency eventually led to premature ovarian failure. These anomalies were associated with cell polarity defects in the follicle. Within the follicle, laminin and type IV collagen assembled ectopic basement membrane-like structures, the cell adherens junction components N-cadherin and β-catenin lost their polarized expression pattern, and expression of the gap junction protein connexin 43 was reduced by ~30% when compared with that of the controls. Besides these changes, expression of antimüllerian hormone (Amh) was inhibited in the absence of Wnt4 signaling in vivo. Consistent with this, Wnt4 signaling up-regulated Amh gene expression in KK1 cells in vitro. Thus, Wnt4 signaling is necessary during maturation of the ovarian follicles, where it coordinates expression of Amh, cell survival, and polarized organization of the follicular cells.

  13. Regulation of Wnt4 in chronic obstructive pulmonary disease

    PubMed Central

    Durham, Andrew L.; McLaren, Alistair; Hayes, Brian P.; Caramori, Gaetano; Clayton, Chris L.; Barnes, Peter J.; Chung, K. Fan; Adcock, Ian M.

    2013-01-01

    Chronic obstructive pulmonary disease (COPD) is associated with persistent inflammation and oxidative stress in susceptible individuals. Using microarray analysis of bronchial biopsy samples from patients with COPD and controls, we identified Wnt4 as being up-regulated in COPD. Analysis of bronchial biopsy samples showed a very strong correlation between Wnt4 and IL8 gene expression, suggesting that Wnt4 plays a role in chronic lung inflammation. In vitro, Wnt4 induced proliferation and inflammation in human epithelial cells (BEAS-2B) and normal primary human bronchial epithelial cells in a concentration-dependent manner. This effect was enhanced in the presence of interleukin-1β (IL-1β) as a result of activation of the p38 and c-Jun NH2-terminal kinase mitogen-activated protein kinase pathways. Hydrogen peroxide, but not proinflammatory stimuli, up-regulated Wnt4 expression in epithelial cells. In monocytic THP-1 and primary airway smooth muscle cells, Wnt4 induced inflammation and enhanced the inflammatory response to lipopolysaccharide and IL-1β but did not induce proliferation. In addition, these other cell types did not have enhanced Wnt4 expression in response to hydrogen peroxide. Our results indicate that airway epithelial activation, due to oxidative stress, may lead to Wnt4 induction. Wnt4, in turn, acts through the noncanonical pathway to activate epithelial cell remodeling and IL8 gene expression, leading to neutrophil infiltration and inflammation.—Durham, A. L., McLaren, A., Hayes, B. P., Caramori, G., Clayton, C. L., Barnes, P. J., Chung, K. F., Adcock, I. M. Regulation of Wnt4 in chronic obstructive pulmonary disease. PMID:23463699

  14. Notch activates Wnt-4 signalling to control medio-lateral patterning of the pronephros.

    PubMed

    Naylor, Richard W; Jones, Elizabeth A

    2009-11-01

    Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. Here, we further the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. Our results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. We show that this novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. We also identified pronephric expression of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, along with data from previous publications, we propose a mechanism by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen.

  15. Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development.

    PubMed

    Shan, Jingdong; Jokela, Tiina; Skovorodkin, Ilya; Vainio, Seppo

    2010-01-01

    The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4(EGFPCre) knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26(LacZ) reporter with the Wnt4(EGFPCre) allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4(EGFPCre)-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26(YFP)-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4(EGFPCre); Rosa26(LacZ)-marked cells with those of the

  16. Negative regulation of the Wnt signal by MM-1 through inhibiting expression of the wnt4 gene.

    PubMed

    Yoshida, Tatsuya; Kitaura, Hirotake; Hagio, Yuko; Sato, Toshiya; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2008-04-01

    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc through TIF1beta/KAP1, a transcriptional corepressor, and that the c-fms gene was a target gene involved in this pathway. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, to further identify target genes of MM-1, DNA microarray analysis was carried out by comparing expression levels of genes in MM-1 knockdown and parental cells, and the wnt4 gene, a member of the Wnt-beta-catenin pathway, was identified as a target gene of MM-1. Increased expression level of the wnt4 gene, accumulation and translocation of beta-catenin to the cytoplasm and nucleus, and upregulation of TCF/Lef-1, a target protein of the Wnt-beta-catenin pathway, were found in MM-1 knockdown cells. Reporter assays using various deletion constructs of the wnt4 gene promoter showed that MM-1 recognized the region spanning -286 to -229 from a transcription start site, and MM-1 complex was found to bind to this region by chromatin immunoprecipitation and gel mobility shift assays. Furthermore, it was found that Egr-1 and MM-1 were bound to this region and that both proteins mutually down-regulate promoter activity of the wnt4 gene. Since the c-myc gene is the target gene of the Wnt-beta-catenin pathway, these findings suggest that MM-1 inhibits c-Myc by a dual mechanism.

  17. Structural dynamics and inhibitor searching for Wnt-4 protein using comparative computational studies

    PubMed Central

    Hammad, Mirza A; Azam, Syed Sikander

    2015-01-01

    Wnt-4 (wingless mouse mammary tumor virus integration site-4) protein is involved in many crucial embryonic pathways regulating essential processes. Aberrant Wnt-4 activity causes various anomalies leading to gastric, colon, or breast cancer. Wnt-4 is a conserved protein in structure and sequence. All Wnt proteins contain an unusual fold comprising of a thumb (or N-terminal domain) and index finger (or C-terminal domain) bifurcated by a palm domain. The aim of this study was to identify the best inhibitors of Wnt-4 that not only interact with Wnt-4 protein but also with the covalently bound acyl group to inhibit aberrant Wnt-4 activity. A systematic computational approach was used to analyze inhibition of Wnt-4. Palmitoleic acid was docked into Wnt-4 protein, followed by ligand-based virtual screening of nearly 209,847 compounds; conformer generation of 271 compounds resulted from extensive virtual screening and comparative docking of 10,531 conformers of 271 unique compounds through GOLD (Genetic Optimization for Ligand Docking), AutoDock-Vina, and FRED (Fast Rigid Exhaustive Docking) was subsequently performed. Linux scripts was used to handle the libraries of compounds. The best compounds were selected on the basis of having maximum interactions to protein with bound palmitoleic acid. These represented lead inhibitors in further experiments. Palmitoleic acid is important for efficient Wnt activity, but aberrant Wnt-4 expression can be inhibited by designing inhibitors interacting with both protein and palmitoleic acid. PMID:25995617

  18. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  19. Plant growth-promoting hormones activate mammalian guanylate cyclase activity.

    PubMed

    Vesely, D L; Hudson, J L; Pipkin, J L; Pack, L D; Meiners, S E

    1985-05-01

    In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.

  20. Sequence Analysis and Molecular Characterization of Wnt4 Gene in Metacestodes of Taenia solium

    PubMed Central

    Hou, Junling; Luo, Xuenong; Wang, Shuai; Yin, Cai; Zhang, Shaohua; Zhu, Xueliang; Dou, Yongxi

    2014-01-01

    Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium. PMID:24850959

  1. Wnt4 is required for ostia development in the Drosophila heart.

    PubMed

    Chen, Zhimin; Zhu, Jun-Yi; Fu, Yulong; Richman, Adam; Han, Zhe

    2016-05-15

    The Drosophila ostia are valve-like structures in the heart with functional similarity to vertebrate cardiac valves. The Wnt/β-catenin signaling pathway is critical for valve development in zebrafish and mouse, but the key ligand(s) for valve induction remains unclear. We observed high levels of Wnt4 gene expression in Drosophila ostia progenitor cells, immediately prior to morphological differentiation of these cells associated with ostia formation. This differentiation was blocked in Wnt4 mutants and in flies expressing canonical Wnt signaling pathway inhibitors but not inhibitors of the planar cell polarity pathway. High levels of Wnt4 dependent activation of a canonical Wnt signaling reporter was observed specifically in ostia progenitor cells. In vertebrate valve formation Wnt signaling is active in cells undergoing early endothelial-mesenchymal transition (EMT) and the Wnt9 homolog of Drosophila Wnt4 is expressed in valve progenitors. In demonstrating an essential role for Wnt4 in ostia development we have identified similarities between molecular and cellular events associated with early EMT during vertebrate valve development and the differentiation and partial delamination of ostia progenitor cells in the process of ostia formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Sequence analysis and molecular characterization of Wnt4 gene in metacestodes of Taenia solium.

    PubMed

    Hou, Junling; Luo, Xuenong; Wang, Shuai; Yin, Cai; Zhang, Shaohua; Zhu, Xueliang; Dou, Yongxi; Cai, Xuepeng

    2014-04-01

    Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

  3. Gateways to the FANTOM5 promoter level mammalian expression atlas

    SciTech Connect

    Lizio, Marina; Harshbarger, Jayson; Shimoji, Hisashi; Severin, Jessica; Kasukawa, Takeya; Sahin, Serkan; Abugessaisa, Imad; Fukuda, Shiro; Hori, Fumi; Ishikawa-Kato, Sachi; Mungall, Christopher J.; Arner, Erik; Baillie, J. Kenneth; Bertin, Nicolas; Bono, Hidemasa; de Hoon, Michiel; Diehl, Alexander D.; Dimont, Emmanuel; Freeman, Tom C.; Fujieda, Kaori; Hide, Winston; Kaliyaperumal, Rajaram; Katayama, Toshiaki; Lassmann, Timo; Meehan, Terrence F.; Nishikata, Koro; Ono, Hiromasa; Rehli, Michael; Sandelin, Albin; Schultes, Erik A.; ‘t Hoen, Peter A. C.; Tatum, Zuotian; Thompson, Mark; Toyoda, Tetsuro; Wright, Derek W.; Daub, Carsten O.; Itoh, Masayoshi; Carninci, Piero; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Kawaji, Hideya

    2015-01-05

    The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). In conclusion, this resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

  4. Analysis of the Bombyx mori nucleopolyhedrovirus ie-1 promoter in insect, mammalian, plant, and bacterial cells.

    PubMed

    Fujita, Ryosuke; Ono, Chikako; Ono, Isamu; Asano, Shin-Ichiro; Bando, Hisanori

    2015-09-04

    The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a β-galactosidase assay in E. coli cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Modulation of mammalian sperm function by fertilization promoting peptide (FPP).

    PubMed

    Fraser, L R

    1998-01-01

    Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2) is produced by the prostate gland and secreted into seminal plasma. When added to uncapacitated mouse and human sperm suspensions, it stimulates capacitation as demonstrated by both cytological changes and increased fertilizing ability in vitro. When added to capacitated suspensions, FPP inhibits spontaneous acrosome loss but cells retain high fertility in vitro. Adenosine elicits similar responses to FPP in both uncapacitated and capacitated cells and FPP + adenosine has a greater effect on uncapacitated cells than either used individually. We have proposed that these two molecules modulate the same pathway (adenylate cyclase/cAMP) but act via different receptors. The structure of FPP is crucial for bioactivity: loss of the terminal amide group abolishes activity and substitution of the central glutamic acid can markedly alter activity. Most recently we have found that stimulation of TCP-11, the product of the mouse t-complex gene Tcp-11, elicits responses indistinguishable from those obtained with FPP and we have hypothesized that the protein TCP-11 is the receptor for FPP. The existence of a human homologue for Tcp-11 suggests that the gene product, in conjunction with FPP, could play an important role in human fertility.

  6. Transposon Dysregulation Modulates dWnt4 Signaling to Control Germline Stem Cell Differentiation in Drosophila.

    PubMed

    Upadhyay, Maitreyi; Martino Cortez, Yesenia; Wong-Deyrup, SiuWah; Tavares, Leticia; Schowalter, Sean; Flora, Pooja; Hill, Corinne; Nasrallah, Mohamad Ali; Chittur, Sridar; Rangan, Prashanth

    2016-03-01

    Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate.

  7. Transposon Dysregulation Modulates dWnt4 Signaling to Control Germline Stem Cell Differentiation in Drosophila

    PubMed Central

    Upadhyay, Maitreyi; Martino Cortez, Yesenia; Wong-Deyrup, SiuWah; Tavares, Leticia; Schowalter, Sean; Flora, Pooja; Hill, Corinne; Nasrallah, Mohamad Ali; Chittur, Sridar; Rangan, Prashanth

    2016-01-01

    Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate. PMID:27019121

  8. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    PubMed

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  9. Wnt-4 expression is increased in fibroblasts after TGF-beta1 stimulation and during fetal and postnatal wound repair.

    PubMed

    Colwell, Amy S; Krummel, Thomas M; Longaker, Michael T; Lorenz, H Peter

    2006-06-01

    Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined. Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction. In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p < 0.05) and peaked with an 11-fold increase at 2 hours (p < 0.05). By 24 hours, expression decreased to 2-fold baseline levels (p < 0.05). In postnatal fibroblasts, Wnt-4 expression also increased after TGF-beta stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p < 0.005). Expression levels at 24 hours were still 4-fold greater than baseline (p < 0.05). In E17 fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p < 0.005). Small increases in Wnt-4 expression (less than 2-fold) occurred during both fetal scarless and postnatal scarring mouse wound repair. The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

  10. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  11. Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

    PubMed Central

    Campos-Sandoval, José A.; Manzanares, Elisa; Lobo, Carolina; Segura, J. A.; Alonso, Francisco J.; Matés, José M.; Márquez, Javier

    2012-01-01

    Background Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene. Methodology/Principal Findings We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed. Conclusions/Significance This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was

  12. Use of viral promoters in mammalian cell-based bioassays: How reliable?

    PubMed Central

    Betrabet, Shrikant S; Choudhuri, Jyoti; Gill-Sharma, Manjit

    2004-01-01

    Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues) specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase), which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10-12 to 10-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for measuring bioactivities of

  13. Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Hu, Qiaomu; Zhu, Ying; Liu, Yang; Wang, Na; Chen, Songlin

    2014-11-24

    Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.

  14. Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis)

    PubMed Central

    Hu, Qiaomu; Zhu, Ying; Liu, Yang; Wang, Na; Chen, Songlin

    2014-01-01

    Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection. PMID:25418599

  15. Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis)

    NASA Astrophysics Data System (ADS)

    Hu, Qiaomu; Zhu, Ying; Liu, Yang; Wang, Na; Chen, Songlin

    2014-11-01

    Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.

  16. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia

    SciTech Connect

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3′, 5’-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram

  17. Orphan CpG islands identify numerous conserved promoters in the mammalian genome.

    PubMed

    Illingworth, Robert S; Gruenewald-Schneider, Ulrike; Webb, Shaun; Kerr, Alastair R W; James, Keith D; Turner, Daniel J; Smith, Colin; Harrison, David J; Andrews, Robert; Bird, Adrian P

    2010-09-23

    CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are "orphans" that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development.

  18. Orphan CpG Islands Identify Numerous Conserved Promoters in the Mammalian Genome

    PubMed Central

    Illingworth, Robert S.; Gruenewald-Schneider, Ulrike; Webb, Shaun; Kerr, Alastair R. W.; James, Keith D.; Turner, Daniel J.; Smith, Colin; Harrison, David J.; Andrews, Robert; Bird, Adrian P.

    2010-01-01

    CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are “orphans” that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development. PMID:20885785

  19. Three Novel Downstream Promoter Elements Regulate MHC Class I Promoter Activity in Mammalian Cells

    PubMed Central

    Lee, Namhoon; Iyer, Shankar S.; Mu, Jie; Weissman, Jocelyn D.; Ohali, Anat; Howcroft, T. Kevin; Lewis, Brian A.; Singer, Dinah S.

    2010-01-01

    Background MHC class I transcription is regulated by two distinct types of regulatory pathways: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. Methodology/Principal Findings We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3′ end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5′ end of the core promoter. Conclusions/Significance Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5′ end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription. PMID:21179443

  20. Sonic hedgehog promotes stem-cell potential of Mueller glia in the mammalian retina

    SciTech Connect

    Wan Jin; Zheng Hua; Xiao Honglei; She Zhenjue; Zhou Guomin

    2007-11-16

    Mueller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Mueller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Mueller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Mueller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Mueller glia-derived cells. Together, these results provide evidences that Mueller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

  1. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

    PubMed

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

  2. Milk--A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation.

    PubMed

    Melnik, Bodo C

    2015-07-27

    Based on own translational research of the biochemical and hormonal effects of cow's milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1) essential branched-chain amino acids (BCAAs); (2) glutamine; (3) palmitic acid; and (4) bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER) stress and drives an aimless quasi-program, which promotes aging and age-related diseases.

  3. Milk—A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation

    PubMed Central

    Melnik, Bodo C.

    2015-01-01

    Based on own translational research of the biochemical and hormonal effects of cow’s milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1) essential branched-chain amino acids (BCAAs); (2) glutamine; (3) palmitic acid; and (4) bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER) stress and drives an aimless quasi-program, which promotes aging and age-related diseases. PMID:26225961

  4. Genetic Screening of WNT4 and WNT5B in Two Populations with Deviating Bone Mineral Densities.

    PubMed

    Hendrickx, Gretl; Boudin, Eveline; Steenackers, Ellen; Nielsen, Torben Leo; Andersen, Marianne; Brixen, Kim; Van Hul, Wim

    2017-03-01

    A role for WNT4 and WNT5B in bone metabolism was indicated by genome-wide association studies (GWAS) and a Wnt4 knockout mouse model. The aim of this study was therefore to replicate and further investigate the causality between genetic variation in WNT4 and WNT5B and deviating bone mineral density (BMD) values. A WNT4 and WNT5B mutation screening was performed in patients with craniotubular hyperostosis using Sanger sequencing. Here, no putative causal mutations were detected. Moreover, a high and low BMD cohort was selected from the Odense Androgen Study population for re-sequencing. In WNT4 we detected four variants (three rare, one common), while in WNT5B we detected five variants (two rare, three common). For the common variants, no significant difference in genotype frequencies between the high and low BMD cohorts was observed. The SNPs associated with the GWAS were genotyped in these cohorts, but again no significant difference in genotype frequencies was observed. Despite the findings of the GWAS, we were not able to replicate or further verify the genetic association of polymorphisms in WNT4 and WNT5B with BMD. In order to do so, the intronic regions of both genes could be investigated more thoroughly in more extended populations (or extremes) with greater power. Future genetic and functional studies toward adjacent genes of WNT4 and WNT5B can also be interesting to figure out whether the signal from GWAS could possibly be attributed to genetic variation in these genes.

  5. Structural homologies and functional similarities between mammalian origins of replication and amplification promoting sequences.

    PubMed

    Stolzenburg, F; Gerwig, R; Dinkl, E; Grummt, F

    1994-06-01

    MuNTS2, a 423 bp sequence isolated from the non-transcribed spacer of murine rDNA stimulates the amplification of cis-linked plasmid DNA in mouse cells under selective conditions. Here we demonstrate that a 180 bp subdomain of muNTS2 is highly homologous (approximately 70%) to three domains of the first well-characterized origin of replication of mammalian chromosomes, i.e. the origin of bidirectional replication (OBR) of the dihydrofolate reductase (DHFR) locus in Chinese hamster ovary (CHO) cells. When subcloned, the 180 bp homology region of muNTS2 was revealed to be essential for the amplification promoting activity of muNTS2. Fragments of the initiation zone of DNA replication from the DHFR locus of hamster cells containing the domains of homology to the mouse muNTS2 element proved also to promote DNA amplification. Thus, the screening system for amplification promoting elements turned out to detect an origin of bidirectional replication.

  6. wnt4 Is associated with the development of ovarian tissue in the protandrous black Porgy, Acanthopagrus schlegeli.

    PubMed

    Wu, Guan-Chung; Chang, Ching-Fong

    2009-12-01

    The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle with sex differentiation at the juvenile stage, mono-male development, and male-to-female sex change (with vitellogenic oocytes) at age 3 yr. In the present study, we investigated the possible roles of wnt4 in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, ovarian growth, and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment had an effect on these processes. Furthermore, a natural sex change (> or =2-yr-old fish) and a nonchemical method to induce an early sex change (> or =1-yr-old fish) via the removal of testicular tissue were examined in this study. We present herein an integrative immunohistochemical, cellular, and molecular data set describing these phenomena. During gonadal sex differentiation, no increase in wnt4 expression was detected. A profile of increased wnt4 expression and decreased cyp19a1a expression was associated with ovarian growth (proliferation of oogonia and development of ovarian lamellae) in > or =1-yr-old fish. Both E2 and AI induced an increase in wnt4 transcripts and resulted in ovarian development in > or =0-yr-old and > or =1-yr-old fish. Increased wnt4 transcripts were found in ovarian tissue undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in > or =2-yr-old fish. Removal of testicular tissue in > or =1-yr-old fish resulted in successful early sex change (with vitellogenic oocytes) 6 mo after the excision. During the process of the early sex change (3 mo after testis excision), the fish ovary became active and had increased diameter of the primary oocytes; this was in accord with increased ovarian wnt4 expression but not sf1, foxl2, and genes in the steroidogenic pathway, including cyp19a1a. Wnt4 staining further confirmed

  7. Placental, Matrilineal, and Epigenetic Mechanisms Promoting Environmentally Adaptive Development of the Mammalian Brain

    PubMed Central

    Broad, Kevin D.; Rocha-Ferreira, Eridan; Hristova, Mariya

    2016-01-01

    The evolution of intrauterine development, vivipary, and placentation in eutherian mammals has introduced new possibilities and constraints in the regulation of neural plasticity and development which promote neural function that is adaptive to the environment that a developing brain is likely to encounter in the future. A range of evolutionary adaptations associated with placentation transfers disproportionate control of this process to the matriline, a period unique in mammalian development in that there are three matrilineal genomes interacting in the same organism at the same time (maternal, foetal, and postmeiotic oocytes). The interactions between the maternal and developing foetal hypothalamus and placenta can provide a template by which a mother can transmit potentially adaptive information concerning potential future environmental conditions to the developing brain. In conjunction with genomic imprinting, it also provides a template to integrate epigenetic information from both maternal and paternal lineages. Placentation also hands ultimate control of genomic imprinting and intergenerational epigenetic information transfer to the matriline as epigenetic markers undergo erasure and reprogramming in the developing oocyte. These developments, in conjunction with an expanded neocortex, provide a unique evolutionary template by which matrilineal transfer of maternal care, resources, and culture can be used to promote brain development and infant survival. PMID:27069693

  8. Human TMEM30a promotes uptake of antitumor and bioactive choline phospholipids into mammalian cells.

    PubMed

    Chen, Rui; Brady, Erin; McIntyre, Thomas M

    2011-03-01

    Antitumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. 1-O-Alkyl-2-carboxymethyl-sn-glycero-3-phosphocholine (Edelfosine) like other short-chained phospholipids--inflammatory platelet-activating factor (PAF) and apoptotic oxidatively truncated phospholipids--are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in a process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show that import of choline phospholipids into S. cerevisiae ΔLem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. Short hairpin RNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [(3)H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show that extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short chain choline phospholipids such as Edelfosine, PAF, and proapoptotic phospholipids.

  9. Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

    PubMed

    Ren, Jiafa; Li, Jianzhong; Feng, Ye; Shu, Bingyan; Gui, Yuan; Wei, Wei; He, Weichun; Yang, Junwei; Dai, Chunsun

    2017-08-01

    Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor(-/-) macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor(+/+) macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  10. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation

    PubMed Central

    Clowney, E. Josephine; Magklara, Angeliki; Colquitt, Bradley M.; Pathak, Nidhi; Lane, Robert P.; Lomvardas, Stavros

    2011-01-01

    The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of “genomic contrast” in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell. PMID:21705439

  11. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    PubMed Central

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-01-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

  12. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    NASA Astrophysics Data System (ADS)

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-06-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

  13. WNT4 acts downstream of BMP2 to mediate the regulation of ATRA signaling on RUNX1 expression: Implications for terminal differentiation of antler chondrocytes.

    PubMed

    Zhang, Hong-Liang; Yang, Zhan-Qing; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng; Guo, Bin

    2017-04-24

    Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1. © 2017 Wiley Periodicals, Inc.

  14. Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

    PubMed Central

    van Deursen, Diederik; Botma, Gert-Jan; Jansen, Hans; Verhoeven, Adrie JM

    2007-01-01

    Background Mammalian hepatic lipase (HL) genes are transcribed almost exclusively in hepatocytes. The basis for this liver-restricted expression is not completely understood. We hypothesized that the responsible cis-acting elements are conserved among mammalian HL genes. To identify these elements, we made a genomic comparison of 30 kb of 5'-flanking region of the rat, mouse, rhesus monkey, and human HL genes. The in silico data were verified by promoter-reporter assays in transfected hepatoma HepG2 and non-hepatoma HeLa cells using serial 5'-deletions of the rat HL (-2287/+9) and human HL (-685/+13) promoter region. Results Highly conserved elements were present at the proximal promoter region, and at 14 and 22 kb upstream of the transcriptional start site. Both of these upstream elements increased transcriptional activity of the human HL (-685/+13) promoter region 2–3 fold. Within the proximal HL promoter region, conserved clusters of transcription factor binding sites (TFBS) were identified at -240/-200 (module A), -80/-40 (module B), and -25/+5 (module C) by the rVista software. In HepG2 cells, modules B and C, but not module A, were important for basal transcription. Module B contains putative binding sites for hepatocyte nuclear factors HNF1α. In the presence of module B, transcription from the minimal HL promoter was increased 1.5–2 fold in HepG2 cells, but inhibited 2–4 fold in HeLa cells. Conclusion Our data demonstrate that searching for conserved non-coding sequences by comparative genomics is a valuable tool in identifying candidate enhancer elements. With this approach, we found two putative enhancer elements in the far upstream region of the HL gene. In addition, we obtained evidence that the -80/-40 region of the HL gene is responsible for enhanced HL promoter activity in hepatoma cells, and for silencing HL promoter activity in non-liver cells. PMID:17428321

  15. Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons

    PubMed Central

    Chen, Meifan; Geoffroy, Cédric G.; Wong, Hetty N.; Tress, Oliver; Nguyen, Mallorie T.; Holzman, Lawrence B.; Jin, Yishi; Zheng, Binhai

    2016-01-01

    Leucine Zipper-bearing Kinase (LZK/MAP3K13) is a member of the mixed lineage kinase family with high sequence identity to Dual Leucine Zipper Kinase (DLK/MAP3K12). While DLK is established as a key regulator of axonal responses to injury, the role of LZK in mammalian neurons is poorly understood. By gain- and loss-of-function analyses in neuronal cultures, we identify LZK as a novel positive regulator of axon growth. LZK signals specifically through MKK4 and JNKs among MAP2Ks and MAPKs respectively in neuronal cells, with JNK activity positively regulating LZK protein levels. Neuronal maturation or activity deprivation activates the LZK-MKK4-JNK pathway. LZK and DLK share commonalities in signaling, regulation, and effects on axon extension. Furthermore, LZK-dependent regulation of DLK protein expression and the lack of additive effects on axon growth upon co-manipulation suggest complex functional interaction and cross-regulation between these two kinases. Together, our data support the possibility for two structurally related MAP3Ks to work in concert to mediate axonal responses to external insult or injury in mammalian CNS neurons. PMID:27511108

  16. Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity.

    PubMed

    Sterkel, Alana K; Lorenzini, Jenna L; Fites, J Scott; Subramanian Vignesh, Kavitha; Sullivan, Thomas D; Wuthrich, Marcel; Brandhorst, Tristan; Hernandez-Santos, Nydiaris; Deepe, George S; Klein, Bruce S

    2016-03-09

    Systemic fungal infections trigger marked immune-regulatory disturbances, but the mechanisms are poorly understood. We report that the pathogenic yeast of Blastomyces dermatitidis elaborates dipeptidyl-peptidase IVA (DppIVA), a close mimic of the mammalian ectopeptidase CD26, which modulates critical aspects of hematopoiesis. We show that, like the mammalian enzyme, fungal DppIVA cleaved C-C chemokines and GM-CSF. Yeast producing DppIVA crippled the recruitment and differentiation of monocytes and prevented phagocyte activation and ROS production. Silencing fungal DppIVA gene expression curtailed virulence and restored recruitment of CCR2(+) monocytes, generation of TipDC, and phagocyte killing of yeast. Pharmacological blockade of DppIVA restored leukocyte effector functions and stemmed infection, while addition of recombinant DppIVA to gene-silenced yeast enabled them to evade leukocyte defense. Thus, fungal DppIVA mediates immune-regulatory disturbances that underlie invasive fungal disease. These findings reveal a form of molecular piracy by a broadly conserved aminopeptidase during disease pathogenesis.

  17. Metabolic preconditioning of mammalian cells: mimetic agents for hypoxia lack fidelity in promoting phosphorylation of pyruvate dehydrogenase.

    PubMed

    Borcar, Apurva; Menze, Michael A; Toner, Mehmet; Hand, Steven C

    2013-01-01

    Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl(2) does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser(293), Ser(300), and Ser(232), respectively) in HepG2 cells. This same effect is also observed for site 1 in mouse NIH/3T3 fibroblasts and J774 macrophages. CoCl(2) unexpectedly decreases the mRNA expression for PDH kinase-2 in HepG2 cells, which likely explains the dephosphorylation of PDH observed. And nor does desferrioxamine promote the expected increase in PDH phosphorylation. Dimethyloxaloylglycine (a prolyl hydroxylase inhibitor) performs better in this regard, but failed to promote the stronger effects seen with hypoxia. Consequently, CoCl(2) and desferrioxamine are unreliable mimics of hypoxia for physiological events downstream of HIF-1α stabilization. Our study demonstrates that mimetic chemicals must be chosen with caution and evaluated thoroughly if bona fide cellular outcomes are to be promoted with fidelity.

  18. The Schizosaccharomyces pombe open promoter bubble: mammalian-like arrangement and properties.

    PubMed

    Choi, Wai S; Lin, Yin C; Gralla, Jay D

    2004-07-23

    The fission yeast Schizosaccharomyces pombe is often used as a genetic system to model processes that apply to higher cells. Here S.pombe was used to study promoter DNA opening and transcription initiation by RNA polymerase II. The melted region within the adh promoter is about 20 bp in size and has the start site near its center. This arrangement is similar to that at the AdML promoter but different from that in Saccharomyces cerevisiae. Although expression of human TFIIB shifts the start site to the nearby human position, it does not change the location of the bubble. The start site shift is directed by the C terminus of human TFIIB, in contrast to expectations from S.cerevisiae. The creation of the bubble requires the ATPase motifs of XPB. Overall, the data show that promoter melting and initiation in fission yeast is much more similar to humans than to budding yeast.

  19. Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

    SciTech Connect

    Al-Dosari, Mohammed; Zhang Guisheng; Knapp, Joseph E.; Liu Dexi . E-mail: dliu@pitt.edu

    2006-01-13

    Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), {alpha}-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken {beta} actin (ACT), nuclear factor {kappa} B (NF{kappa}B), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.

  20. Short Promoters in Viral Vectors Drive Selective Expression in Mammalian Inhibitory Neurons, but do not Restrict Activity to Specific Inhibitory Cell-Types

    PubMed Central

    Nathanson, Jason L.; Jappelli, Roberto; Scheeff, Eric D.; Manning, Gerard; Obata, Kunihiko; Brenner, Sydney; Callaway, Edward M.

    2009-01-01

    Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown. PMID:19949461

  1. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers

    PubMed Central

    Core, Leighton J.; Martins, André L.; Danko, Charles G.; Waters, Colin; Siepel, Adam; Lis, John T.

    2014-01-01

    Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here, we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B-cell (GM12878) and chronic myelogenous leukemic (K562) tier 1, ENCODE cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses reveal a common architecture of initiation, including tightly spaced (110 bp) divergent initiation, similar frequencies of core-promoter sequence elements, highly positioned flanking nucleosomes, and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modifications, transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers. PMID:25383968

  2. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    PubMed Central

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  3. Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

    PubMed Central

    Wu, Jun; Roman, Angel-Carlos; Carvajal-Gonzalez, Jose Maria; Mlodzik, Marek

    2013-01-01

    Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled(Fz)/PCP factors and although their molecular interactions are beginning to be understood, the upstream input providing directional bias/polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with the extra-cellular domain of Fz acting as a ligand for Van-Gogh (Vang). We demonstrate in Drosophila wings that Wg and dWnt4 provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg/dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process. PMID:23912125

  4. Wnt4 inhibits β-catenin/TCF signalling by redirecting β-catenin to the cell membrane

    PubMed Central

    Bernard, Pascal; Fleming, Alice; Lacombe, Arnaud; Harley, Vincent R.; Vilain, Eric

    2009-01-01

    Background information During embryonic development, β-catenin is central both to the transcriptional activation of Wnt [wingless-type MMTV (murine-mammary-tumour virus) integration site family] target genes and as a mediator of cell–cell adhesion. Signals that regulate its levels and subcellular localization are critical. One mechanism of Wnt signalling results in stabilization of β-catenin protein, which leads to its translocation into the nucleus, where it interacts with TCF (T-cell factor, HMG box) and activates transcription of target genes. Less well understood are mechanisms of Wnt signalling that do not involve β-catenin stabilization and result in inhibition of β-catenin-mediated transcription. Results Here, we show that a member of the Wnt protein family, Wnt4 (Wnt, member 4), regulates the subcellular localization of β-catenin, redirecting it to the cell membrane. Unique among Wnts, this action does not affect the stability of β-catenin but does prohibit its involvement in TCF gene transactivation. Conclusions This novel mechanism suggests that Wnt4 acts as a switch between the two modes of β-catenin function, transcriptional activation and cell–cell adhesion. PMID:17976036

  5. Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila.

    PubMed

    Wu, Jun; Roman, Angel-Carlos; Carvajal-Gonzalez, Jose Maria; Mlodzik, Marek

    2013-09-01

    Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). We demonstrate in Drosophila melanogaster wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process.

  6. Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation and survival under hypoxia.

    PubMed

    Xiong, Weipeng; Knox, Aaron J; Xu, Mei; Kiseljak-Vassiliades, Katja; Colgan, Sean P; Brodsky, Kelley S; Kleinschmidt-Demasters, Bette K; Lillehei, Kevin O; Wierman, Margaret E

    2015-03-01

    The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LβT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target.

  7. GSK3β Inhibition Promotes Synaptogenesis in Drosophila and Mammalian Neurons

    PubMed Central

    Enriquez-Barreto, Lilian; Ferrús, Alberto; Morales, Miguel; Acebes, Ángel

    2015-01-01

    The PI3K-dependent activation of AKT results in the inhibition of GSK3β in most signaling pathways. These kinases regulate multiple neuronal processes including the control of synapse number as shown for Drosophila and rodents. Alzheimer disease’s patients exhibit high levels of circulating GSK3β and, consequently, pharmacological strategies based on GSK3β antagonists have been designed. The approach, however, has yielded inconclusive results so far. Here, we carried out a comparative study in Drosophila and rats addressing the role of GSK3β in synaptogenesis. In flies, the genetic inhibition of the shaggy-encoded GSK3β increases the number of synapses, while its upregulation leads to synapse loss. Likewise, in three weeks cultured rat hippocampal neurons, the pharmacological inhibition of GSK3β increases synapse density and Synapsin expression. However, experiments on younger cultures (12 days) yielded an opposite effect, a reduction of synapse density. This unexpected finding seems to unveil an age- and dosage-dependent differential response of mammalian neurons to the stimulation/inhibition of GSK3β, a feature that must be considered in the context of human adult neurogenesis and pharmacological treatments for Alzheimer’s disease based on GSK3β antagonists. PMID:25764078

  8. Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

    PubMed Central

    O’Keefe, Rachel A.; Blice-Baum, Anna; Gong, Xuan; Karaca, Esra

    2016-01-01

    Abstract The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes’ transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation. PMID:27957526

  9. Mammalian Ste20-Like Kinase 4 Promotes Pituitary Cell Proliferation and Survival Under Hypoxia

    PubMed Central

    Xiong, Weipeng; Knox, Aaron J.; Xu, Mei; Kiseljak-Vassiliades, Katja; Colgan, Sean P.; Brodsky, Kelley S.; Kleinschmidt-Demasters, Bette K.; Lillehei, Kevin O.

    2015-01-01

    The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LβT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target. PMID:25650755

  10. SNP-SNP interactions between WNT4 and WNT5A were associated with obesity related traits in Han Chinese Population

    PubMed Central

    Dong, Shan-Shan; Hu, Wei-Xin; Yang, Tie-Lin; Chen, Xiao-Feng; Yan, Han; Chen, Xiang-Ding; Tan, Li-Jun; Tian, Qing; Deng, Hong-Wen; Guo, Yan

    2017-01-01

    Considering the biological roles of WNT4 and WNT5A involved in adipogenesis, we aimed to investigate whether SNPs in WNT4 and WNT5A contribute to obesity related traits in Han Chinese population. Targeted genomic sequence for WNT4 and WNT5A was determined in 100 Han Chinese subjects and tag SNPs were selected. Both single SNP and SNP × SNP interaction association analyses with body mass index (BMI) were evaluated in the 100 subjects and another independent sample of 1,627 Han Chinese subjects. Meta-analyses were performed and multiple testing corrections were carried out using the Bonferroni method. Consistent with the Genetic Investigation of ANthropometric Traits (GIANT) dataset results, we didn’t detect significant association signals in single SNP association analyses. However, the interaction between rs2072920 and rs11918967, was associated with BMI after multiple testing corrections (combined P = 2.20 × 10−4). The signal was also significant in each contributing data set. SNP rs2072920 is located in the 3′-UTR of WNT4 and SNP rs11918967 is located in the intron of WNT5A. Functional annotation results revealed that both SNPs might be involved in transcriptional regulation of gene expression. Our results suggest that a combined effect of SNPs via WNT4-WNT5A interaction may affect the variation of BMI in Han Chinese population. PMID:28272483

  11. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation.

    PubMed

    Zhang, Yichi; Aguilar, Oscar A; Storey, Kenneth B

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser(319) and Thr(24), as well as p-Foxo3a Thr(32) decreased by at least 45% throughout torpor. MyoG was

  12. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation

    PubMed Central

    Zhang, Yichi; Aguilar, Oscar A.

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319 and Thr24, as well as p-Foxo3a Thr32 decreased by at least 45% throughout torpor. MyoG was upregulated only

  13. Transcriptional and structural impact of TATA-initiation site spacing in mammalian core promoters

    PubMed Central

    Ponjavic, Jasmina; Lenhard, Boris; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Sandelin, Albin

    2006-01-01

    Background The TATA box, one of the most well studied core promoter elements, is associated with induced, context-specific expression. The lack of precise transcription start site (TSS) locations linked with expression information has impeded genome-wide characterization of the interaction between TATA and the pre-initiation complex. Results Using a comprehensive set of 5.66 × 106 sequenced 5' cDNA ends from diverse tissues mapped to the mouse genome, we found that the TATA-TSS distance is correlated with the tissue specificity of the downstream transcript. To achieve tissue-specific regulation, the TATA box position relative to the TSS is constrained to a narrow window (-32 to -29), where positions -31 and -30 are the optimal positions for achieving high tissue specificity. Slightly larger spacings can be accommodated only when there is no optimally spaced initiation signal; in contrast, the TATA box like motifs found downstream of position -28 are generally nonfunctional. The strength of the TATA binding protein-DNA interaction plays a subordinate role to spacing in terms of tissue specificity. Furthermore, promoters with different TATA-TSS spacings have distinct features in terms of consensus sequence around the initiation site and distribution of alternative TSSs. Unexpectedly, promoters that have two dominant, consecutive TSSs are TATA depleted and have a novel GGG initiation site consensus. Conclusion In this report we present the most comprehensive characterization of TATA-TSS spacing and functionality to date. The coupling of spacing to tissue specificity at the transcriptome level provides important clues as to the function of core promoters and the choice of TSS by the pre-initiation complex. PMID:16916456

  14. Induction of mutation and differentiation in mammalian cells by chemicals which initiate or promote tumor formation

    SciTech Connect

    Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, cells with appropriate markers for mutagenesis, such as the Chinese hamster V79 cells, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons, nitrosamines and aflatoxins, and to establish means to study organ specificity of some chemical carcinogens. However, most short term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  15. Control of mammalian cell mutagenesis and differentiation by chemicals which initiate or promote tumor formation

    SciTech Connect

    Jones, C. A.; Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, Chinese hamster V79 cells, which are susceptible to mutagenesis, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons and nitrosamines and to study the organ specificity exhibited by some chemical carcinogens. However, most short-term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  16. Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells.

    PubMed

    White, M R; Masuko, M; Amet, L; Elliott, G; Braddock, M; Kingsman, A J; Kingsman, S M

    1995-02-01

    The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.

  17. Promoter Decommissioning by the NuRD Chromatin Remodeling Complex Triggers Synaptic Connectivity in the Mammalian Brain

    PubMed Central

    Yamada, Tomoko; Yang, Yue; Hemberg, Martin; Yoshida, Toshimi; Cho, Ha Young; Murphy, J. Patrick; Fioravante, Diasynou; Regehr, Wade G.; Gygi, Steven P.; Georgopoulos, Katia; Bonni, Azad

    2014-01-01

    SUMMARY Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-Seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally-regulated programming mechanism that drives synaptic connectivity in the mammalian brain. PMID:24991957

  18. Promoter decommissioning by the NuRD chromatin remodeling complex triggers synaptic connectivity in the mammalian brain.

    PubMed

    Yamada, Tomoko; Yang, Yue; Hemberg, Martin; Yoshida, Toshimi; Cho, Ha Young; Murphy, J Patrick; Fioravante, Diasynou; Regehr, Wade G; Gygi, Steven P; Georgopoulos, Katia; Bonni, Azad

    2014-07-02

    Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Canonical Poly(A) Polymerase Activity Promotes the Decay of a Wide Variety of Mammalian Nuclear RNAs.

    PubMed

    Bresson, Stefan M; Hunter, Olga V; Hunter, Allyson C; Conrad, Nicholas K

    2015-10-01

    The human nuclear poly(A)-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs). In addition, PABPN1 promotes hyperadenylation by stimulating poly(A)-polymerases (PAPα/γ), but this activity has not previously been linked to the decay of endogenous transcripts. Moreover, the mechanisms underlying target specificity have remained elusive. Here, we inactivated PAP-dependent hyperadenylation in cells by two independent mechanisms and used an RNA-seq approach to identify endogenous targets. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and promoter upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD). Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. Additional investigation showed that a genetically-encoded poly(A) tail is sufficient to drive decay, suggesting that degradation occurs independently of the canonical cleavage and polyadenylation reaction. Surprisingly, treatment with transcription inhibitors uncouples polyadenylation from decay, leading to runaway hyperadenylation of nuclear decay targets. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts.

  20. Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures

    PubMed Central

    Santos, Anderson K.; Parreira, Ricardo C.; Resende, Rodrigo R.

    2016-01-01

    Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology. PMID:27582737

  1. Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells1

    PubMed Central

    Chen, Rui; Brady, Erin; McIntyre, Thomas M.

    2010-01-01

    Anti-tumor alkylphospholipids initiate apoptosis in transformed HL-60 and Jurkat cells while sparing their progenitors. Edelfosine like other short-chained phospholipids—inflammatory Platelet-activating Factor (PAF) and apoptotic oxidatively-truncated phospholipids—are proposed to have intracellular sites of action, yet a conduit for these choline phospholipids into mammalian cells is undefined. Edelfosine is also accumulated by Saccharomyces cerevisiae in process requiring the membrane protein Lem3p, and the human genome contains a Lem3p homolog TMEM30a. We show import of choline phospholipids into S. cerevisiae ⊗Lem3 is partially reconstituted by human TMEM30a and by Lem3p-TMEM30a chimeras, showing the proteins are orthologous. TMEM30a-GFP chimeras expressed in mammalian cells localized in plasma membranes, as well as internal organelles, and ectopic TMEM30a expression promoted uptake of exogenous choline and ethanolamine phospholipids. shRNA knockdown of TMEM30a reduced fluorescent choline phospholipid and [3H]PAF import. This knockdown also reduced mitochondrial depolarization from exogenous Edelfosine or the mitotoxic oxidatively truncated phospholipid azelaoyl phosphatidylcholine, and the knockdown reduced apoptosis in response to these two phospholipids. These results show extracellular choline phospholipids with short sn-2 residues can have intracellular roles and sites of metabolism because they are transport substrates for a TMEM30a phospholipid import system. Variation in this mechanism could limit sensitivity to short-chain choline phospholipids such as Edelfosine, PAF, and pro-apoptotic phospholipids. PMID:21289302

  2. ATRA Signaling Regulates the Expression of COL9A1 through BMP2-WNT4-RUNX1 Pathway in Antler Chondrocytes.

    PubMed

    Zhang, Hong-Liang; Guo, Bin; Yang, Zhan-Qing; Duan, Cui-Cui; Geng, Shuang; Wang, Kai; Yu, Hai-Fan; Yue, Zhan-Peng

    2017-09-01

    Although all-trans retinoic acid (ATRA) is involved in the regulation of cartilage growth and development, its regulatory mechanisms remain unknown. Here, we showed that ATRA could induce the expression of COL9A1 in antler chondrocytes. Silencing of cellular retinoic acid binding protein 2 (CRABP2) could impede the ATRA-induced upregulation of COL9A1, whereas overexpression of CRABP2 presented the opposite effect. RARα agonist Am80 induced the expression of COL9A1, whereas treatment with RARα antagonist Ro 41-5253 or RXRα small-interfering RNA (siRNA) caused an obvious blockage of ATRA on COL9A1. In antler chondrocytes, CYP26A1 and CYP26B1 weakened the sensitivity of ATRA to COL9A1. Simultaneously, Bone morphogenetic protein 2 (BMP2) and WNT4 mediated the regulation of ATRA on COL9A1 expression. Knockdown of WNT4 could abrogate the inhibitory effect of BMP2 overexpression on COL9A1. Conversely, constitutive expression of WNT4 reversed the upregulation of COL9A1 elicited by BMP2 siRNA. Together these data indicated that WNT4 might act downstream of BMP2 to mediate the effect of ATRA on COL9A1 expression. Further analysis evidenced that attenuation of runt-related transcription factor 1 (RUNX1) could prevent the stimulation of ATRA on COL9A1 expression, while exogenous rRUNX1 further enhanced this effectiveness. Moreover, RUNX1 might serve as an intermediate to mediate the regulation of BMP2 and WNT4 on COL9A1 expression. Collectively, ATRA signaling might regulate the expression of COL9A1 through BMP2-WNT4-RUNX1 pathway. © 2017 Wiley Periodicals, Inc.

  3. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  4. Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

    PubMed

    Lyulcheva, Ekaterina; Taylor, Eleanor; Michael, Magdalene; Vehlow, Anne; Tan, Shengjiang; Fletcher, Adam; Krause, Matthias; Bennett, Daimark

    2008-11-01

    MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

  5. Isolation and characterization of mammalian cells expressing the Arf promoter during eye development.

    PubMed

    Iqbal, Nida S; Xu, Lin; Devitt, Caitlin C; Skapek, Stephen X

    2014-05-01

    Although many researchers have successfully uncovered novel functions of the tumor suppressor p19(Arf) utilizing various types of cultured cancer cells and immortalized fibroblasts, these systems do not accurately reflect the endogenous environment in which Arf is developmentally expressed. We addressed this by isolating perivascular cells (PVCs) from the primary vitreous of the mouse eye. This rare cell type normally expresses the p19(Arf) tumor suppressor in a non-pathological, developmental context. We utilized fluorescence activated cell sorting (FACS) to purify the cells by virtue of a GFP reporter driven by the native Arf promoter and then characterized their morphology and gene expression pattern. We further examined the effects of reintroduction of Arf expression in the Arf(GFP/GFP) PVCs to verify expected downstream effectors of p19(Arf) as well as uncover novel functions of Arf as a regulator of vasculogenesis. This methodology and cell culture model should serve as a useful tool to examine p19(Arf) biology.

  6. Role of fertilization promoting peptide (FPP) in modulating mammalian sperm function.

    PubMed

    Fraser, L R

    1998-12-01

    Fertilization promoting peptide (FPP), structurally similar to thyrotrophin releasing hormone, is produced by the prostate gland and secreted into seminal plasma. Recent in vitro studies have provided evidence that FPP elicits biologically relevant responses in mouse, human and boar spermatozoa. In the presence of nanomolar concentrations of FPP, spermatozoa become fertilizing more quickly and then are inhibited from undergoing spontaneous acrosome loss, an event that would make them non-fertilizing. In vivo, these responses would be very important in maximizing the availability of potentially fertilizing spermatozoa. Adenosine, which can elicit the same responses as FPP, is known to modulate the adenylyl cyclase(AC)/cAMP signal transduction pathway; current evidence indicates that FPP and adenosine act via separate receptors on the same signal transduction pathway. Mouse spermatozoa are known to have adenosine receptors and a putative receptor for FPP (TCP-11) has been identified. Unlike many surface receptors, TCP-11 has no obvious transmembrane regions whereby modulation of AC could occur. Recent evidence suggests that FPP receptors may dimerize with adenosine receptors to activate the signaling pathway, with stimulatory adenosine receptors involved in the stimulation of capacitation, but inhibitory receptors involved in inhibition of spontaneous acrosome loss. These results indicate that FPP plays an important role in normal sperm function and that it might be used in new therapeutic strategies designed to alleviate some causes of sperm dysfunction.

  7. Crumbs 3b promotes tight junctions in an ezrin-dependent manner in mammalian cells

    PubMed Central

    Tilston-Lünel, Andrew M.; Haley, Kathryn E.; Schlecht, Nicolas F.; Wang, Yanhua; Chatterton, Abigail L.D.; Moleirinho, Susana; Watson, Ailsa; Hundal, Harinder S.; Prystowsky, Michael B.; Gunn-Moore, Frank J.; Reynolds, Paul A.

    2016-01-01

    Crumbs 3 (CRB3) is a component of epithelial junctions, which has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion, and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, we describe novel data demonstrating that, as with previous studies on CRB3a, CRB3b also promotes the formation of tight junctions (TJs). However, significantly we demonstrate that the 4.1-ezrin–radixin–moesin-binding motif of CRB3b is required for CRB3b functionality and that ezrin binds to the FBM of CRB3b. Furthermore, we show that ezrin contributes to CRB3b functionality and the correct distribution of TJ proteins. We demonstrate that both CRB3 isoforms are required for the production of functionally mature TJs and also the localization of ezrin to the plasma membrane. Finally, we demonstrate that reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and shows evidence that while CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC. PMID:27190314

  8. CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons

    PubMed Central

    Farina, Margherita; van de Bospoort, Rhea; He, Enqi; Persoon, Claudia M; van Weering, Jan RT; Broeke, Jurjen H; Verhage, Matthijs; Toonen, Ruud F

    2015-01-01

    Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role. DOI: http://dx.doi.org/10.7554/eLife.05438.001 PMID:25719439

  9. CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons.

    PubMed

    Farina, Margherita; van de Bospoort, Rhea; He, Enqi; Persoon, Claudia M; van Weering, Jan R T; Broeke, Jurjen H; Verhage, Matthijs; Toonen, Ruud F

    2015-02-26

    Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role.

  10. Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation.

    PubMed

    Rebl, Alexander; Rebl, Henrike; Liu, Shuzhen; Goldammer, Tom; Seyfert, Hans-Martin

    2011-01-01

    Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Sry and SoxE genes: How they participate in mammalian sex determination and gonadal development?

    PubMed

    She, Zhen-Yu; Yang, Wan-Xi

    2017-03-01

    In mammals, sex determination defines the differentiation of the bipotential genital ridge into either testes or ovaries. Sry, the mammalian Y-chromosomal testis-determining gene, is a master regulator of male sex determination. It acts to switch the undifferentiated genital ridge towards testis development, triggering the adoption of a male fate. Sry initiates a cascade of gene networks through the direct regulation of Sox9 expression and promotes supporting cell differentiation, Leydig cell specification, vasculature formation and testis cord development. In the absence of Sry, alternative genetic cascades, including female sex-determining genes RSPO1, Wnt4/β-catenin and Foxl2, are involved in the formation of female genitalia and the maintenance of female ovarian development. The mutual antagonisms between male and female sex-determining pathways are crucial in not just the initiation but also the maintenance of the somatic sex of the gonad throughout the organism's lifetime. Any imbalances in above sex-determining genes can cause disorders of sex development in humans and mice. In this review, we provide a detailed summary of the expression profiles, biochemical properties and developmental functions of Sry and SoxE genes in embryonic testis development and adult gonadal development. We also briefly summarize the dedicate balances between male and female sex-determining genes in mammalian sex development, with particular highlights on the molecular actions of Sry and Sox9 transcription factors.

  12. Search for the sex-determining switch in monotremes: mapping WT1, SF1, LHX1, LHX2, FGF9, WNT4, RSPO1 and GATA4 in platypus.

    PubMed

    Grafodatskaya, Daria; Rens, Willem; Wallis, Mary C; Trifonov, Vladimir; O'Brien, Patricia C M; Clarke, Oliver; Graves, Jennifer A M; Ferguson-Smith, Malcolm A

    2007-01-01

    The duck-billed platypus has five pairs of sex chromosomes, but there is no information about the primary sex-determining switch in this species. As there is no apparent SRY orthologue in platypus, another gene must acquire the function of a key regulator of the gonadal male or female fate. SOX9 was ruled out from being this key regulator as it maps to an autosome in platypus. To check whether other genes in mammalian gonadogenesis could be the primary switch in monotremes, we have mapped a number of candidates in platypus. We report here the autosomal location of WT1, SF1, LHX1, LHX9, FGF9, WNT4 and RSPO1 in platypus, thus excluding these from being key regulators of sex determination in this species. We found that GATA4 maps to sex chromosomes Y1 and X2; however, it lies in the pairing region shown by chromosome painting to be homologous, so is unlikely to be either male-specific or differentially dosed in male and female.

  13. Conserved POU/OCT- and GATA-binding sites in 5'-flanking promoter region of mammalian WNT8B orthologs.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-05-01

    WNT family members are secreted-type glycoproteins regulating cell fate, planar cell polarity, cell adhesion, and cell movement. WNT signals are context-dependently transduced to the canonical pathway for the transcriptional up-regulation of MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, and also to the non-canonical pathway for the activation of RHOA, JNK, PKC, NFAT and NLK signaling cascades. We cloned and characterized the wild-type human WNT8B, while another group the aberrant human WNT8B with Gly230Ala and Arg284Leu amino-acid substitutions. Although WNT8B is undetectable in normal adult tissues by using Northern blot analyses, WNT8B is expressed in gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, and embryonal tumors. Here, comparative integromics on WNT8B orthologs were investigated by using bioinformatics (Techint) and human intelligence (Humint). Cow Wnt8b gene was identified within NW_001494361.1 genome sequence. Predicted sequence XM_582222.3 was an artificial cow Wnt8b with aberrant prediction for the first exon. Cow Wnt8b complete coding sequence was found to encode a 350-amino-acid protein, which showed 96.9% total-amino-acid identity with human WNT8B. Comparative proteomics revealed that N-terminal signal peptide, 22 Cys residues, two Asn-linked glycosylation sites, Gly230, and Arg284 of human WNT8B were conserved among mammalian WNT8B orthologs. Comparative genomics revealed that POU/OCT- and GATA-binding sites in the 5'-flanking promoter region were conserved among human, chimpanzee, cow, mouse, and rat WNT8B orthologs. In silico expression analyses revealed that human WNT8B was expressed in embryoid body derived from embryonic stem (ES) cells, hepatocyte progenitors derived from ES cells, fetal brain, diffuse-type gastric cancer, colorectal cancer, prostate cancer, and ovarian fibrotheoma. Based on the expression profiles of POU and GATA family transcription factors, it was revealed that WNT8B expression in hepatocyte

  14. Chemical Modification of Reactive Multilayered Films Fabricated from Poly(2-Alkenyl Azlactone)s: Design of Surfaces that Prevent or Promote Mammalian Cell Adhesion and Bacterial Biofilm Growth

    PubMed Central

    Buck, Maren E.; Breitbach, Anthony S.; Belgrade, Sonja K.; Blackwell, Helen E.; Lynn, David M.

    2009-01-01

    We report an approach to the design of reactive polymer films that can be functionalized post-fabrication to either prevent or promote the attachment and growth of cells. Our approach is based on the reactive layer-by-layer assembly of covalently crosslinked thin films using a synthetic polyamine and a polymer containing reactive azlactone functionality. Our results demonstrate (i) that the residual azlactone functionality in these films can be exploited to immobilize amine-functionalized chemical motifs similar to those that promote or prevent cell and protein adhesion when assembled as self-assembled monolayers on gold-coated surfaces, and (ii) that the immobilization of these motifs changes significantly the behaviors and interactions of cells with the surfaces of these polymer films. We demonstrate that films treated with the hydrophobic molecule decylamine support the attachment and growth of mammalian cells in vitro. In contrast, films treated with the hydrophilic carbohydrate D-glucamine prevent cell adhesion and growth almost completely. The results of additional experiments suggest that these large differences in cell behavior can be understood, at least in part, in terms of differences in the abilities of these two different chemical motifs to promote or prevent the adsorption of protein onto film coated surfaces. We demonstrate further that this approach can be used to pattern regions of these reactive films that resist the initial attachment and subsequent invasion of mammalian cells for periods of at least one month in the presence of serum-containing cell culture media. Finally, we report that films that prevent the adhesion and growth of mammalian cells also prevent the initial formation of bacterial biofilms when incubated in the presence of the clinically relevant pathogen Pseudomonas aeruginosa. The results of these studies, collectively, suggest the basis of general approaches to the fabrication and functionalization of thin films that prevent

  15. A possibility of nutriceuticals as an anti-aging intervention: Activation of sirtuins by promoting mammalian NAD biosynthesis

    PubMed Central

    Imai, Shin-ichiro

    2010-01-01

    Aging science has recently drawn much attention, and discussions on the possibility of anti-aging medicine have multiplied. One potential target for the development of anti-aging drugs is the SIR2 (silent information regulator 2) family of NAD-dependent deacetylases/ADP-ribosyltransferases, called “sirtuins.” Sirtuins regulate many fundamental biological processes in response to a variety of environmental and nutritional stimuli. In mammals, the mammalian SIR2 ortholog SIRT1 has been most studied, and small molecule SIRT1 activators (STACs), including a plant-derived polyphenolic compound resveratrol, have been developed. On the other hand, sirtuin activity is regulated by NAD biosynthetic pathways, and nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in the regulation of mammalian sirtuin activity. Recent studies have provided a proof of concept for the idea that nicotinamide mononucleotide (NMN), the NAMPT reaction product, can be used a nutriceutical to activate SIRT1 activity. Based on these recent findings, the possibility of sirtuin-targeted nutriceutical development will be discussed. PMID:20085812

  16. 1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells.

    PubMed

    Soares, Chrislaine O; Colli, Walter; Bechara, Etelvino J H; Alves, Maria Julia M

    2012-12-15

    The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 μM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 μM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.

  17. MicroRNA-24 Attenuates Neointimal Hyperplasia in the Diabetic Rat Carotid Artery Injury Model by Inhibiting Wnt4 Signaling Pathway.

    PubMed

    Yang, Jian; Fan, Zhixing; Yang, Jun; Ding, Jiawang; Yang, Chaojun; Chen, Lihua

    2016-05-24

    The long-term stimulation of hyperglycemia greatly increases the incidence of vascular restenosis (RS) after angioplasty. Neointimal hyperplasia after vascular injury is the pathological cause of RS, but its mechanism has not been elucidated. MicroRNA-24 (miR-24) has low expression in the injured carotid arteries of diabetic rats. However, the role of miR-24 in the vascular system is unknown. In this study, we explore whether over-expression of miR-24 could attenuate neointimal formation in streptozotocin (STZ)-induced diabetic rats. Adenovirus (Ad-miR-24-GFP) was used to deliver the miR-24 gene to injured carotid arteries in diabetic rats. The level of neointimal hyperplasia was examined by hematoxylin-eosin (HE) staining. Vascular smooth muscle cell (VSMC) proliferation in the neointima was evaluated by immunostaining for proliferating cell nuclear antigen (PCNA). The mRNA levels of miR-24, PCNA, wingless-type MMTV integration site family member 4 (Wnt4), disheveled-1 (Dvl-1), β-catenin and cell cycle-associated molecules (Cyclin D1, p21) were determined by Quantitative Real-Time PCR (qRT-PCR). PCNA, Wnt4, Dvl-1, β-catenin, Cyclin D1 and p21 protein levels were measured by Western blotting analysis. STZ administration decreased plasma insulin and increased fasting blood glucose in Sprague-Dawley (SD) rats. The expression of miR-24 was decreased in the carotid artery after a balloon injury in diabetic rats, and adenoviral transfection (Ad-miR-24-GFP) increased the expression of miR-24. Over-expression of miR-24 suppressed VSMC proliferation and neointimal hyperplasia in diabetic rats at 14 days. Furthermore, compared with Sham group, the mRNA and protein levels of PCNA, Wnt4, Dvl-1, β-catenin, and Cyclin D1 were strikingly up-regulated in the carotid arteries of diabetic rats after a balloon injury. Interestingly, up-regulation of miR-24 significantly reduced the mRNA and protein levels of these above molecules. In contrast, the change trend in p21 mRNA and

  18. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells

    PubMed Central

    Mantsoki, Anna; Devailly, Guillaume; Joshi, Anagha

    2015-01-01

    In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a ‘poised’ state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. PMID:26582124

  19. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells.

    PubMed

    Mantsoki, Anna; Devailly, Guillaume; Joshi, Anagha

    2015-11-19

    In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a 'poised' state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a 'TCCCC' sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development.

  20. Macrolide- and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives.

    PubMed

    Malphettes, Laetitia; Fussenegger, Martin

    2004-11-20

    RNA interference has emerged as a powerful technology for downregulation of specific genes in cells and animals. We have pioneered macrolide- and tetracycline-adjustable short interfering RNA (siRNA) expression for conditional target gene translation fine-tuning in mammalian/human cell lines based on modified RNA polymerase II promoters. Established macrolide- and tetracycline-dependent transactivators/trans-silencers bound and activated modified target promoters tailored for optimal siRNA expression in response to clinical antibiotics' dosing regimes and modulated desired target genes in Chinese hamster ovary (CHO-K1) and human fibrosarcoma (HT-1080) cells with high precision. Further optimization of adjustable RNA polymerase II-based siRNA-specific promoters as well as their combination with various transmodulators enabled near-perfect regulation configurations in specific cell types. Devoid of major genetic constraints compared to basic RNA polymerase III-based siRNA-specific promoters, we expect RNA polymerase II counterparts to significantly advance siRNA-based molecular interventions in biopharmaceutical manufacturing and gene-function analysis as well as gene therapy and tissue engineering.

  1. Do Sirtuins Promote Mammalian Longevity?: A Critical Review on Its Relevance to the Longevity Effect Induced by Calorie Restriction

    PubMed Central

    Park, Seongjoon; Mori, Ryoichi; Shimokawa, Isao

    2013-01-01

    Sirtuins (SIRTs), a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, are emerging as key molecules that regulate aging and age-related diseases including cancers, metabolic disorders, and neurodegenerative diseases. Seven isoforms of SIRT (SIRT1–7) have been identified in mammals. SIRT1 and 6, mainly localized in the nucleus, regulate transcription of genes and DNA repair. SIRT3 in the mitochondria regulates mitochondrial bioenergetics. Initial studies in yeasts, nematodes, and flies indicated a strong connection of SIRT with the life-prolonging effects of calorie restriction (CR), a robust experimental intervention for longevity in a range of organisms. However, subsequent studies reported controversial findings regarding SIRT roles in the effect of CR. This review describes the functional roles of mammalian SIRTs and discusses their relevance to mechanisms underlying the longevity effect of CR. PMID:23661364

  2. The Planar Cell Polarity Transmembrane Protein Vangl2 Promotes Dendrite, Spine and Glutamatergic Synapse Formation in the Mammalian Forebrain

    PubMed Central

    Okerlund, Nathan D.; Stanley, Robert E.; Cheyette, Benjamin N.R.

    2016-01-01

    The transmembrane protein Vangl2, a key regulator of the Wnt/planar cell polarity (PCP) pathway, is involved in dendrite arbor elaboration, dendritic spine formation and glutamatergic synapse formation in mammalian central nervous system neurons. Cultured forebrain neurons from Vangl2 knockout mice have simpler dendrite arbors, fewer total spines, less mature spines and fewer glutamatergic synapse inputs on their dendrites than control neurons. Neurons from mice heterozygous for a semidominant Vangl2 mutation have similar but not identical phenotypes, and these phenotypes are also observed in Golgi-stained brain tissue from adult mutant mice. Given increasing evidence linking psychiatric pathophysiology to these subneuronal sites and structures, our findings underscore the relevance of core PCP proteins including Vangl2 to the underlying biology of major mental illnesses and their treatment. PMID:27606324

  3. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    PubMed Central

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  4. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    SciTech Connect

    Wang, Shengchao; Kawashima, Nobuyuki; Sakamoto, Kei; Katsube, Ken-ichi; Umezawa, Akihiro; Suda, Hideaki

    2010-09-10

    Research highlights: {yields} High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. {yields} Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. {yields} Rbpj upregulated promoter activities of Runx2 and Ose2. {yields} Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  5. Repeated Recruitment of LTR Retrotransposons as Promoters by the Anti-Apoptotic Locus NAIP during Mammalian Evolution

    PubMed Central

    Romanish, Mark T; Lock, Wynne M; van de Lagemaat, Louie N.; Dunn, Catherine A; Mager, Dixie L

    2007-01-01

    Neuronal apoptosis inhibitory protein (NAIP, also known as BIRC1) is a member of the conserved inhibitor of apoptosis protein (IAP) family. Lineage-specific rearrangements and expansions of this locus have yielded different copy numbers among primates and rodents, with human retaining a single functional copy and mouse possessing several copies, depending on the strain. Roles for this gene in disease have been documented, but little is known about transcriptional regulation of NAIP. We show here that NAIP has multiple promoters sharing no similarity between human and rodents. Moreover, we demonstrate that multiple, domesticated long terminal repeats (LTRs) of endogenous retroviral elements provide NAIP promoter function in human, mouse, and rat. In human, an LTR serves as a tissue-specific promoter, active primarily in testis. However, in rodents, our evidence indicates that an ancestral LTR common to all rodent genes is the major, constitutive promoter for these genes, and that a second LTR found in two of the mouse genes is a minor promoter. Thus, independently acquired LTRs have assumed regulatory roles for orthologous genes, a remarkable evolutionary scenario. We also demonstrate that 5′ flanking regions of IAP family genes as a group, in both human and mouse are enriched for LTR insertions compared to average genes. We propose several potential explanations for these findings, including a hypothesis that recruitment of LTRs near NAIP or other IAP genes may represent a host-cell adaptation to modulate apoptotic responses. PMID:17222062

  6. CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1

    PubMed Central

    Tong, Xin; Zhang, Deqiang; Guha, Anirvan; Arthurs, Blake; Cazares, Victor; Gupta, Neil; Yin, Lei

    2015-01-01

    The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1. PMID:26431207

  7. Evolution of CpG island promoter function underlies changes in KChIP2 potassium channel subunit gene expression in mammalian heart.

    PubMed

    Yan, Qinghong; Masson, Rajeev; Ren, Yi; Rosati, Barbara; McKinnon, David

    2012-01-31

    Scaling of cardiac electrophysiology with body mass requires large changes in the ventricular action potential duration and heart rate in mammals. These changes in cellular electrophysiological function are produced by systematic and coordinated changes in the expression of multiple ion channel and transporter genes. Expression of one important potassium current, the transient outward current (I(to)), changes significantly during mammalian evolution. Changes in I(to) expression are determined, in part, by variation in the expression of an obligatory auxiliary subunit encoded by the KChIP2 gene. The KChIP2 gene is expressed in both cardiac myocytes and neurons and transcription in both cell types is initiated from the same CpG island promoter. Species-dependent variation of KChIP2 expression in heart is mediated by the evolution of the cis-regulatory function of this gene. Surprisingly, the major locus of evolutionary change for KChIP2 gene expression in heart lies within the CpG island core promoter. The results demonstrate that CpG island promoters are not simply permissive for gene expression but can also contribute to tissue-selective expression and, as such, can function as an important locus for the evolution of cis-regulatory function. More generally, evolution of the cis-regulatory function of voltage-gated ion channel genes appears to be an effective and efficient way to modify channel expression levels to optimize electrophysiological function.

  8. Wnt8a and Wnt3a Cooperate in the Axial Stem Cell Niche to Promote Mammalian Body Axis Extension

    PubMed Central

    Cunningham, Thomas J.; Kumar, Sandeep; Yamaguchi, Terry P.; Duester, Gregg

    2015-01-01

    Background Vertebrate body axis extension occurs in a head-to-tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/β-catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a. Results We examined mouse Wnt8a−/− and Wnt3a−/− single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 upregulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to establish its expression boundaries and possesses an upstream RA response element that binds RA receptors. Conclusions These findings provide new insight into interaction of caudal Wnt-FGF-RA signals required for body axis extension. PMID:25809880

  9. Analysis of hairless corepressor mutants to characterize molecular cooperation with the vitamin D receptor in promoting the mammalian hair cycle.

    PubMed

    Hsieh, Jui-Cheng; Slater, Stephanie A; Whitfield, G Kerr; Dawson, Jamie L; Hsieh, Grace; Sheedy, Craig; Haussler, Carol A; Haussler, Mark R

    2010-06-01

    The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion DeltaAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M, and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G, and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N, and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109-111, 134-201, and 202-303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle. (c) 2010 Wiley-Liss, Inc.

  10. Analysis of Hairless Corepressor Mutants to Characterize Molecular Cooperation with the Vitamin D Receptor in Promoting the Mammalian Hair Cycle

    PubMed Central

    Hsieh, Jui-Cheng; Slater, Stephanie A.; Whitfield, G. Kerr; Dawson, Jamie L.; Hsieh, Grace; Sheedy, Craig; Haussler, Carol A.; Haussler, Mark R.

    2010-01-01

    The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion ΔAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109–111, 134–201, and 202–303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle. PMID:20512927

  11. Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP.

    PubMed

    Wickramasinghe, Vihandha O; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B; Stewart, Murray; Venkitaraman, Ashok R; Laskey, Ronald A

    2014-04-01

    The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.

  12. Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

    PubMed Central

    Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

    2014-01-01

    The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

  13. Molecular cloning, characterization and expression analysis of Wnt4, Wnt5, Wnt6, Wnt7, Wnt10 and Wnt16 from Litopenaeus vannamei.

    PubMed

    Zhang, Shuang; Li, Chao-Zheng; Yang, Qi-Hui; Dong, Xiao-Hui; Chi, Shu-Yan; Liu, Hong-Yu; Shi, Li-Li; Tan, Bei-Ping

    2016-07-01

    The Wnt (Wg-type MMTV integration site) signaling represents as the negative regulator of virus-induced innate immune responses. Wnt genes act as ligands to activate the Wnt signaling. To know more about the information of Wnt genes in invertebrates, Litopenaeus vannamei Wnt genes (LvWnts) were identified and characterized. In this study, Six Wnt genes (LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16) were obtained in L. vannamei. The complete cDNAs open reading frames (ORF) of LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16 were 1077 bp, 1107 bp, 1350 bp, 1047 bp, 1509 bp and 1158 bp (GenBank accession no. KU169896, KU169897, KU169898, KU169899, KU169900 and KU169901), encoding 358, 368, 449, 348, 502 and 385 amino acid (aa) residues respectively. All the six members of LvWnts contain a Wnt1 domain, which is considered as an important feature of Wnt gene family. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was more than 48% for all the LvWnts except LvWnt10 (36-41%). The phylogenetic relationship analysis illustrated that different subtype of Wnts formed their own separate branches and were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvWnts were confirmed by RT-PCR in all the examined five developmental stages and eleven tissues of L. vannamei with different express patterns. LvWnt4, LvWnt5 and LvWnt10 were expressed highest in nerve while LvWnt6, LvWnt7 and LvWnt16 were expressed highest in intestine, stomach and gill, respectively. In addition, all the LvWnts were regulated by white spot syndrome virus (WSSV) challenges at different levels in hepatopancreas, gill and hemocytes, suggesting that Wnt genes may play a role in the defense against pathogenic virus infection in innate immune of L. vannamei.

  14. Expression of Rickettsia Adr2 protein in E. coli is sufficient to promote resistance to complement-mediated killing, but not adherence to mammalian cells.

    PubMed

    Garza, Daniel A; Riley, Sean P; Martinez, Juan J

    2017-01-01

    Bacteria exposed to host serum are subject to the antibacterial effects to the complement system. However, pathogenic microorganisms have evolved mechanisms of evading this immune attack. We have previously demonstrated that at least two R. conorii antigens, RC1281/Adr1 and OmpB β-peptide, contribute to the evasion of complement-mediated killing by binding the complement regulatory proteins vitronectin and factor H. RC1282/Adr2, a protein related to Adr1, is predicted to share similar structural features, suggesting that this protein may also contribute to evasion of complement-mediated killing. Interestingly, the R. prowazekii Adr1 and Adr2(RP828) proteins were originally found to interact with host cell surface proteins, suggesting their putative roles as adhesins in this pathogenic rickettsial species. In this study, we expressed both R. conorii and R. prowazekii Adr2 on the surface of a non-adherent, serum-sensitive strain of E. coli to examine the potential role of this protein to mediate evasion of complement-mediated killing and adherence to host cells. We demonstrate that, similar to R. conorii Adr1, R. conorii and R. prowazekii Adr2 are sufficient to mediate serum resistance and to promote interaction with the host complement regulator vitronectin. Furthermore, we demonstrate that expression of Adr2 in a non-adherent strain of E. coli is insufficient to mediate adherence to cultured mammalian endothelial cells. Together, our data demonstrate that the R. conorii and R. prowazekii Adr2 protein does not participate in the interactions with mammalian cells, but rather, participates in the evasion of killing by complement.

  15. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

    PubMed

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  16. Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function.

    PubMed

    Howard, Marybeth; Fischer, Horst; Roux, Jeremie; Santos, Bento C; Gullans, Steven R; Yancey, Paul H; Welch, William J

    2003-09-12

    In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to

  17. A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the β-actin promoter and enhancer.

    PubMed

    Lupold, Shawn E; Johnson, Tamara; Chowdhury, Wasim H; Rodriguez, Ronald

    2012-01-01

    Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.

  18. The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells.

    PubMed

    Pabis, Marta; Neufeld, Noa; Steiner, Michaela C; Bojic, Teodora; Shav-Tal, Yaron; Neugebauer, Karla M

    2013-08-01

    The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

  19. Mammalian target of rapamycin inhibitors, temsirolimus and torin 1, attenuate stemness-associated properties and expression of mesenchymal markers promoted by phorbol-myristate-acetate and oncostatin-M in glioblastoma cells.

    PubMed

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2017-03-01

    The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

  20. Mammalian Polo-like kinase 1 (Plk1) promotes proper chromosome segregation by phosphorylating and delocalizing the PBIP1·CENP-Q complex from kinetochores.

    PubMed

    Park, Chi Hoon; Park, Jung-Eun; Kim, Tae-Sung; Kang, Young Hwi; Soung, Nak-Kyun; Zhou, Ming; Kim, Nam-Hyung; Bang, Jeong Kyu; Lee, Kyung S

    2015-03-27

    Mammalian Plk1 is critically required for proper M phase progression. Plk1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating Plk1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. Plk1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine Plk1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although Plk1 did not alter the level of PBIP1 and CENP-Q ubiquitination, Plk1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that Plk1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting Plk1-dependent mitotic progression, but also for resetting the timing of Plk1 recruitment to prekinetochores in the next cell cycle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Mammalian Polo-like Kinase 1 (Plk1) Promotes Proper Chromosome Segregation by Phosphorylating and Delocalizing the PBIP1·CENP-Q Complex from Kinetochores

    PubMed Central

    Park, Chi Hoon; Park, Jung-Eun; Kim, Tae-Sung; Kang, Young Hwi; Soung, Nak-Kyun; Zhou, Ming; Kim, Nam-Hyung; Bang, Jeong Kyu; Lee, Kyung S.

    2015-01-01

    Mammalian Plk1 is critically required for proper M phase progression. Plk1 is self-recruited to prekinetochores/kinetochores by phosphorylating and binding to the Thr-78 motif of a kinetochore scaffold protein, PBIP1 (also called CENP-U/50), which forms a stable complex with another kinetochore component, CENP-Q. However, the mechanism regulating Plk1 localization to this site remains largely unknown. Here, we demonstrate that the PBIP1·CENP-Q complex became hyperphosphorylated and rapidly delocalized from kinetochores as cells entered mitosis. Plk1 phosphorylated the CENP-Q subunit of the PBIP1·CENP-Q complex at multiple sites, and mutation of nine Plk1-dependent phosphorylation sites to Ala (9A) enhanced CENP-Q association with chromatin and prolonged CENP-Q localization to kinetochores. Conversely, mutation of the nine sites to phospho-mimicking Asp/Glu (9D/E) residues dissociated CENP-Q from chromatin and kept the CENP-Q(9D/E) mutant from localizing to interphase prekinetochores. Strikingly, both the 9A and 9D/E mutants induced a defect in proper chromosome segregation, suggesting that both timely localization of the PBIP1·CENP-Q complex to prekinetochores and delocalization from kinetochores are critical for normal M phase progression. Notably, although Plk1 did not alter the level of PBIP1 and CENP-Q ubiquitination, Plk1-dependent phosphorylation and delocalization of these proteins from kinetochores appeared to indirectly lead to their degradation in the cytosol. Thus, we propose that Plk1 regulates the timing of the delocalization and ultimate destruction of the PBIP1·CENP-Q complex and that these processes are important not only for promoting Plk1-dependent mitotic progression, but also for resetting the timing of Plk1 recruitment to prekinetochores in the next cell cycle. PMID:25670858

  2. TGF-β/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression

    PubMed Central

    Hayashida, Tomoko; Hubchak, Susan C.; Hanna, Christian; Platanias, Leonidas C.; William Schnaper, H.

    2013-01-01

    Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis. PMID:23761672

  3. TGF-β/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1α expression.

    PubMed

    Rozen-Zvi, Benaya; Hayashida, Tomoko; Hubchak, Susan C; Hanna, Christian; Platanias, Leonidas C; Schnaper, H William

    2013-08-15

    Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

  4. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors.

  5. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  6. PB2-588 V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses

    PubMed Central

    Xiao, Chencheng; Ma, Wenjun; Sun, Na; Huang, Lihong; Li, Yaling; Zeng, Zhaoyong; Wen, Yijun; Zhang, Zaoyue; Li, Huanan; Li, Qian; Yu, Yuandi; Zheng, Yi; Liu, Shukai; Hu, Pingsheng; Zhang, Xu; Ning, Zhangyong; Qi, Wenbao; Liao, Ming

    2016-01-01

    Human infections with avian influenza H7N9 or H10N8 viruses have been reported in China, raising concerns that they might cause human epidemics and pandemics. However, how these viruses adapt to mammalian hosts is unclear. Here we show that besides the commonly recognized viral polymerase subunit PB2 residue 627 K, other residues including 87E, 292 V, 340 K, 588 V, 648 V, and 676 M in PB2 also play critical roles in mammalian adaptation of the H10N8 virus. The avian-origin H10N8, H7N9, and H9N2 viruses harboring PB2-588 V exhibited higher polymerase activity, more efficient replication in mammalian and avian cells, and higher virulence in mice when compared to viruses with PB2-588 A. Analyses of available PB2 sequences showed that the proportion of avian H9N2 or human H7N9 influenza isolates bearing PB2-588 V has increased significantly since 2013. Taken together, our results suggest that the substitution PB2-A588V may be a new strategy for an avian influenza virus to adapt mammalian hosts. PMID:26782141

  7. PB2-588 V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses.

    PubMed

    Xiao, Chencheng; Ma, Wenjun; Sun, Na; Huang, Lihong; Li, Yaling; Zeng, Zhaoyong; Wen, Yijun; Zhang, Zaoyue; Li, Huanan; Li, Qian; Yu, Yuandi; Zheng, Yi; Liu, Shukai; Hu, Pingsheng; Zhang, Xu; Ning, Zhangyong; Qi, Wenbao; Liao, Ming

    2016-01-19

    Human infections with avian influenza H7N9 or H10N8 viruses have been reported in China, raising concerns that they might cause human epidemics and pandemics. However, how these viruses adapt to mammalian hosts is unclear. Here we show that besides the commonly recognized viral polymerase subunit PB2 residue 627 K, other residues including 87E, 292 V, 340 K, 588 V, 648 V, and 676 M in PB2 also play critical roles in mammalian adaptation of the H10N8 virus. The avian-origin H10N8, H7N9, and H9N2 viruses harboring PB2-588 V exhibited higher polymerase activity, more efficient replication in mammalian and avian cells, and higher virulence in mice when compared to viruses with PB2-588 A. Analyses of available PB2 sequences showed that the proportion of avian H9N2 or human H7N9 influenza isolates bearing PB2-588 V has increased significantly since 2013. Taken together, our results suggest that the substitution PB2-A588V may be a new strategy for an avian influenza virus to adapt mammalian hosts.

  8. Sirtuins: Guardians of Mammalian Healthspan

    PubMed Central

    Giblin, William; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    The first link between sirtuins and longevity was made 15 years ago in yeast. These initial studies sparked efforts by many laboratories working in diverse model organisms to elucidate the relationships between sirtuins, lifespan, and age-associated dysfunction. Here we discuss the current understanding of how sirtuins relate to aging. We focus primarily on mammalian sirtuins SIRT1, SIRT3, and SIRT6, the three sirtuins for which the most relevant data are available. Strikingly, a large body of evidence now indicates that these and other mammalian sirtuins suppress a variety of age-related pathologies and promote healthspan. Moreover, increased expression of SIRT1 or SIRT6 extends mouse lifespan. Overall, these data point to important roles for sirtuins in promoting mammalian health, and perhaps in modulating the aging process. PMID:24877878

  9. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  10. U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

    PubMed Central

    Cohen, J B; Broz, S D; Levinson, A D

    1993-01-01

    Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells. Images PMID:7682651

  11. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  12. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

    PubMed

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E; Arons, Elena; Zaman, Paula; Polach, Kevin J; Matar, Majed; Yung, Lai-Ming; Yu, Paul B; Bowman, Frederick P; Opotowsky, Alexander R; Waxman, Aaron B; Loscalzo, Joseph; Leopold, Jane A; Maron, Bradley A

    2016-07-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth

  13. Mammalian Eps15 homology domain 1 promotes metastasis in non-small cell lung cancer by inducing epithelial-mesenchymal transition.

    PubMed

    Meng, Qingwei; Xing, Ying; Ren, Tingting; Lu, Hailing; Xi, Yuhui; Jiang, Zhijun; Hu, Jing; Li, Chunhong; Sun, Lichun; Sun, Dianjun; Cai, Li

    2017-04-04

    The identification of the earliest molecular events responsible for the metastatic dissemination of non-small cell lung cancer (NSCLC) remains critical for early detection, prevention, and treatment interventions. In this study, we hypothesized that Mammalian Eps15 homology domain 1 (EHD1) might be responsible for the metastatic behavior of cells in NSCLC. We demonstrated that upregulation of EHD1 is associated with lymph nodes metastasis and unfavorable survival in patients with NSCLC. EHD1 knockdown inhibited the invasion and migration of human NSCLC cells, and overexpression of EHD1 increased the metastatic potential of lung cancer cells. Using the Affymetrix Human Gene 1.0 ST platform, microarray analysis revealed that an association between EHD1 and epithelial-mesenchymal transition (EMT), supported by downregulation of mesenchymal markers and upregulation of epithelial markers following knockdown of EHD1 in cell lines. Moreover, overexpression of EHD1 induced the EMT and increased the metastatic potential of lung cancer cells in vitro and in vivo. These results provide a model to illustrate the relationship between EHD1 expression and lung cancer metastasis, opening up new avenues for the prognosis and therapy of lung cancer.

  14. Antagonism of microRNA-99a promotes cell invasion and down-regulates E-cadherin expression in pancreatic cancer cells by regulating mammalian target of rapamycin.

    PubMed

    Li, Dan; Li, Xiaohan; Cao, Wei; Qi, Yafei; Yang, Xianghong

    2014-06-01

    MicroRNA-99a (miRNA-99a), a potential tumor suppressor, has been implicated in tumorigenesis of many human malignancies. However, the role of miRNA-99a in pancreatic cancer remains unclear. In the present study, we transfected miRNA-99a antagonism into human pancreatic cancer AsPC-1 cells to inhibit miRNA-99a expression and investigated its influence on cell migration and invasion as well as the underlying possible mechanisms. We found that miRNA-99a antagonism significantly increased proliferation, migration and invasion abilities of AsPC-1 cells, which was accompanied by increased expression of mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and α-SMA), and decreased expression of epithelial phenotype cell biomarker (E-cadherin). Interestingly, small interfering RNA (siRNA)-mediated knockdown of mammalian target of rapamycin (mTOR) remarkably restored miRNA-99a antagonism-induced down-regulation of E-cadherin. In conclusion, our data suggest that miRNA-99a is involved in pancreatic cancer migration and invasion by regulating mTOR, and may provide a target for effective therapies against pancreatic cancer.

  15. Isoflurane Promotes Non-Small Cell Lung Cancer Malignancy by Activating the Akt-Mammalian Target of Rapamycin (mTOR) Signaling Pathway

    PubMed Central

    Zhang, Wenhua; Shao, Xueqian

    2016-01-01

    Background Lung cancer is one of the leading causes of cancer mortalities worldwide, and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Surgery remains one of the front-line treatment options for NSCLC, but events within the perioperative period were found to affect cancer prognosis, such as anesthesia procedures. Isoflurane, a commonly used volatile anesthetic, enhances the malignant potential of renal, prostate, and ovarian cancer cells, but its effects on NSCLC development have not been previously reported. Material/Methods CCK-8 and MTT cell proliferation assays were used to analyze NSCLC cell proliferation. Metastatic ability was examined by wound healing and transwell assays. We used Western blot analysis to study the mechanism of effect of Isoflurane in NSCLC development. Results We demonstrated that isoflurane promotes proliferation, migration and invasiveness of NSCLC cells, as well as upregulation of the Akt-mTOR signaling pathway in NSCLC cells. Pharmacological inhibition of Akt-mTOR signaling abolished the ability of isoflurane to promote proliferation, migration, and invasion of NSCLC cells, indicating that isoflurane promotes NSCLC cell malignancy by activating the Akt-mTOR signaling pathway. Conclusions Isoflurane promotes NSCLC proliferation, migration and invasion by activating the Akt-mTOR signaling pathway. PMID:27897153

  16. Pathways of mammalian replication fork restart.

    PubMed

    Petermann, Eva; Helleday, Thomas

    2010-10-01

    Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.

  17. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension

    PubMed Central

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E.; Arons, Elena; Zaman, Paula; Polach, Kevin J.; Matar, Majed; Yung, Lai-Ming; Yu, Paul B.; Bowman, Frederick P.; Opotowsky, Alexander R.; Waxman, Aaron B.; Loscalzo, Joseph; Leopold, Jane A.; Maron, Bradley A.

    2016-01-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10−9 to 10−7 M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor–small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro. Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo. Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.—Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery

  18. Zebrafish Noxa promotes mitosis in early embryonic development and regulates apoptosis in subsequent embryogenesis.

    PubMed

    Zhong, J-X; Zhou, L; Li, Z; Wang, Y; Gui, J-F

    2014-06-01

    Noxa functions in apoptosis and immune system of vertebrates, but its activities in embryo development remain unclear. In this study, we have studied the role of zebrafish Noxa (zNoxa) by using zNoxa-specifc morpholino knockdown and overexpression approaches in developing zebrafish embryos. Expression pattern analysis indicates that zNoxa transcript is of maternal origin, which displays a uniform distribution in early embryonic development until shield stage, and the zygote zNoxa transcription is initiated from this stage and mainly localized in YSL of the embryos. The zNoxa expression alterations result in strong embryonic development defects, demonstrating that zNoxa regulates apoptosis from 75% epiboly stage of development onward, in which zNoxa firstly induces the expression of zBik, and then cooperates with zBik to regulate apoptosis. Moreover, zNoxa knockdown also causes a reduction in number of mitotic cells before 8 h.p.f., suggesting that zNoxa also promotes mitosis before 75% epiboly stage. The effect of zNoxa on mitosis is mediated by zWnt4b in early embryos, whereas zMcl1a and zMcl1b suppress the ability of zNoxa to regulate mitosis and apoptosis at different developmental stages. In addition, mammalian mouse Noxa (mNoxa) mRNA was demonstrated to rescue the arrest of mitosis when zNoxa was knocked down, suggesting that mouse and zebrafish Noxa might have similar dual functions. Therefore, the current findings indicate that Noxa is a novel regulator of early mitosis before 75% epiboly stage when it translates into a key mediator of apoptosis in subsequent embryogenesis.

  19. Comparative integromics on FZD7 orthologs: conserved binding sites for PU.1, SP1, CCAAT-box and TCF/LEF/SOX transcription factors within 5'-promoter region of mammalian FZD7 orthologs.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-03-01

    that the binding sites for PU.1, SP1/Krüppel-like, CCAAT-box, and TCF/LEF/SOX transcription factors were conserved among 5'-promoter regions of mammalian FZD7 orthologs.

  20. Modeling the mammalian sleep cycle.

    PubMed

    Weber, Franz

    2017-08-24

    During sleep, the mammalian brain transitions through repeated cycles of non-rapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep. The physiological implementation of this slow ultradian brain rhythm is largely unknown. Two differing dynamical mechanisms have been proposed to underlie the NREM-REM cycle. The first model type relies on reciprocal interactions between inhibitory and excitatory neural populations resulting in stable limit cycle oscillations. Recent experimental findings instead favor a model, in which mutually inhibitory interactions between REM sleep-promoting (REM-on) and REM sleep-suppressing (REM-off) neural populations stabilize the brain state. Slow modulations in the neural excitability, that are hypothesized to reflect the homeostatic need for REM sleep, abruptly switch the brain in and out of REM sleep. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  2. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  3. Mammalian development in space.

    PubMed

    Ronca, April E

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  4. Mammalian touch catches up

    PubMed Central

    Walsh, Carolyn M.; Bautista, Diana M.; Lumpkin, Ellen A.

    2015-01-01

    An assortment of touch receptors innervate the skin and encode different tactile features of the environment. Compared with invertebrate touch and other sensory systems, our understanding of the molecular and cellular underpinnings of mammalian touch lags behind. Two recent breakthroughs have accelerated progress. First, an arsenal of cell-type-specific molecular markers allowed the functional and anatomical properties of sensory neurons to be matched, thereby unraveling a cellular code for touch. Such markers have also revealed key roles of non-neuronal cell types, such as Merkel cells and keratinocytes, in touch reception. Second, the discovery of Piezo genes as a new family of mechanically activated channels has fueled the discovery of molecular mechanisms that mediate and mechanotransduction in mammalian touch receptors. PMID:26100741

  5. Phospholipid synthesis and transport in mammalian cells.

    PubMed

    Vance, Jean E

    2015-01-01

    Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter-organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria-associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter-organelle phospholipid transport in mammalian cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Mammalian glycosylation in immunity.

    PubMed

    Marth, Jamey D; Grewal, Prabhjit K

    2008-11-01

    Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.

  7. Mammalian sperm morphometry.

    PubMed Central

    Gage, M J

    1998-01-01

    Understanding the adaptive significance of sperm form and function has been a challenge to biologists because sperm are highly specialized cells operating at a microscopic level in a complex environment. A fruitful course of investigation has been to use the comparative approach. This comparative study attempts to address some fundamental questions of the evolution of mammalian sperm morphometry. Data on sperm morphometry for 445 mammalian species were collated from published sources. I use contemporary phylogenetic analysis to control for the inherent non-independence of species and explore relationships between the morphometric dimensions of the three essential spermatozoal components: head, mid-piece and flagellum. Energy for flagellar action is metabolized by the mitochondrial-dense mid-piece and these combine to propel the sperm head, carrying the male haplotype, to the ovum. I therefore search for evolutionary associations between sperm morphometry and body mass, karyotype and the duration of oestrus. In contrast to previous findings, there is no inverse correlation between body weight and sperm length. Sperm mid-piece and flagellum lengths are positively associated with both head length and area, and the slopes of these relationships are discussed. Flagellum length is positively associated with mid-piece length but, in contrast to previous research and after phylogenetic control, I find no relationship between flagellum length and the volume of the mitochondrial sheath. Sperm head dimensions are not related to either genome mass or chromosome number, and there are no relationships between sperm morphometry and the duration of oestrus. PMID:9474794

  8. Mammalian Molecular Clocks

    PubMed Central

    Kwon, Ilmin; Choe, Han Kyoung; Son, Gi Hoon

    2011-01-01

    As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock. PMID:22110358

  9. The mammalian blastocyst.

    PubMed

    Frankenberg, Stephen R; de Barros, Flavia R O; Rossant, Janet; Renfree, Marilyn B

    2016-01-01

    The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website.

  10. Mammalian phospholipase C.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

    Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

  11. New Mammalian Expression Systems.

    PubMed

    Zhu, Jie; Hatton, Diane

    2017-06-06

    There are an increasing number of recombinant antibodies and proteins in preclinical and clinical development for therapeutic applications. Mammalian expression systems are key to enabling the production of these molecules, and Chinese hamster ovary (CHO) cell platforms continue to be central to delivery of the stable cell lines required for large-scale production. Increasing pressure on timelines and efficiency, further innovation of molecular formats and the shift to new production systems are driving developments of these CHO cell line platforms. The availability of genome and transcriptome data coupled with advancing gene editing tools are increasing the ability to design and engineer CHO cell lines to meet these challenges. This chapter aims to give an overview of the developments in CHO expression systems and some of the associated technologies over the past few years.

  12. The transcriptional landscape of the mammalian genome.

    PubMed

    Carninci, P; Kasukawa, T; Katayama, S; Gough, J; Frith, M C; Maeda, N; Oyama, R; Ravasi, T; Lenhard, B; Wells, C; Kodzius, R; Shimokawa, K; Bajic, V B; Brenner, S E; Batalov, S; Forrest, A R R; Zavolan, M; Davis, M J; Wilming, L G; Aidinis, V; Allen, J E; Ambesi-Impiombato, A; Apweiler, R; Aturaliya, R N; Bailey, T L; Bansal, M; Baxter, L; Beisel, K W; Bersano, T; Bono, H; Chalk, A M; Chiu, K P; Choudhary, V; Christoffels, A; Clutterbuck, D R; Crowe, M L; Dalla, E; Dalrymple, B P; de Bono, B; Della Gatta, G; di Bernardo, D; Down, T; Engstrom, P; Fagiolini, M; Faulkner, G; Fletcher, C F; Fukushima, T; Furuno, M; Futaki, S; Gariboldi, M; Georgii-Hemming, P; Gingeras, T R; Gojobori, T; Green, R E; Gustincich, S; Harbers, M; Hayashi, Y; Hensch, T K; Hirokawa, N; Hill, D; Huminiecki, L; Iacono, M; Ikeo, K; Iwama, A; Ishikawa, T; Jakt, M; Kanapin, A; Katoh, M; Kawasawa, Y; Kelso, J; Kitamura, H; Kitano, H; Kollias, G; Krishnan, S P T; Kruger, A; Kummerfeld, S K; Kurochkin, I V; Lareau, L F; Lazarevic, D; Lipovich, L; Liu, J; Liuni, S; McWilliam, S; Madan Babu, M; Madera, M; Marchionni, L; Matsuda, H; Matsuzawa, S; Miki, H; Mignone, F; Miyake, S; Morris, K; Mottagui-Tabar, S; Mulder, N; Nakano, N; Nakauchi, H; Ng, P; Nilsson, R; Nishiguchi, S; Nishikawa, S; Nori, F; Ohara, O; Okazaki, Y; Orlando, V; Pang, K C; Pavan, W J; Pavesi, G; Pesole, G; Petrovsky, N; Piazza, S; Reed, J; Reid, J F; Ring, B Z; Ringwald, M; Rost, B; Ruan, Y; Salzberg, S L; Sandelin, A; Schneider, C; Schönbach, C; Sekiguchi, K; Semple, C A M; Seno, S; Sessa, L; Sheng, Y; Shibata, Y; Shimada, H; Shimada, K; Silva, D; Sinclair, B; Sperling, S; Stupka, E; Sugiura, K; Sultana, R; Takenaka, Y; Taki, K; Tammoja, K; Tan, S L; Tang, S; Taylor, M S; Tegner, J; Teichmann, S A; Ueda, H R; van Nimwegen, E; Verardo, R; Wei, C L; Yagi, K; Yamanishi, H; Zabarovsky, E; Zhu, S; Zimmer, A; Hide, W; Bult, C; Grimmond, S M; Teasdale, R D; Liu, E T; Brusic, V; Quackenbush, J; Wahlestedt, C; Mattick, J S; Hume, D A; Kai, C; Sasaki, D; Tomaru, Y; Fukuda, S; Kanamori-Katayama, M; Suzuki, M; Aoki, J; Arakawa, T; Iida, J; Imamura, K; Itoh, M; Kato, T; Kawaji, H; Kawagashira, N; Kawashima, T; Kojima, M; Kondo, S; Konno, H; Nakano, K; Ninomiya, N; Nishio, T; Okada, M; Plessy, C; Shibata, K; Shiraki, T; Suzuki, S; Tagami, M; Waki, K; Watahiki, A; Okamura-Oho, Y; Suzuki, H; Kawai, J; Hayashizaki, Y

    2005-09-02

    This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

  13. Adrenomedullin in mammalian embryogenesis.

    PubMed

    Garayoa, Mercedes; Bodegas, Elena; Cuttitta, Frank; Montuenga, Luis M

    2002-04-01

    Here are summarized data supporting that adrenomedullin (AM) is a multifunctional factor involved in the complex regulatory mechanisms of mammalian development. During rodent embryogenesis, AM is first expressed in the heart, followed by a broader but also defined spatio-temporal pattern of expression in vascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs. AM pattern of expression is suggestive of its involvement in the control of embryonic invasion, proliferation, and differentiation processes, probably through autocrine or paracrine modes of action. AM levels in fetoplacental tissues, uterus, maternal and umbilical plasma are highly increased during normal gestation. These findings in addition to other physiological and gene targeting studies support the importance of AM as a vasorelaxant factor implicated in the regulation of maternal vascular adaptation to pregnancy, as well as of fetal and fetoplacental circulations. AM is also present in amniotic fluid and milk, which is suggestive of additional functions in the maturation and immunological protection of the fetus. Altered expression of AM has been found in some gestational pathologies, although it is not yet clear whether this corresponds to causative or compensatory mechanisms. Future studies in regard to the distribution and expression levels of the molecules known to function as AM receptors, together with data on the action of complement factor H (an AM binding protein), may help to better define the roles of AM during embryonic development. Copyright 2002 Wiley-Liss, Inc.

  14. The Mammalian Septin Interactome

    PubMed Central

    Neubauer, Katharina; Zieger, Barbara

    2017-01-01

    Septins are GTP-binding and membrane-interacting proteins with a highly conserved domain structure involved in various cellular processes, including cytoskeleton organization, cytokinesis, and membrane dynamics. To date, 13 different septin genes have been identified in mammals (SEPT1 to SEPT12 and SEPT14), which can be classified into four distinct subgroups based on the sequence homology of their domain structure (SEPT2, SEPT3, SEPT6, and SEPT7 subgroup). The family members of these subgroups have a strong affinity for other septins and form apolar tri-, hexa-, or octameric complexes consisting of multiple septin polypeptides. The first characterized core complex is the hetero-trimer SEPT2-6-7. Within these complexes single septins can be exchanged in a subgroup-specific manner. Hexamers contain SEPT2 and SEPT6 subgroup members and SEPT7 in two copies each whereas the octamers additionally comprise two SEPT9 subgroup septins. The various isoforms seem to determine the function and regulation of the septin complex. Septins self-assemble into higher-order structures, including filaments and rings in orders, which are typical for different cell types. Misregulation of septins leads to human diseases such as neurodegenerative and bleeding disorders. In non-dividing cells such as neuronal tissue and platelets septins have been associated with exocytosis. However, many mechanistic details and roles attributed to septins are poorly understood. We describe here some important mammalian septin interactions with a special focus on the clinically relevant septin interactions. PMID:28224124

  15. Mammalian clock output mechanisms.

    PubMed

    Kalsbeek, Andries; Yi, Chun-Xia; Cailotto, Cathy; la Fleur, Susanne E; Fliers, Eric; Buijs, Ruud M

    2011-06-30

    In mammals many behaviours (e.g. sleep-wake, feeding) as well as physiological (e.g. body temperature, blood pressure) and endocrine (e.g. plasma corticosterone concentration) events display a 24 h rhythmicity. These 24 h rhythms are induced by a timing system that is composed of central and peripheral clocks. The highly co-ordinated output of the hypothalamic biological clock not only controls the daily rhythm in sleep-wake (or feeding-fasting) behaviour, but also exerts a direct control over many aspects of hormone release and energy metabolism. First, we present the anatomical connections used by the mammalian biological clock to enforce its endogenous rhythmicity on the rest of the body, especially the neuro-endocrine and energy homoeostatic systems. Subsequently, we review a number of physiological experiments investigating the functional significance of this neuro-anatomical substrate. Together, this overview of experimental data reveals a highly specialized organization of connections between the hypothalamic pacemaker and neuro-endocrine system as well as the pre-sympathetic and pre-parasympathetic branches of the autonomic nervous system.

  16. The Mammalian Septin Interactome.

    PubMed

    Neubauer, Katharina; Zieger, Barbara

    2017-01-01

    Septins are GTP-binding and membrane-interacting proteins with a highly conserved domain structure involved in various cellular processes, including cytoskeleton organization, cytokinesis, and membrane dynamics. To date, 13 different septin genes have been identified in mammals (SEPT1 to SEPT12 and SEPT14), which can be classified into four distinct subgroups based on the sequence homology of their domain structure (SEPT2, SEPT3, SEPT6, and SEPT7 subgroup). The family members of these subgroups have a strong affinity for other septins and form apolar tri-, hexa-, or octameric complexes consisting of multiple septin polypeptides. The first characterized core complex is the hetero-trimer SEPT2-6-7. Within these complexes single septins can be exchanged in a subgroup-specific manner. Hexamers contain SEPT2 and SEPT6 subgroup members and SEPT7 in two copies each whereas the octamers additionally comprise two SEPT9 subgroup septins. The various isoforms seem to determine the function and regulation of the septin complex. Septins self-assemble into higher-order structures, including filaments and rings in orders, which are typical for different cell types. Misregulation of septins leads to human diseases such as neurodegenerative and bleeding disorders. In non-dividing cells such as neuronal tissue and platelets septins have been associated with exocytosis. However, many mechanistic details and roles attributed to septins are poorly understood. We describe here some important mammalian septin interactions with a special focus on the clinically relevant septin interactions.

  17. A Rosetta stone of mammalian genetics.

    PubMed

    Nadeau, J H; Grant, P L; Mankala, S; Reiner, A H; Richardson, J E; Eppig, J T

    1995-01-26

    The Mammalian Comparative Database provides genetic maps of mammalian species. Comparative maps are valuable aids for predicting linkages, developing animal models and studying genome organization and evolution.

  18. Stem Cells in Mammalian Gonads.

    PubMed

    Wu, Ji; Ding, Xinbao; Wang, Jian

    Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.

  19. Mammalian Interphase Cdks

    PubMed Central

    2012-01-01

    Cyclin-dependent kinases (Cdks) drive cell cycle progression in all eukaryotes. Yeasts have a single major Cdk that mediates distinct cell cycle transitions via association with different cyclins. The closest homolog in mammals, Cdk1, drives mitosis. Mammals have additional Cdks—Cdk2, Cdk4, and Cdk6—that represent the major Cdks activated during interphase (iCdks). A large body of evidence has accrued that suggests that activation of iCdks dictates progression though interphase. In apparent contradiction, deficiency in each individual iCdk, respectively, in knockout mice proved to be compatible with live birth and in some instances fertility. Moreover, murine embryos could be derived with Cdk1 as the only functional Cdk. Thus, none of the iCdks is strictly essential for mammalian cell cycle progression, raising the possibility that Cdk1 is the dominant regulator in interphase. However, an absence of iCdks has been accompanied by major shifts in cyclin association to Cdk1, suggesting gain in function. After considerable tweaking, a chemical genetic approach has recently been able to examine the impact of acute inhibition of Cdk2 activity without marked distortion of cyclin/Cdk complex formation. The results suggest that, when expressed at its normal levels, Cdk2 performs essential roles in driving human cells into S phase and maintaining genomic stability. These new findings appear to have restored order to the cell cycle field, bringing it full circle to the view that iCdks indeed play important roles. They also underscore the caveat in knockdown and knockout approaches that protein underexpression can significantly perturb a protein interaction network. We discuss the implications of the new synthesis for future cell cycle studies and anti–Cdk-based therapy of cancer and other diseases. PMID:23634250

  20. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  1. Maturation of the mammalian secretome

    PubMed Central

    Simpson, Jeremy C; Mateos, Alvaro; Pepperkok, Rainer

    2007-01-01

    A recent use of quantitative proteomics to determine the constituents of the endoplasmic reticulum and Golgi complex is discussed in the light of other available methodologies for cataloging the proteins associated with the mammalian secretory pathway. PMID:17472737

  2. Bursty gene expression in the intact mammalian liver

    PubMed Central

    Halpern, Keren Bahar; Tanami, Sivan; Landen, Shanie; Chapal, Michal; Szlak, Liran; Hutzler, Anat; Nizhberg, Anna; Itzkovitz, Shalev

    2015-01-01

    Summary Bursts of nascent mRNA have been shown to lead to substantial cell-cell variation in unicellular organisms, facilitating diverse responses to environmental challenges. It is unknown whether similar bursts and gene-expression noise occur in mammalian tissues. To address this, we combine single molecule transcript counting with dual-color labeling and quantification of nascent mRNA to characterize promoter states, transcription rates and transcript lifetimes in the intact mouse liver. We find that liver gene expression is highly bursty, with promoters stochastically switching between transcriptionally active and inactive states. Promoters of genes with short mRNA lifetimes are active longer, facilitating rapid response while reducing burst-associated noise. Moreover, polyploid hepatocytes exhibit less noise than diploid hepatocytes, suggesting a possible benefit to liver polyploidy. Thus temporal averaging and liver polyploidy dampen the intrinsic variability associated with transcriptional bursts. Our approach can be used to study transcriptional bursting in diverse mammalian tissues. PMID:25728770

  3. Chronobiology in mammalian health.

    PubMed

    Liu, Zhihua; Chu, Guiyan

    2013-03-01

    Circadian rhythms are daily cycles of physiology and behavior that are driven by an endogenous oscillator with a period of approximately one day. In mammals, the hypothalamic suprachiasmatic nuclei are our principal circadian oscillators which influences peripheral tissue clocks via endocrine, autonomic and behavioral cues, and other brain regions and most peripheral tissues contain circadian clocks as well. The circadian molecular machinery comprises a group of circadian genes, namely Clock, Bmal1, Per1, Per2, Per3, Cry1 and Cry2. These circadian genes drive endogenous oscillations which promote rhythmically expression of downstream genes and thereby physiological and behavioral processes. Disruptions in circadian homeostasis have pronounced impact on physiological functioning, overall health and disease susceptibility. This review introduces the general profile of circadian gene expression and tissue-specific circadian regulation, highlights the connection between the circadian rhythms and physiological processes, and discusses the role of circadian rhythms in human disease.

  4. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  5. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  6. Mammalian sex hormones in plants.

    PubMed

    Janeczko, Anna; Skoczowski, Andrzej

    2005-01-01

    The occurrence of mammalian sex hormones and their physiological role in plants is reviewed. These hormones, such as 17beta-estradiol, androsterone, testosterone or progesterone, were present in 60-80% of the plant species investigated. Enzymes responsible for their biosynthesis and conversion were also found in plants. Treatment of the plants with sex hormones or their precursors influenced plant development: cell divisions, root and shoot growth, embryo growth, flowering, pollen tube growth and callus proliferation. The regulatory abilities of mammalian sex hormones in plants makes possible their use in practice, especially in plant in vitro culture.

  7. [Comparative embryology and mammalian cloning].

    PubMed

    Sakharova, N Iu; Chaĭlakhian, L M

    2010-01-01

    A hypothesis has been advanced that logically combines "contradictory" facts concerning the early mammalian development and shows a natural relationship between the embryos developing from a fertilized ovum and from cells of the inner cell mass of blastocyst. When studying the theoretical questions of cloning, it is necessary to take into consideration the peculiarities of prenatal mammalian ontogenesis, which make themselves evident upon comparison with other animals. The absence of yolk in the mammalian ovum defines sharp differences in the early development between mammals and other Amniota. The whole asynchronic cleavage results in the formation of the morula followed by the blastocyst, which hatches from zona pellucida and is implanted into the uterus tissue. This fact allows us to consider the blastocyst as a mammalian larva, which is fed thanks to maternal organism. It is known that, in the body of a larva (blastocyst), a new embryo develops from some somatic cells. This process is known as a polyembryony, which is typical for the development of some parasitic insects. The polyembryony in turn is a variant of somatic embryogenesis, which is a form of asexual reproduction. Thus, two different embryos, "conceptus" and "embryo proper", have different origin: the first forms by the sexual way and the second, by the asexual. The investigation of the mechanisms of somatic embryogenesis in mammals will help us to find conditions necessary for the full reprograming of donor somatic nuclei and provide the successful development of reconstructed embryos.

  8. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  9. Inhibition of miR-29c promotes proliferation, and inhibits apoptosis and differentiation in P19 embryonic carcinoma cells.

    PubMed

    Chen, Bin; Song, Guixian; Liu, Ming; Qian, Lingmei; Wang, Lihua; Gu, Haitao; Shen, Yahui

    2016-03-01

    In our previous study, the upregulation of microRNA (miR)-29c was identified in the mother of a fetus with a congenital heart defect. However, the functional and regulatory mechanisms of miR‑29c in the development of the heart remain to be elucidated. In the present study, the role and mechanism of miR‑29c inhibition in heart development were investigated in an embryonic carcinoma cell model. Inhibition of miR‑29c promoted proliferation, and suppressed the apoptosis and differentiation of P19 cells. It was also demonstrated that Wingless‑related MMTV integration site 4 (Wnt4) was a target of miR‑29c, determined using bioinformatic analysis combined with luciferase assays. The inhibition of miR‑29c stimulated the WNT4/β‑catenin pathway, promoting proliferation of the P19 cells, but suppressing their differentiation into cardiomyocytes. Furthermore, the inhibition of miR‑29c promoted the expression of B cell lymphoma‑2 and inhibited cell apoptosis. These results demonstrate the significance of miR‑29c in the process of cardiac development and suggest that miR-29c dysregulation may be associated with the occurrence of CHD. Thus, miR-29c may have therapeutic potential in the future.

  10. A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead.

    PubMed

    Wilanowski, Tomasz; Tuckfield, Annabel; Cerruti, Loretta; O'Connell, Sinead; Saint, Robert; Parekh, Vishwas; Tao, Jianning; Cunningham, John M; Jane, Stephen M

    2002-06-01

    The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.

  11. Mechanisms of mammalian iron homeostasis

    PubMed Central

    Pantopoulos, Kostas; Porwal, Suheel Kumar; Tartakoff, Alan; Devireddy, L.

    2012-01-01

    Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in acquisition or distribution of the metal causes anemia; whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways, as well as in mechanisms underlying intracellular iron trafficking, an important but less-studied area of mammalian iron homeostasis. PMID:22703180

  12. Architecture of the Mammalian Golgi

    PubMed Central

    Klumperman, Judith

    2011-01-01

    Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described. PMID:21502307

  13. Bioenergetics of Mammalian Sperm Capacitation

    PubMed Central

    Ferramosca, Alessandra; Zara, Vincenzo

    2014-01-01

    After ejaculation, the mammalian male gamete must undergo the capacitation process, which is a prerequisite for egg fertilization. The bioenergetics of sperm capacitation is poorly understood despite its fundamental role in sustaining the biochemical and molecular events occurring during gamete activation. Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for spermatozoa. Since recent data suggest that spermatozoa have the ability to use different metabolic substrates, the main aim of this work is to present a broad overview of the current knowledge on the energy-producing metabolic pathways operating inside sperm mitochondria during capacitation in different mammalian species. Metabolism of glucose and of other energetic substrates, such as pyruvate, lactate, and citrate, is critically analyzed. Such knowledge, besides its obvious importance for basic science, could eventually translate into the development of novel strategies for treatment of male infertility, artificial reproduction, and sperm selection methods. PMID:24791005

  14. Ceramide signaling in mammalian epidermis

    PubMed Central

    Uchida, Yoshikazu

    2013-01-01

    Ceramide, the backbone structure of all sphingolipids, as well as a minor component of cellular membranes, has a unique role in the skin, by forming the epidermal permeability barrier at the extracellular domains of the outermost layer of skin, the stratum corneum, which is required for terrestrial mammalian survival. In contrast to the role of ceramide in forming the permeability barrier, the signaling roles of ceramide and its metabolites have not yet been recognized. Ceramide and/or its metabolites regulate proliferation, differentiation, and apoptosis in epidermal keratinocytes. Recent studies have further demonstrated that a ceramide metabolite, sphingosine-1-phosphate, modulates innate immune function. Ceramide already has been applied to therapeutic approaches for treatment of eczema associated with attenuated epidermal permeability barrier function. Pharmacological modulation of ceramide and its metabolites signaling can also be applied to cutaneous disease prevention and therapy. The author here describes the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including epidermis. PMID:24055887

  15. Mammalian Polyamine Metabolism and Function

    PubMed Central

    Pegg, Anthony E.

    2009-01-01

    Summary Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism. PMID:19603518

  16. Evaluating and treating mammalian bites.

    PubMed

    Rasmussen, Donna; Landon, Alexandra; Powell, Jennifer; Brown, Gina R

    2017-03-01

    Mammalian bites, typically from dogs, cats, or humans, are a common presentation in EDs and family practice settings, and patients present with varying degrees of complexity. Injuries can range from local to systemic, including aggressive bacterial infections and permanent limb impairment. Using a systematic approach to initial wound assessment, followed by appropriate diagnostic testing and treatment, is critical to improved long-term patient outcomes.

  17. GLUTs and mammalian sperm metabolism.

    PubMed

    Bucci, Diego; Rodriguez-Gil, Juan Enrique; Vallorani, Claudia; Spinaci, Marcella; Galeati, Giovanna; Tamanini, Carlo

    2011-01-01

    Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

  18. Scalable architecture in mammalian brains.

    PubMed

    Clark, D A; Mitra, P P; Wang, S S

    2001-05-10

    Comparison of mammalian brain parts has often focused on differences in absolute size, revealing only a general tendency for all parts to grow together. Attempts to find size-independent effects using body weight as a reference variable obscure size relationships owing to independent variation of body size and give phylogenies of questionable significance. Here we use the brain itself as a size reference to define the cerebrotype, a species-by-species measure of brain composition. With this measure, across many mammalian taxa the cerebellum occupies a constant fraction of the total brain volume (0.13 +/- 0.02), arguing against the hypothesis that the cerebellum acts as a computational engine principally serving the neocortex. Mammalian taxa can be well separated by cerebrotype, thus allowing the use of quantitative neuroanatomical data to test evolutionary relationships. Primate cerebrotypes have progressively shifted and neocortical volume fractions have become successively larger in lemurs and lorises, New World monkeys, Old World monkeys, and hominoids, lending support to the idea that primate brain architecture has been driven by directed selection pressure. At the same time, absolute brain size can vary over 100-fold within a taxon, while maintaining a relatively uniform cerebrotype. Brains therefore constitute a scalable architecture.

  19. Mammalian Alphaherpesvirus miRNAs

    PubMed Central

    Jurak, Igor; Griffiths, Anthony; Coen, Donald M.

    2012-01-01

    Mammalian alphaherpesviruses are major causes of human and veterinary disease. During productive infection, these viruses exhibit complex and robust patterns of gene expression. These viruses also form latent infections in neurons of sensory ganglia in which productive cycle gene expression is highly repressed. Both modes of infection provide advantageous opportunities for regulation by microRNAs. Thus far, published data regarding microRNAs are available for six mammalian alphaherpesviruses. No microRNAs have yet been detected from varicella zoster virus. The five other viruses -- herpes simplex viruses-1 and -2, herpes B virus, bovine herpesvirus-1, and pseudorabies virus -- representing both genera of mammalian alphaherpesviruses have been shown to express microRNAs. In this article, we discuss these microRNAs in terms of where they are encoded in the viral genome relative to other viral transcripts; whether they are expressed during productive or latent infection; their potential targets; what little is known about their actual targets and functions during viral infection; and what little is known about the interactions of these viruses with the host microRNA machinery. PMID:21736960

  20. Functional characterization of mammalian Wntless homolog in mammalian system.

    PubMed

    Wang, Li-Ting; Wang, Shih-Jong; Hsu, Shih-Hsien

    2012-07-01

    Wntless (GPR177) protein is a newly identified regulator of Wnt signals in Drosophila, but its cellular function in mammals is still unclear. In this study, we explored the expression pattern and potential cellular function of Wntless in mammalian cells. Wntless mRNA was expressed in many mouse tissues, including the spleen, lung, kidney, thymus, and stomach, and lower levels of expression were detected in the mouse brain and testis. Expression of Wntless protein analyzed by Western blot and immunohistochemical staining was only detected in the submucosa, muscle, ganglia, and nerve cells of murine large intestines. Both immunofluorescence staining and subcellular fraction extraction analysis revealed that endogenous Wntless protein was expressed predominantly in the cytoplasmic organelles with a morphologically dot-shaped distribution. Furthermore, overexpression of Wntless could be corrected by and may activate the nuclear factor-κB (NF-κB) signaling pathway in cancer (HeLa) cells. These results suggest that Wntless plays a role in signaling regulation during the formation of cancer in addition to its role as a retromer protein in mammalian systems.

  1. Evolutionary paths to mammalian cochleae.

    PubMed

    Manley, Geoffrey A

    2012-12-01

    Evolution of the cochlea and high-frequency hearing (>20 kHz; ultrasonic to humans) in mammals has been a subject of research for many years. Recent advances in paleontological techniques, especially the use of micro-CT scans, now provide important new insights that are here reviewed. True mammals arose more than 200 million years (Ma) ago. Of these, three lineages survived into recent geological times. These animals uniquely developed three middle ear ossicles, but these ossicles were not initially freely suspended as in modern mammals. The earliest mammalian cochleae were only about 2 mm long and contained a lagena macula. In the multituberculate and monotreme mammalian lineages, the cochlea remained relatively short and did not coil, even in modern representatives. In the lineage leading to modern therians (placental and marsupial mammals), cochlear coiling did develop, but only after a period of at least 60 Ma. Even Late Jurassic mammals show only a 270 ° cochlear coil and a cochlear canal length of merely 3 mm. Comparisons of modern organisms, mammalian ancestors, and the state of the middle ear strongly suggest that high-frequency hearing (>20 kHz) was not realized until the early Cretaceous (~125 Ma). At that time, therian mammals arose and possessed a fully coiled cochlea. The evolution of modern features of the middle ear and cochlea in the many later lineages of therians was, however, a mosaic and different features arose at different times. In parallel with cochlear structural evolution, prestins in therian mammals evolved into effective components of a new motor system. Ultrasonic hearing developed quite late-the earliest bat cochleae (~60 Ma) did not show features characteristic of those of modern bats that are sensitive to high ultrasonic frequencies.

  2. Mammalian cloning: advances and limitations.

    PubMed

    Solter, D

    2000-12-01

    For many years, researchers cloning mammals experienced little success, but recent advances have led to the successful cloning of several mammalian species. However, cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not clear what technical and biological factors underlie this. Our understanding of the molecular basis of reprogramming remains extremely limited and affects experimental approaches towards increasing the success rate of cloning. Given the future practical benefits that cloning can offer, the time has come to address what should be done to resolve this problem.

  3. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  4. Mammalian pheromones: emerging properties and mechanisms of detection.

    PubMed

    Stowers, Lisa; Kuo, Tsung-Han

    2015-10-01

    The concept of mammalian pheromones was established decades before the discovery of any bioactive ligands. Therefore, their molecular identity, native sources, and the meaning of their detection has been largely speculative. There has been recent success in identifying a variety of candidate mouse pheromones and other specialized odors. These discoveries reveal that mammalian pheromones come in a variety of ligand types and they are detected by sensory neurons that are pre-set to promote an array of social and survival behaviors. Importantly, recent findings show that they activate molecularly diverse sensory neurons that differ from canonical odorant detectors. These novel sensory neurons hold future promise to unlock the mystery of how their detection is hardwired to generate behavior. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Mammalian pheromones; emerging properties and mechanisms of detection

    PubMed Central

    Stowers, Lisa; Kuo, Tsung-Han

    2015-01-01

    The concept of mammalian pheromones was established decades before the discovery of any bioactive ligands. Therefore, their molecular identity, native sources, and the meaning of their detection has been largely speculative. There has been recent success in identifying a variety of candidate mouse pheromones and other specialized odors. These discoveries reveal that mammalian pheromones come in a variety of ligand types and they are detected by sensory neurons that are pre-set to promote an array of social and survival behaviors. Importantly, recent findings show that they activate molecularly diverse sensory neurons that differ from canonical odorant detectors. These novel sensory neurons hold future promise to unlock the mystery of how their detection is hardwired to generate behavior. PMID:25747731

  6. Ceramide signaling in mammalian epidermis.

    PubMed

    Uchida, Yoshikazu

    2014-03-01

    Ceramide, the backbone structure of all sphingolipids, as well as a minor component of cellular membranes, has a unique role in the skin, by forming the epidermal permeability barrier at the extracellular domains of the outermost layer of the skin, the stratum corneum, which is required for terrestrial mammalian survival. In contrast to the role of ceramide in forming the permeability barrier, the signaling roles of ceramide and its metabolites have not yet been recognized. Ceramide and/or its metabolites regulate proliferation, differentiation, and apoptosis in epidermal keratinocytes. Recent studies have further demonstrated that a ceramide metabolite, sphingosine-1-phosphate, modulates innate immune function. Ceramide has already been applied to therapeutic approaches for treatment of eczema associated with attenuated epidermal permeability barrier function. Pharmacological modulation of ceramide and its metabolites' signaling can also be applied to cutaneous disease prevention and therapy. The author here describes the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including the epidermis. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

  7. The non-mammalian MIF superfamily.

    PubMed

    Sparkes, Amanda; De Baetselier, Patrick; Roelants, Kim; De Trez, Carl; Magez, Stefan; Van Ginderachter, Jo A; Raes, Geert; Bucala, Richard; Stijlemans, Benoît

    2017-03-01

    Macrophage migration inhibitory factor (MIF) was first described as a cytokine 50 years ago, and emerged in mammals as a pleiotropic protein with pro-inflammatory, chemotactic, and growth-promoting activities. In addition, MIF has gained substantial attention as a pivotal upstream mediator of innate and adaptive immune responses and with pathologic roles in several diseases. Of less importance in mammals is an intrinsic but non-physiologic enzymatic activity that points to MIF's evolution from an ancient defense molecule. Therefore, it is not surprising that mif-like genes also have been found across a range of different organisms including bacteria, plants, ‎protozoa, helminths, molluscs, arthropods, fish, amphibians and birds. While Genebank analysis identifying mif-like genes across species is extensive, contained herein is an overview of the non-mammalian MIF-like proteins that have been most well studied experimentally. For many of these organisms, MIF contributes to an innate defense system or plays a role in development. For parasitic organisms however, MIF appears to function as a virulence factor aiding in the establishment or persistence of infection by modulating the host immune response. Consequently, a combined targeting of both parasitic and host MIF could lead to more effective treatment strategies for parasitic diseases of socioeconomic importance.

  8. Programmed cell senescence during mammalian embryonic development.

    PubMed

    Muñoz-Espín, Daniel; Cañamero, Marta; Maraver, Antonio; Gómez-López, Gonzalo; Contreras, Julio; Murillo-Cuesta, Silvia; Rodríguez-Baeza, Alfonso; Varela-Nieto, Isabel; Ruberte, Jesús; Collado, Manuel; Serrano, Manuel

    2013-11-21

    Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  10. Polarity in Mammalian Epithelial Morphogenesis

    PubMed Central

    Roignot, Julie; Peng, Xiao; Mostov, Keith

    2013-01-01

    Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires the intervention of several fundamental cell processes, whose integration in space and time is only starting to be elucidated. To understand what governs the building of epithelial tissues during development, it is essential to consider the polarization process in the context of the whole tissue. To this end, the development of three-dimensional organotypic cell culture models has brought new insights into the mechanisms underlying the establishment and maintenance of higher-order epithelial tissue architecture, and in the dynamic remodeling of cell polarity that often occurs during development of epithelial organs. Here we discuss some important aspects of mammalian epithelial morphogenesis, from the establishment of cell polarity to epithelial tissue generation. PMID:23378592

  11. Producing Newborn Synchronous Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Helmstetter, Charles E.; Thornton, Maureen

    2008-01-01

    A method and bioreactor for the continuous production of synchronous (same age) population of mammalian cells have been invented. The invention involves the attachment and growth of cells on an adhesive-coated porous membrane immersed in a perfused liquid culture medium in a microgravity analog bioreactor. When cells attach to the surface divide, newborn cells are released into the flowing culture medium. The released cells, consisting of a uniform population of synchronous cells are then collected from the effluent culture medium. This invention could be of interest to researchers investigating the effects of the geneotoxic effects of the space environment (microgravity, radiation, chemicals, gases) and to pharmaceutical and biotechnology companies involved in research on aging and cancer, and in new drug development and testing.

  12. Mammalian glutaminase isozymes in brain.

    PubMed

    Márquez, Javier; Cardona, Carolina; Campos-Sandoval, José A; Peñalver, Ana; Tosina, Marta; Matés, José M; Martín-Rufián, Mercedes

    2013-06-01

    Glutamine/glutamate homeostasis must be exquisitely regulated in mammalian brain and glutaminase (GA, E.C. 3.5.1.2) is one of the main enzymes involved. The products of GA reaction, glutamate and ammonia, are essential metabolites for energy and biosynthetic purposes but they are also hazardous compounds at concentrations beyond their normal physiological thresholds. The classical pattern of GA expression in mammals has been recently challenged by the discovery of novel transcript variants and protein isoforms. Furthermore, the interactome of brain GA is also starting to be uncovered adding a new level of regulatory complexity. GA may traffic in brain and unexpected locations, like cytosol and nucleus, have been found for GA isoforms. Finally, the expression of GA in glial cells has been reported and its potential implications in ammonia homeostasis are discussed.

  13. Interaction theory of mammalian mitochondria.

    PubMed

    Nakada, K; Inoue, K; Hayashi, J

    2001-11-09

    We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit.

  14. Determinants of Mammalian Nucleolar Architecture

    PubMed Central

    Farley, Katherine I.; Surovtseva, Yulia; Merkel, Janie; Baserga, Susan J.

    2015-01-01

    The nucleolus is responsible for the production of ribosomes, essential machines which synthesize all proteins needed by the cell. The structure of human nucleoli is highly dynamic and is directly related to its functions in ribosome biogenesis. Despite the importance of this organelle, the intricate relationship between nucleolar structure and function remains largely unexplored. How do cells control nucleolar formation and function? What are the minimal requirements for making a functional nucleolus? Here we review what is currently known regarding mammalian nucleolar formation at nucleolar organizer regions (NORs), which can be studied by observing the dissolution and reformation of the nucleolus during each cell division. Additionally, the nucleolus can be examined by analyzing how alterations in nucleolar function manifest in differences in nucleolar architecture. Furthermore, changes in nucleolar structure and function are correlated with cancer, highlighting the importance of studying the determinants of nucleolar formation. PMID:25670395

  15. Pharmacology of mammalian olfactory receptors.

    PubMed

    Smith, Richard S; Peterlin, Zita; Araneda, Ricardo C

    2013-01-01

    Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.

  16. Body Size in Mammalian Paleobiology

    NASA Astrophysics Data System (ADS)

    Damuth, John; MacFadden, Bruce J.

    1990-11-01

    This valuable collection of essays presents and evaluates techniques of body-mass estimation and reviews current and potential applications of body-size estimates in paleobiology. Papers discuss explicitly the errors and biases of various regression techniques and predictor variables, and the identification of functionally similar groups of species for improving the accuracy of estimates. At the same time other chapters review and discuss the physiological, ecological, and behavioral correlates of body size in extant mammals; the significance of body-mass distributions in mammalian faunas; and the ecology and evolution of body size in particular paleofaunas. Coverage is particularly detailed for carnivores, primates, and ungulates, but information is also presented on marsupials, rodents, and proboscideans.

  17. Mammalian skin evolution: a reevaluation.

    PubMed

    Maderson, P F A

    2003-06-01

    A 1972 model for the evolutionary origin of hair suggested a primary mechanoreceptor role improving behavioral thermoregulation contributed to the success of late Paleozoic mammal-like reptiles. An insulatory role appeared secondarily subsequent to protohair multiplication. That model is updated in light of new data on (a) palaeoecology of mammalian ancestors; (b) involvement of HRPs in keratinization; (c) lipogenic lamellar bodies that form the barrier to cutaneous water loss; and (d) growth factors involved in hair follicle embryogenesis and turnover. It is now proposed that multiplication of sensory protohairs caused by mutations in patterning genes initially protected the delicate barrier tissues and eventually produced the minimal morphology necessary for an insulatory pelage. The latter permitted Mesozoic mammals to occupy the nocturnal niche 'in the shadow of dinosaurs'. When the giant reptiles became extinct, mammals underwent rapid radiation and reemerged as the dominant terrestrial vertebrates.

  18. The mammalian Cretaceous cochlear revolution.

    PubMed

    Manley, Geoffrey A

    2016-12-19

    The hearing organs of amniote vertebrates show large differences in their size and structure between the species' groups. In spite of this, their performance in terms of hearing sensitivity and the frequency selectivity of auditory-nerve units shows unexpectedly small differences. The only substantial difference is that therian, defined as live-bearing, mammalian groups are able to hear ultrasonic frequencies (above 15-20 kHz), whereas in contrast monotreme (egg laying) mammals and all non-mammalian amniotes cannot. This review compares the structure and physiology of the cochleae of the main groups and asks the question as to why the many structural differences seen in therian mammals arose, yet did not result in greater differences in physiology. The likely answers to this question are found in the history of the mammals during the Cretaceous period that ended 65 million years ago. During that period, the therian cochlea lost its lagenar macula, leading to a fall in endolymph calcium levels. This likely resulted in a small revolution and an auditory crisis that was compensated for by a subsequent series of structural and physiological adaptations. The end result was a system of equivalent performance to that independently evolved in other amniotes but with the additional - and of course "unforeseen" - advantage that ultrasonic-frequency responses became an available option. That option was not always availed of, but in most groups of therian mammals it did evolve and is used for communication and orientation based on improved sound localization, with micro-bats and toothed whales relying on it for prey capture.

  19. Genome regulation in mammalian cells.

    PubMed

    Puck, T T; Krystosek, A; Chan, D C

    1990-05-01

    A theory is presented proposing that genetic regulation in mammalian cells is at least a two-tiered effect; that one level of regulation involves the transition between gene exposure and sequestration; that normal differentiation requires a different spectrum of genes to be exposed in each separate state of differentiation; that the fiber systems of the cell cytoskeleton and the nuclear matrix together control the degree of gene exposure; that specific phosphorylation of these elements causes them to assume a different organizational network and to impose a different pattern of sequestration and exposure on the elements of the genome; that the varied gene phosphorylation mechanisms in the cell are integrated in this function; that attachment of this network system to specific parts of the chromosomes brings about sequestration or exposure of the genes in their neighborhood in a fashion similar to that observed when microtubule elements attach through the kinetochore to the centromeric DNA; that one function of repetitive sequences is to serve as elements for the final attachment of this fibrous network to the specific chromosomal loci; and that at least an important part of the calcium manifestation as a metabolic trigger of different differentiation states involves its acting as a binding agent to centers of electronegativity, in particular proteins and especially phosphorylated groups, so as to change the conformation of the fiber network that ultimately controls gene exposure in the mammalian cell. It would appear essential to determine what abnormal gene exposures and sequestrations are characteristic of each type of cancer; which agonists, if any, will bring about reverse transformation; and whether these considerations can be used in therapy.

  20. Mammalian Mitochondrial ncRNA Database.

    PubMed

    Anandakumar, Shanmugam; Vijayakumar, Saravanan; Arumugam, Nagarajan; Gromiha, M Michael

    2015-01-01

    Mammalian Mitochondrial ncRNA is a web-based database, which provides specific information on non-coding RNA in mammals. This database includes easy searching, comparing with BLAST and retrieving information on predicted structure and its function about mammalian ncRNAs. The database is available for free at http://www.iitm.ac.in/bioinfo/mmndb/.

  1. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  2. Photodynamic inactivation of mammalian viruses and bacteriophages.

    PubMed

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  3. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  4. The APC/C in female mammalian meiosis I.

    PubMed

    Homer, Hayden

    2013-08-01

    The anaphase-promoting complex or cyclosome (APC/C) orchestrates a meticulously controlled sequence of proteolytic events critical for proper cell cycle progression, the details of which have been most extensively elucidated during mitosis. It has become apparent, however, that the APC/C, particularly when acting in concert with its Cdh1 co-activator (APC/C(Cdh1)), executes a staggeringly diverse repertoire of functions that extend its remit well outside the bounds of mitosis. Findings over the past decade have not only earmarked mammalian oocyte maturation as one such case in point but have also begun to reveal a complex pattern of APC/C regulation that underpins many of the oocyte's unique developmental attributes. This review will encompass the latest findings pertinent to the APC/C, especially APC/C(Cdh1), in mammalian oocytes and how its activity and substrates shape the stop-start tempo of female mammalian first meiotic division and the challenging requirement for assembling spindles in the absence of centrosomes.

  5. Tissue-specific gene targeting by the multiprotein mammalian DREAM complex.

    PubMed

    Flowers, Stephen; Beck, George R; Moran, Elizabeth

    2011-08-12

    The mammalian DP, RB-like, E2F, and MuvB-like proteins (DREAM) complex, whose key components include p130 and E2F4, plays a fundamental role in repression of cell cycle-specific genes during growth arrest. Mammalian DREAM is well conserved with Drosophila and Caenorhabditis elegans complexes that repress pivotal developmental genes, but the mammalian complex has been thought to exist only in quiescent cells and not to be linked with development. However, new findings here identify tissue-specific promoters repressed by DREAM in proliferating precursors, revealing a new connection between control of growth arrest and terminal differentiation. Mechanistically, tissue-specific promoter occupation by DREAM is dependent on the integrity of a repressor form of the SWI/SNF chromatin-remodeling complex.

  6. Promising roles of mammalian E2Fs in hepatocellular carcinoma.

    PubMed

    Zhan, Lei; Huang, Cheng; Meng, Xiao Ming; Song, Yang; Wu, Xiao Qin; Miu, Cheng Gui; Zhan, Xiang Shu; Li, Jun

    2014-05-01

    In mammalian cells, E2F family of transcription factors (E2Fs) traditionally modulates assorted cellular functions related to cell cycle progression, proliferation, apoptosis and differentiation. Eight members, E2F1 E2F8 have been recognized of this family so far, and the members of this family are generally divided into activator E2F (E2F1--E2F3a), repressor E2F (E2F3b--E2F5) and inhibitor E2F (E2F6--E2F8) subclasses based on their structur-e and function. Studies have showed that the mammalian E2F family members represent a recent evolutionary adaptation to malignancies besides hepatocellular carcinoma (HCC), and a growing body of evidence has validated that the individual members of the family develop a close relationship with HCC. E2F1 was identified to play overlapping roles in HCC, while E2F2--E2F8 (except E2F6 and E2F7) showed to be tumor-promoter in HCC. However, the mechanism underlying the mammalian E2Fs associated with HCC is still unknown and needs further research. The aim of this review is to sum up the collective knowledge of E2F family and the roles of each member of this family in HCC. Moreover, we will discuss some novel therapeutic target for HCC based on the complicated functions of mammalian E2Fs. Copyright © 2014. Published by Elsevier Inc.

  7. Autophagosome formation in mammalian cells.

    PubMed

    Burman, Chloe; Ktistakis, Nicholas T

    2010-12-01

    Autophagy is a fundamental intracellular trafficking pathway conserved from yeast to mammals. It is generally thought to play a pro-survival role, and it can be up regulated in response to both external and intracellular factors, including amino acid starvation, growth factor withdrawal, low cellular energy levels, endoplasmic reticulum (ER) stress, hypoxia, oxidative stress, pathogen infection, and organelle damage. During autophagy initiation a portion of the cytosol is surrounded by a flat membrane sheet known as the isolation membrane or phagophore. The isolation membrane then elongates and seals itself to form an autophagosome. The autophagosome fuses with normal endocytic traffic to mature into a late autophagosome, before fusing with lysosomes. The molecular machinery that enables formation of an autophagosome in response to the various autophagy stimuli is almost completely identified in yeast and-thanks to the observed conservation-is also being rapidly elucidated in higher eukaryotes including mammals. What are less clear and currently under intense investigation are the mechanism by which these various autophagy components co-ordinate in order to generate autophagosomes. In this review, we will discuss briefly the fundamental importance of autophagy in various pathophysiological states and we will then review in detail the various players in early autophagy. Our main thesis will be that a conserved group of heteromeric protein complexes and a relatively simple signalling lipid are responsible for the formation of autophagosomes in mammalian cells.

  8. Mitochondrial dysfunction in mammalian ageing.

    PubMed

    Terzioglu, Mügen; Larsson, Nils-Göran

    2007-01-01

    Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. Increasing age in mammals correlates with increased levels of mitochondrial DNA (mtDNA) mutations and deteriorating respiratory chain function. Mosaic respiratory chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. Experimental evidence in the mouse has linked increased levels of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, greying of the hair, weight reduction and decreased fertility. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and increased cell loss is therefore likely of importance in age-associated mitochondrial dysfunction. There is a tendency to automatically link mitochondrial dysfunction to increased production of reactive oxygen species (ROS). However, the experimental support for this concept is rather weak. Mouse models with respiratory chain deficiency induced by tissue-specific mtDNA depletion or by massive increase of point mutations in mtDNA have very minor or no increase of oxidative stress. Future studies are needed to address the relative importance of mitochondrial dysfunction and ROS in mammalian ageing.

  9. Structure of the mammalian kinetochore.

    PubMed

    Ris, H; Witt, P L

    1981-01-01

    The structure of the mammalian trilaminar kinetochore was investigated using stereo electron microscopy of chromosomes in hypotonic solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed wihout pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were found not to be attached to the kinetochore out layer, but were situated in the fibrous corona on the external surface of the outer layer. This was verified by observation of thick sections in stereo which made it possible to identify microtubules ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached to the outer layer.

  10. Nuclear Organization of Mammalian Genomes

    PubMed Central

    Sadoni, Nicolas; Langer, Sabine; Fauth, Christine; Bernardi, Giorgio; Cremer, Thomas; Turner, Bryan M.; Zink, Daniele

    1999-01-01

    We investigated the nuclear higher order compartmentalization of chromatin according to its replication timing (Ferreira et al. 1997) and the relations of this compartmentalization to chromosome structure and the spatial organization of transcription. Our aim was to provide a comprehensive and integrated view on the relations between chromosome structure and functional nuclear architecture. Using different mammalian cell types, we show that distinct higher order compartments whose DNA displays a specific replication timing are stably maintained during all interphase stages. The organizational principle is clonally inherited. We directly demonstrate the presence of polar chromosome territories that align to build up higher order compartments, as previously suggested (Ferreira et al. 1997). Polar chromosome territories display a specific orientation of early and late replicating subregions that correspond to R- or G/C-bands of mitotic chromosomes. Higher order compartments containing G/C-bands replicating during the second half of the S phase display no transcriptional activity detectable by BrUTP pulse labeling and show no evidence of transcriptional competence. Transcriptionally competent and active chromatin is confined to a coherent compartment within the nuclear interior that comprises early replicating R-band sequences. As a whole, the data provide an integrated view on chromosome structure, nuclear higher order compartmentalization, and their relation to the spatial organization of functional nuclear processes. PMID:10491386

  11. Technology of mammalian cell encapsulation.

    PubMed

    Uludag, H; De Vos, P; Tresco, P A

    2000-08-20

    Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates a cell mass from an outside environment and aims to maintain normal cellular physiology within a desired permeability barrier. Numerous encapsulation techniques have been developed over the years. These techniques are generally classified as microencapsulation (involving small spherical vehicles and conformally coated tissues) and macroencapsulation (involving larger flat-sheet and hollow-fiber membranes). This review is intended to summarize techniques of cell encapsulation as well as methods for evaluating the performance of encapsulated cells. The techniques reviewed include microencapsulation with polyelectrolyte complexation emphasizing alginate-polylysine capsules, thermoreversible gelation with agarose as a prototype system, interfacial precipitation and interfacial polymerization, as well as the technology of flat sheet and hollow fiber-based macroencapsulation. Four aspects of encapsulated cells that are critical for the success of the technology, namely the capsule permeability, mechanical properties, immune protection and biocompatibility, have been singled out and methods to evaluate these properties were summarized. Finally, speculations regarding future directions of cell encapsulation research and device development are included from the authors' perspective.

  12. Mammalian cell cultivation in space

    NASA Astrophysics Data System (ADS)

    Gmünder, Felix K.; Suter, Robert N.; Kiess, M.; Urfer, R.; Nordau, C.-G.; Cogoli, A.

    Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 μl. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

  13. [Chemical defense of plant to mammalian herbivore].

    PubMed

    Li, J; Liu, J

    2001-06-01

    The research progress in the chemical defense of plant to mammalian herbivore was reviewed in this paper. The plant secondary compounds mainly are phenolics, terpenoids and nitrogen-containing compounds. The defense efficiency of plant to mammalian herbivores is different with the types and content of secondary compounds in plant. Secondary compounds inhibited the foraging of mammalian herbivores by affecting the intake, digestion, metabolites and reproduction of animal. It is the main trends to study the mode of coevolution of plant and animals mediated by plant secondary compounds.

  14. Ghrelin Receptors in Non-Mammalian Vertebrates

    PubMed Central

    Kaiya, Hiroyuki; Kangawa, Kenji; Miyazato, Mikiya

    2012-01-01

    The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates. PMID:23882259

  15. High resolution thermal denaturation of mammalian DNAs.

    PubMed Central

    Guttmann, T; Vítek, A; Pivec, L

    1977-01-01

    High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed. PMID:840642

  16. Labeling proteins on live mammalian cells using click chemistry.

    PubMed

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

  17. Synthetic mammalian trigger-controlled bipartite transcription factors

    PubMed Central

    Folcher, Marc; Xie, Mingqi; Spinnler, Andrea; Fussenegger, Martin

    2013-01-01

    Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in

  18. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  19. Transcriptional regulation of mammalian miRNA genes

    PubMed Central

    Schanen, Brian C.; Li, Xiaoman

    2010-01-01

    MicroRNAs (miRNAs) are members of a growing family of non-coding transcripts, 21-23 nucleotides long, which regulate a diverse collection of biological processes and various diseases by RNA-mediated gene-silencing mechanisms. While currently many studies focus on defining the regulatory functions of miRNAs, few are directed towards how miRNA genes are themselves transcriptionally regulated. Recent studies of miRNA transcription have elucidated RNA polymerase II as the major polymerase of miRNAs, however, little is known of the structural features of miRNA promoters, especially those of mammalian miRNAs. Here, we review the current literature regarding features conserved among miRNA promoters useful for their detection and the current novel methodologies available to enable researchers to advance our understanding of the transcriptional regulation of miRNA genes. PMID:20977933

  20. Viral Cyclins Mediate Separate Phases of Infection by Integrating Functions of Distinct Mammalian Cyclins

    PubMed Central

    Lee, Katherine S.; Suarez, Andrea L.; Claypool, David J.; Armstrong, Taylor K.; Buckingham, Erin M.; van Dyk, Linda F.

    2012-01-01

    Gammaherpesvirus cyclins have expanded biochemical features relative to mammalian cyclins, and promote infection and pathogenesis including acute lung infection, viral persistence, and reactivation from latency. To define the essential features of the viral cyclin, we generated a panel of knock-in viruses expressing various viral or mammalian cyclins from the murine gammaherpesvirus 68 cyclin locus. Viral cyclins of both gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus supported all cyclin-dependent stages of infection, indicating functional conservation. Although mammalian cyclins could not restore lung replication, they did promote viral persistence and reactivation. Strikingly, distinct and non-overlapping mammalian cyclins complemented persistence (cyclin A, E) or reactivation from latency (cyclin D3). Based on these data, unique biochemical features of viral cyclins (e.g. enhanced kinase activation) are not essential to mediate specific processes during infection. What is essential for, and unique to, the viral cyclins is the integration of the activities of several different mammalian cyclins, which allows viral cyclins to mediate multiple, discrete stages of infection. These studies also demonstrated that closely related stages of infection, that are cyclin-dependent, are in fact genetically distinct, and thus predict that cyclin requirements may be used to tailor potential therapies for virus-associated diseases. PMID:22319441

  1. Enzymology of Mammalian DNA Methyltransferases.

    PubMed

    Jurkowska, Renata Z; Jeltsch, Albert

    2016-01-01

    DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications.

  2. Chemosignals, Hormones and Mammalian Reproduction

    PubMed Central

    Petrulis, Aras

    2013-01-01

    Many mammalian species use chemosignals to coordinate reproduction by altering the physiology and behavior of both sexes. Chemosignals prime reproductive physiology so that individuals become sexually mature and active at times when mating is most probable and suppress it when it is not. Once in reproductive condition, odors produced and deposited by both males and females are used to find and select individuals for mating. The production, dissemination and appropriate responses to these cues are modulated heavily by organizational and activational effects of gonadal sex steroids and thereby intrinsically link chemical communication to the broader reproductive context. Many compounds have been identified as “pheromones” but very few have met the expectations of that term: a unitary, species-typical substance that is both necessary and sufficient for an experience-independent behavioral or physiological response. In contrast, most responses to chemosignals are dependent or heavily modulated by experience, either in adulthood or during development. Mechanistically, chemosignals are perceived by both main and accessory (vomeronasal) olfactory systems with the importance of each system tied strongly to the nature of the stimulus rather than to the response. In the central nervous system, the vast majority of responses to chemosignals are mediated by cortical and medial amygdala connections with hypothalamic and other forebrain structures. Despite the importance of chemosignals in mammals, many details of chemical communication differ even among closely related species and defy clear categorization. Although generating much research and public interest, strong evidence for the existence of a robust chemical communication among humans is lacking. PMID:23545474

  3. Chemosignals, hormones and mammalian reproduction.

    PubMed

    Petrulis, Aras

    2013-05-01

    Many mammalian species use chemosignals to coordinate reproduction by altering the physiology and behavior of both sexes. Chemosignals prime reproductive physiology so that individuals become sexually mature and active at times when mating is most probable and suppress it when it is not. Once in reproductive condition, odors produced and deposited by both males and females are used to find and select individuals for mating. The production, dissemination and appropriate responses to these cues are modulated heavily by organizational and activational effects of gonadal sex steroids and thereby intrinsically link chemical communication to the broader reproductive context. Many compounds have been identified as "pheromones" but very few have met the expectations of that term: a unitary, species-typical substance that is both necessary and sufficient for an experience-independent behavioral or physiological response. In contrast, most responses to chemosignals are dependent or heavily modulated by experience, either in adulthood or during development. Mechanistically, chemosignals are perceived by both main and accessory (vomeronasal) olfactory systems with the importance of each system tied strongly to the nature of the stimulus rather than to the response. In the central nervous system, the vast majority of responses to chemosignals are mediated by cortical and medial amygdala connections with hypothalamic and other forebrain structures. Despite the importance of chemosignals in mammals, many details of chemical communication differ even among closely related species and defy clear categorization. Although generating much research and public interest, strong evidence for the existence of a robust chemical communication among humans is lacking. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. The life history of retrocopies illuminates the evolution of new mammalian genes.

    PubMed

    Carelli, Francesco Nicola; Hayakawa, Takashi; Go, Yasuhiro; Imai, Hiroo; Warnefors, Maria; Kaessmann, Henrik

    2016-03-01

    New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution.

  5. The life history of retrocopies illuminates the evolution of new mammalian genes

    PubMed Central

    Carelli, Francesco Nicola; Hayakawa, Takashi; Go, Yasuhiro; Imai, Hiroo; Warnefors, Maria; Kaessmann, Henrik

    2016-01-01

    New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88–280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the “life history” of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution. PMID:26728716

  6. Bats and Rodents Shape Mammalian Retroviral Phylogeny.

    PubMed

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-11-09

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general.

  7. Mammalian synthetic biology: emerging medical applications.

    PubMed

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

    2015-05-06

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes.

  8. Mammalian Response to Cenozoic Climatic Change

    NASA Astrophysics Data System (ADS)

    Blois, Jessica L.; Hadly, Elizabeth A.

    2009-05-01

    Multiple episodes of rapid and gradual climatic changes influenced the evolution and ecology of mammalian species and communities throughout the Cenozoic. Climatic change influenced the abundance, genetic diversity, morphology, and geographic ranges of individual species. Within communities these responses interacted to catalyze immigration, speciation, and extinction. Combined they affected long-term patterns of community stability, functional turnover, biotic turnover, and diversity. Although the relative influence of climate on particular evolutionary processes is oft debated, an understanding of processes at the root of biotic change yields important insights into the complexity of mammalian response. Ultimately, all responses trace to events experienced by populations. However, many such processes emerge as patterns above the species level, where shared life history traits and evolutionary history allow us to generalize about mammalian response to climatic change. These generalizations provide the greatest power to understand and predict mammalian responses to current and future global change.

  9. Mammalian synthetic biology: emerging medical applications

    PubMed Central

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  10. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  11. Circadian Plasticity of Mammalian Inhibitory Interneurons

    PubMed Central

    2017-01-01

    Inhibitory interneurons participate in all neuronal circuits in the mammalian brain, including the circadian clock system, and are indispensable for their effective function. Although the clock neurons have different molecular and electrical properties, their main function is the generation of circadian oscillations. Here we review the circadian plasticity of GABAergic interneurons in several areas of the mammalian brain, suprachiasmatic nucleus, neocortex, hippocampus, olfactory bulb, cerebellum, striatum, and in the retina. PMID:28367335

  12. Simplified Bioreactor For Growing Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F.

    1995-01-01

    Improved bioreactor for growing mammalian cell cultures developed. Designed to support growth of dense volumes of mammalian cells by providing ample, well-distributed flows of nutrient solution with minimal turbulence. Cells relatively delicate and, unlike bacteria, cannot withstand shear forces present in turbulent flows. Bioreactor vessel readily made in larger sizes to accommodate greater cell production quantities. Molding equipment presently used makes cylinders up to 30 centimeters long. Alternative sintered plastic techniques used to vary pore size and quantity, as necessary.

  13. Hacking the genetic code of mammalian cells.

    PubMed

    Schwarzer, Dirk

    2009-07-06

    A genetic shuttle: The highlighted article, which was recently published by Schultz, Geierstanger and co-workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line.

  14. PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator.

    PubMed

    Brown, Steven A; Ripperger, Juergen; Kadener, Sebastian; Fleury-Olela, Fabienne; Vilbois, Francis; Rosbash, Michael; Schibler, Ueli

    2005-04-29

    The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. We identified two PER1-associated factors, NONO and WDR5, that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuated circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele were nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augmented PER-mediated transcriptional repression, and its reduction by RNAi diminished circadian histone methylations at the promoter of a clock gene.

  15. Archetype, adaptation and the mammalian heart.

    PubMed

    Meijler, F L; Meijler, T D

    2011-03-01

    Forty years ago, we started our quest for 'The Holy Grail' of understanding ventricular rate control and rhythm in atrial fibrillation (AF). We therefore studied the morphology and function of a wide range of mammalian hearts. From mouse to whale, we found that all hearts show similar structural and functional characteristics. This suggests that the mammalian heart remained well conserved during evolution and in this aspect it differs from other organs and parts of the mammalian body. The archetype of the mammalian heart was apparently so successful that adaptation by natural selection (evolution) caused by varying habitat demands, as occurred in other organs and many other aspects of mammalian anatomy, bypassed the heart. The structure and function of the heart of placental mammals have thus been strikingly conserved throughout evolution. The changes in the mammalian heart that did take place were mostly adjustments (scaling), to compensate for variations in body size and shape. A remarkable scaling effect is, for instance, the difference in atrioventricular (AV) conduction time, which is vital for optimal cardiac function in all mammals, small and large. Scaling of AV conduction takes place in the AV node (AVN), but its substrate is unknown. This sheds new light on the vital role of the AVN in health and disease. The AVN is master and servant of the heart at the same time and is of salient importance for our understanding of supraventricular arrhythmias in humans, especially AF. In Information Technology a software infra-structure called 'enterprise service bus' (ESB) may provide understanding of the mammalian heart's conservation during evolution. The ESB is quite unspecific (and thus general) when compared with the specialised components it has to support. For instance, one of the functions of an ESB is the routing of messages between system nodes. This routing is independent and unaware of the content of the messages. The function of the heart is likewise

  16. Baculoviruses deficient in ie1 gene function abrogate viral gene expression in transduced mammalian cells

    SciTech Connect

    Efrose, Rodica; Swevers, Luc; Iatrou, Kostas

    2010-10-25

    One of the newest niches for baculoviruses-based technologies is their use as vectors for mammalian cell transduction and gene therapy applications. However, an outstanding safety issue related to such use is the residual expression of viral genes in infected mammalian cells. Here we show that infectious baculoviruses lacking the major transcriptional regulator, IE1, can be produced in insect host cells stably transformed with IE1 expression constructs lacking targets of homologous recombination that could promote the generation of wt-like revertants. Such ie1-deficient baculoviruses are unable to direct viral gene transcription to any appreciable degree and do not replicate in normal insect host cells. Most importantly, the residual viral gene expression, which occurs in mammalian cells infected with wt baculoviruses is reduced 10 to 100 fold in cells infected with ie1-deficient baculoviruses. Thus, ie1-deficient baculoviruses offer enhanced safety features to baculovirus-based vector systems destined for use in gene therapy applications.

  17. Evolution of mammalian diving capacity traced by myoglobin net surface charge.

    PubMed

    Mirceta, Scott; Signore, Anthony V; Burns, Jennifer M; Cossins, Andrew R; Campbell, Kevin L; Berenbrink, Michael

    2013-06-14

    Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

  18. Fowlpox virus host range restriction: gene expression, DNA replication, and morphogenesis in nonpermissive mammalian cells.

    PubMed

    Somogyi, P; Frazier, J; Skinner, M A

    1993-11-01

    Fowlpox virus (FPV), type species of the Avipoxvirus genus, causes a slow-spreading pox disease of chickens. Following infection of mammalian cells there is no evidence of productive replication of FPV although cytopathic effects are induced and FPV recombinants have been shown to express foreign genes from vaccinia virus early/late promoters. Here we report results of a study to investigate the expression of FPV genes, the replication of FPV genomic DNA, and any ultrastructural changes in mammalian cells infected by wild-type virus, undertaken as a first step in elucidating the nature of the block (or blocks) to productive replication of FPV in mammalian cells. Early and late gene expression as well as genomic DNA replication was observed in fibroblast-like cell lines of monkey and human origin. Furthermore, viral morphogenesis was observed in monkey cells, with the production mainly of immature particles though smaller numbers of apparently mature virus particles were observed.

  19. Mammalian Cell-Based Sensor System

    NASA Astrophysics Data System (ADS)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  20. A Comparative Study of Mammalian Diversification Pattern

    PubMed Central

    Yu, Wenhua; Xu, Junxiao; Wu, Yi; Yang, Guang

    2012-01-01

    Although mammals have long been regarded as a successful radiation, the diversification pattern among the clades is still poorly known. Higher-level phylogenies are conflicting and comprehensive comparative analyses are still lacking. Using a recently published supermatrix encompassing nearly all extant mammalian families and a novel comparative likelihood approach (MEDUSA), the diversification pattern of mammalian groups was examined. Both order- and family-level phylogenetic analyses revealed the rapid radiation of Boreoeutheria and Euaustralidelphia in the early mammalian history. The observation of a diversification burst within Boreoeutheria at approximately 100 My supports the Long Fuse model in elucidating placental diversification progress, and the rapid radiation of Euaustralidelphia suggests an important role of biogeographic dispersal events in triggering early Australian marsupial rapid radiation. Diversification analyses based on family-level diversity tree revealed seven additional clades with exceptional diversification rate shifts, six of which represent accelerations in net diversification rate as compared to the background pattern. The shifts gave origin to the clades Muridae+Cricetidae, Bovidae+Moschidae+Cervidae, Simiiformes, Echimyidae, Odontoceti (excluding Physeteridae+Kogiidae+Platanistidae), Macropodidae, and Vespertilionidae. Moderate to high extinction rates from background and boreoeutherian diversification patterns indicate the important role of turnovers in shaping the heterogeneous taxonomic richness observed among extant mammalian groups. Furthermore, the present results emphasize the key role of extinction on erasing unusual diversification signals, and suggest that further studies are needed to clarify the historical radiation of some mammalian groups for which MEDUSA did not detect exceptional diversification rates. PMID:22457604

  1. Mammalian African trypanosome VSG coat enhances tsetse's vector competence.

    PubMed

    Aksoy, Emre; Vigneron, Aurélien; Bing, XiaoLi; Zhao, Xin; O'Neill, Michelle; Wu, Yi-Neng; Bangs, James D; Weiss, Brian L; Aksoy, Serap

    2016-06-21

    Tsetse flies are biological vectors of African trypanosomes, the protozoan parasites responsible for causing human and animal trypanosomiases across sub-Saharan Africa. Currently, no vaccines are available for disease prevention due to antigenic variation of the Variant Surface Glycoproteins (VSG) that coat parasites while they reside within mammalian hosts. As a result, interference with parasite development in the tsetse vector is being explored to reduce disease transmission. A major bottleneck to infection occurs as parasites attempt to colonize tsetse's midgut. One critical factor influencing this bottleneck is the fly's peritrophic matrix (PM), a semipermeable, chitinous barrier that lines the midgut. The mechanisms that enable trypanosomes to cross this barrier are currently unknown. Here, we determined that as parasites enter the tsetse's gut, VSG molecules released from trypanosomes are internalized by cells of the cardia-the tissue responsible for producing the PM. VSG internalization results in decreased expression of a tsetse microRNA (mir-275) and interferes with the Wnt-signaling pathway and the Iroquois/IRX transcription factor family. This interference reduces the function of the PM barrier and promotes parasite colonization of the gut early in the infection process. Manipulation of the insect midgut homeostasis by the mammalian parasite coat proteins is a novel function and indicates that VSG serves a dual role in trypanosome biology-that of facilitating transmission through its mammalian host and insect vector. We detail critical steps in the course of trypanosome infection establishment that can serve as novel targets to reduce the tsetse's vector competence and disease transmission.

  2. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

  3. Effect of Microgravity on Mammalian Lymphocytes

    NASA Technical Reports Server (NTRS)

    Banerjee, H.; Blackshear, M.; Mahaffey, K.; Knight, C.; Khan, A. A.; Delucas, L.

    2004-01-01

    The effect of microgravity on mammalian system is an important and interesting topic for scientific investigation, since NASA s objective is to send manned flights to planets like Mars and eventual human colonization.The Astronauts will be exposed to microgravity environment for a long duration of time during these flights.Our objective of research is to conduct in vitro studies for the effect of microgravity on mammalian immune system.We did our preliminary investigations by exposing mammalian lymphocytes to a microgravity simulator cell bioreactor designed by NASA and manufactured at Synthecon Inc (USA).Our initial results showed no significant change in cytokine expression in these cells for a time period of forty eight hours exposure.Our future experiments will involve exposure for a longer period of time.

  4. Effect of Microgravity on Mammalian Lymphocytes

    NASA Technical Reports Server (NTRS)

    Banerjee, H.; Blackshear, M.; Mahaffey, K.; Khan, A. A.; Delucas, L.

    2004-01-01

    The effect of microgravity on mammalian system is an important and interesting topic for scientific investigation, since NASA s objective is to send manned flights to planets like Mars and eventual human colonization. The Astronauts will be exposed to microgravity environment for a long duration of time during these flights. Our objective of research is to conduct in vitro studies for the effect of microgravity on mammalian immune system and nervous system. We did our preliminary investigations by exposing mammalian lymphocytes and astrocyte cells to a microgravity simulator cell bioreactor designed by NASA and manufactured at Synthecon, Inc. (USA).Our initial results showed no significant change in cytokine expression in these cells up to a time period of 120 hours exposure. Our future experiments will involve exposure for a longer period of time.

  5. Involvement of opsins in mammalian sperm thermotaxis

    PubMed Central

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-01-01

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways—the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors. PMID:26537127

  6. Mammalian diversity: gametes, embryos and reproduction.

    PubMed

    Behringer, Richard R; Eakin, Guy S; Renfree, Marilyn B

    2006-01-01

    The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.

  7. Involvement of opsins in mammalian sperm thermotaxis.

    PubMed

    Pérez-Cerezales, Serafín; Boryshpolets, Sergii; Afanzar, Oshri; Brandis, Alexander; Nevo, Reinat; Kiss, Vladimir; Eisenbach, Michael

    2015-11-05

    A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways-the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors.

  8. Requirement of mammalian Timeless for circadian rhythmicity.

    PubMed

    Barnes, Jessica W; Tischkau, Shelley A; Barnes, Jeffrey A; Mitchell, Jennifer W; Burgoon, Penny W; Hickok, Jason R; Gillette, Martha U

    2003-10-17

    Despite a central circadian role in Drosophila for the transcriptional regulator Timeless (dTim), the relevance of mammalian Timeless (mTim) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.

  9. Synthetic therapeutic gene circuits in mammalian cells.

    PubMed

    Ye, Haifeng; Fussenegger, Martin

    2014-08-01

    In the emerging field of synthetic biology, scientists are focusing on designing and creating functional devices, systems, and organisms with novel functions by engineering and assembling standardised biological building blocks. The progress of synthetic biology has significantly advanced the design of functional gene networks that can reprogram metabolic activities in mammalian cells and provide new therapeutic opportunities for future gene- and cell-based therapies. In this review, we describe the most recent advances in synthetic mammalian gene networks designed for biomedical applications, including how these synthetic therapeutic gene circuits can be assembled to control signalling networks and applied to treat metabolic disorders, cancer, and immune diseases. We conclude by discussing the various challenges and future prospects of using synthetic mammalian gene networks for disease therapy. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Synthetic mammalian gene circuits for biomedical applications.

    PubMed

    Ye, Haifeng; Aubel, Dominique; Fussenegger, Martin

    2013-12-01

    Synthetic biology is the science of reassembling cataloged and standardized biological items in a systematic and rational manner to create and engineer functional biological designer devices, systems and organisms with novel and useful, preferably therapeutic functions. Synthetic biology has significantly advanced the design of complex genetic networks that can reprogram metabolic activities in mammalian cells and provide novel therapeutic strategies for future gene-based and cell-based therapies. Synthetic biology-inspired therapeutic strategies provide new opportunities for improving human health in the 21st century. This review covers the most recent synthetic mammalian circuits designed for therapy of diseases such as metabolic disorders, cancer, and immune disorders. We conclude by discussing current challenges and future perspectives for biomedical applications of synthetic mammalian gene networks.

  11. Phase resetting of the mammalian circadian clock by DNA damage.

    PubMed

    Oklejewicz, Małgorzata; Destici, Eugin; Tamanini, Filippo; Hut, Roelof A; Janssens, Roel; van der Horst, Gijsbertus T J

    2008-02-26

    To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

  12. Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

    PubMed Central

    Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

    2014-01-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

  13. Modular construction of mammalian gene circuits using TALE transcriptional repressors

    PubMed Central

    Liao, Weixi; Li, Zhihua; Weiss, Ron; Xie, Zhen

    2014-01-01

    An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator-like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches employing feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation. PMID:25643171

  14. Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

    PubMed

    Forristal, Chantal; Henley, Shauna A; MacDonald, James I; Bush, Jason R; Ort, Carley; Passos, Daniel T; Talluri, Srikanth; Ishak, Charles A; Thwaites, Michael J; Norley, Chris J; Litovchick, Larisa; DeCaprio, James A; DiMattia, Gabriel; Holdsworth, David W; Beier, Frank; Dick, Frederick A

    2014-06-01

    Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Better Smelling Through Genetics: Mammalian Odor Perception

    PubMed Central

    Keller, Andreas; Vosshall, Leslie B.

    2008-01-01

    SUMMARY The increasing availability of genomic and genetic tools to study olfaction—the sense of smell—has brought important new insights into how this chemosensory modality functions in different species. Newly sequenced mammalian genomes—from platypus to dog—have made it possible to infer how smell has evolved to suit the needs of a given species and how variation within a species may affect individual olfactory perception. This review will focus on recent advances in the genetics and genomics of mammalian smell, with a primary focus on rodents and humans. PMID:18938244

  16. A new modification for mammalian messenger RNA.

    PubMed

    Liu, Fange; He, Chuan

    2017-09-01

    The discovery of multiple RNA modifications in the past few years has broadened our views of the structures and potential functions of RNA species, but deciphering which modifications are made where and how remains a challenge. A new study by Xu et al. applies a combination of mass spectrometry, biochemistry, genetics, and cellular biology tools to reveal the two mammalian methyltransferases that are responsible for m(3)C installation in tRNA and a third that mediates the previously unknown installation of m(3)C in mammalian mRNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Mammalian cell cultures for biologics manufacturing.

    PubMed

    Kantardjieff, Anne; Zhou, Weichang

    2014-01-01

    Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations.

  18. The mammalian blastema: regeneration at our fingertips

    PubMed Central

    Simkin, Jennifer; Sammarco, Mimi C.; Dawson, Lindsay A.; Schanes, Paula P.; Yu, Ling

    2015-01-01

    Abstract In the mouse, digit tip regeneration progresses through a series of discrete stages that include inflammation, histolysis, epidermal closure, blastema formation, and redifferentiation. Recent studies reveal how each regenerative stage influences subsequent stages to establish a blastema that directs the successful regeneration of a complex mammalian structure. The focus of this review is on early events of healing and how an amputation wound transitions into a functional blastema. The stepwise formation of a mammalian blastema is proposed to provide a model for how specific targeted treatments can enhance regenerative performance in humans. PMID:27499871

  19. Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

    PubMed Central

    Dorris, David R.; Struhl, Kevin

    2000-01-01

    In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme. PMID:10825198

  20. Non-mammalian model systems for studying neuro-immune interactions after spinal cord injury.

    PubMed

    Bloom, Ona

    2014-08-01

    Mammals exhibit poor recovery after injury to the spinal cord, where the loss of neurons and neuronal connections can be functionally devastating. In contrast, it has long been appreciated that many non-mammalian vertebrate species exhibit significant spontaneous functional recovery after spinal cord injury (SCI). Identifying the biological responses that support an organism's inability or ability to recover function after SCI is an important scientific and medical question. While recent advances have been made in understanding the responses to SCI in mammals, we remain without an effective clinical therapy for SCI. A comparative biological approach to understanding responses to SCI in non-mammalian vertebrates will yield important insights into mechanisms that promote recovery after SCI. Presently, mechanistic studies aimed at elucidating responses, both intrinsic and extrinsic to neurons, that result in different regenerative capacities after SCI across vertebrates are just in their early stages. There are several inhibitory mechanisms proposed to impede recovery from SCI in mammals, including reactive gliosis and scarring, myelin associated proteins, and a suboptimal immune response. One hypothesis to explain the robust regenerative capacity of several non-mammalian vertebrates is a lack of some or all of these inhibitory signals. This review presents the current knowledge of immune responses to SCI in several non-mammalian species that achieve anatomical and functional recovery after SCI. This subject is of growing interest, as studies increasingly show both beneficial and detrimental roles of the immune response following SCI in mammals. A long-term goal of biomedical research in all experimental models of SCI is to understand how to promote functional recovery after SCI in humans. Therefore, understanding immune responses to SCI in non-mammalian vertebrates that achieve functional recovery spontaneously may identify novel strategies to modulate immune

  1. Transplantation of prokaryotic two-component signaling pathways into mammalian cells.

    PubMed

    Hansen, Jonathan; Mailand, Erik; Swaminathan, Krishna Kumar; Schreiber, Joerg; Angelici, Bartolomeo; Benenson, Yaakov

    2014-11-04

    Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.

  2. Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries.

    PubMed

    Gitzinger, Marc; Parsons, Juliana; Reski, Ralf; Fussenegger, Martin

    2009-01-01

    Plants and mammals are separated by a huge evolutionary distance. Consequently, biotechnology and genetics have traditionally been divided into 'green' and 'red'. Here, we provide comprehensive evidence that key components of the mammalian transcription, translation and secretion machineries are functional in the model plant Physcomitrella patens. Cross-kingdom compatibility of different expression modalities originally designed for mammalian cells, such as native and synthetic promoters and polyadenylation sites, viral and cellular internal ribosome entry sites, secretion signal peptides and secreted product proteins, and synthetic transactivators and transrepressors, was established. This mammalian expression portfolio enabled constitutive, conditional and autoregulated expression of different product genes in a multicistronic expression format, optionally adjusted by various trigger molecules, such as butyrolactones, macrolide antibiotics and ethanol. Capitalizing on a cross-kingdom-compatible expression platform, we pioneered a prototype biopharmaceutical manufacturing scenario using microencapsulated transgenic P. patens protoplasts cultivated in a Wave Bioreactor. Vascular endothelial growth factor 121 (VEGF(121)) titres matched those typically achieved by standard protonema populations grown in stirred-tank bioreactors. The full compatibility of mammalian expression systems in P. patens further promotes the use of moss as a cost-effective alternative for the manufacture of complex biopharmaceuticals, and as a valuable host system to advance synthetic biology in plants.

  3. Limited Lifespan of Fragile Regions in Mammalian Evolution

    NASA Astrophysics Data System (ADS)

    Alekseyev, Max A.; Pevzner, Pavel A.

    An important question in genome evolution is whether there exist fragile regions (rearrangement hotspots) where chromosomal rearrangements are happening over and over again. Although nearly all recent studies supported the existence of fragile regions in mammalian genomes, the most comprehensive phylogenomic study of mammals (Ma et al. (2006) Genome Research 16, 1557-1565) raised some doubts about their existence. We demonstrate that fragile regions are subject to a "birth and death" process, implying that fragility has limited evolutionary lifespan. This finding implies that fragile regions migrate to different locations in different mammals, explaining why there exist only a few chromosomal breakpoints shared between different lineages. The birth and death of fragile regions phenomenon reinforces the hypothesis that rearrangements are promoted by matching segmental duplications and suggests putative locations of the currently active fragile regions in the human genome.

  4. Noncoding RNAs: Regulators of the Mammalian Transcription Machinery.

    PubMed

    Eidem, Tess M; Kugel, Jennifer F; Goodrich, James A

    2016-06-19

    Transcription by RNA polymerase II (Pol II) is required to produce mRNAs and some noncoding RNAs (ncRNAs) within mammalian cells. This coordinated process is precisely regulated by multiple factors, including many recently discovered ncRNAs. In this perspective, we will discuss newly identified ncRNAs that facilitate DNA looping, regulate transcription factor binding, mediate promoter-proximal pausing of Pol II, and/or interact with Pol II to modulate transcription. Moreover, we will discuss new roles for ncRNAs, as well as a novel Pol II RNA-dependent RNA polymerase activity that regulates an ncRNA inhibitor of transcription. As the multifaceted nature of ncRNAs continues to be revealed, we believe that many more ncRNA species and functions will be discovered. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Mammalian mitogenomic relationships and the root of the eutherian tree

    PubMed Central

    Arnason, Ulfur; Adegoke, Joseph A.; Bodin, Kristina; Born, Erik W.; Esa, Yuzine B.; Gullberg, Anette; Nilsson, Maria; Short, Roger V.; Xu, Xiufeng; Janke, Axel

    2002-01-01

    The strict orthology of mitochondrial (mt) coding sequences has promoted their use in phylogenetic analyses at different levels. Here we present the results of a mitogenomic study (i.e., analysis based on the set of protein-coding genes from complete mt genomes) of 60 mammalian species. This number includes 11 new mt genomes. The sampling comprises all but one of the traditional eutherian orders. The previously unrepresented order Dermoptera (flying lemurs) fell within Primates as the sister group of Anthropoidea, making Primates paraphyletic. This relationship was strongly supported. Lipotyphla (“insectivores”) split into three distinct lineages: Erinaceomorpha, Tenrecomorpha, and Soricomorpha. Erinaceomorpha was the basal eutherian lineage. Sirenia (dugong) and Macroscelidea (elephant shrew) fell within the African clade. Pholidota (pangolin) joined the Cetferungulata as the sister group of Carnivora. The analyses identified monophyletic Pinnipedia with Otariidae (sea lions, fur seals) and Odobenidae (walruses) as sister groups to the exclusion of Phocidae (true seals). PMID:12034869

  6. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  7. Crossroads between Bacterial and Mammalian Glycosyltransferases

    PubMed Central

    Brockhausen, Inka

    2014-01-01

    Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

  8. The cytogenetics of mammalian autosomal rearrangements

    SciTech Connect

    Daniel, A.

    1988-01-01

    Combining data from animal and clinical studies with classical cytogenetic observations, the volume provides information on various aspects of mammalian autosomal rearrangements. Topics range from the reproductive consequences to carriers of autosomal rearrangements to the application of structural rearrangements and DNA probes to gene mapping. In addition, the book presents an overview of new perspectives and future directions for research.

  9. Architecture of mammalian respiratory complex I.

    PubMed

    Vinothkumar, Kutti R; Zhu, Jiapeng; Hirst, Judy

    2014-11-06

    Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.

  10. Architecture of mammalian respiratory complex I

    PubMed Central

    Hirst, Judy

    2014-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The fourteen conserved ‘core’ subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 ‘supernumerary’ subunits are unknown. Here, we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we significantly advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases. PMID:25209663

  11. Medical and experimental mammalian genetics: A perspective

    SciTech Connect

    McKusick, V.A.; Roderick, T.H.; Mori, J.; Paul, N.W.

    1987-01-01

    This book contains 14 papers. Some of the titles are: Structure and Organization of Mammalian Chromosomes: Normal and Abnormal; Globin Gene Structure and the Nature of Mutation; Retroviral DNA Content of the Mouse Genome; Maternal Genes: Mitochondrial Diseases; Human Evolution; and Prospects for Gene Replacement Therapy.

  12. Structure of mammalian respiratory complex I

    PubMed Central

    Hirst, Judy

    2016-01-01

    Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner membrane. Mammalian complex I1 contains 45 subunits, comprising 14 core subunits that house the catalytic machinery and are conserved from bacteria to humans, and a mammalian-specific cohort of 31 supernumerary subunits1,2. Knowledge about the structures and functions of the supernumerary subunits is fragmentary. Here, we describe a 4.2 Å resolution single-particle cryoEM structure of complex I from Bos taurus. We locate and model all 45 subunits to provide the entire structure of the mammalian complex. Furthermore, computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally-dynamic regions and match biochemical descriptions of the ‘active-to-deactive’ enzyme transition that occurs during hypoxia3,4. Thus, our structures provide a foundation for understanding complex I assembly5 and the effects of mutations that cause clinically-relevant complex I dysfunctions6, insights into the structural and functional roles of the supernumerary subunits, and new information on the mechanism and regulation of catalysis. PMID:27509854

  13. Ticks Take Cues from Mammalian Interferon.

    PubMed

    de Silva, Aravinda M

    2016-07-13

    Interferons are considered a first line of immune defense restricted to vertebrates. In this issue of Cell Host & Microbe, Smith et al. (2016) demonstrate that mammalian interferon γ activates an antimicrobial response within ticks feeding on blood. The study suggests that arthropods have a parallel interferon-like defense system.

  14. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  15. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  16. Database construction for PromoterCAD: synthetic promoter design for mammals and plants.

    PubMed

    Nishikata, Koro; Cox, Robert Sidney; Shimoyama, Sayoko; Yoshida, Yuko; Matsui, Minami; Makita, Yuko; Toyoda, Tetsuro

    2014-03-21

    Synthetic promoters can control a gene's timing, location, and expression level. The PromoterCAD web server ( http://promotercad.org ) allows the design of synthetic promoters to control plant gene expression, by novel arrangement of cis-regulatory elements. Recently, we have expanded PromoterCAD's scope with additional plant and animal data: (1) PLACE (Plant Cis-acting Regulatory DNA Elements), including various sized sequence motifs; (2) PEDB (Mammalian Promoter/Enhancer Database), including gene expression data for mammalian tissues. The plant PromoterCAD data now contains 22 000 Arabidopsis thaliana genes, 2 200 000 microarray measurements in 20 growth conditions and 79 tissue organs and developmental stages, while the new mammalian PromoterCAD data contains 679 Mus musculus genes and 65 000 microarray measurements in 96 tissue organs and cell types ( http://promotercad.org/mammal/ ). This work presents step-by-step instructions for adding both regulatory motif and gene expression data to PromoterCAD, to illustrate how users can expand PromoterCAD functionality for their own applications and organisms.

  17. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina

    NASA Astrophysics Data System (ADS)

    Ooto, Sotaro; Akagi, Tadamichi; Kageyama, Ryoichiro; Akita, Joe; Mandai, Michiko; Honda, Yoshihito; Takahashi, Masayo

    2004-09-01

    It has long been believed that the retina of mature mammals is incapable of regeneration. In this study, using the N-methyl-D-aspartate neurotoxicity model of adult rat retina, we observed that some Müller glial cells were stimulated to proliferate in response to a toxic injury and produce bipolar cells and rod photoreceptors. Although these newly produced neurons were limited in number, retinoic acid treatment promoted the number of regenerated bipolar cells. Moreover, misexpression of basic helix-loop-helix and homeobox genes promoted the induction of amacrine, horizontal, and rod photoreceptor specific phenotypes. These findings demonstrated that retinal neurons regenerated even in adult mammalian retina after toxic injury. Furthermore, we could partially control the fate of the regenerated neurons with extrinsic factors or intrinsic genes. The Müller glial cells constitute a potential source for the regeneration of adult mammalian retina and can be a target for drug delivery and gene therapy in retinal degenerative diseases.

  18. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

    PubMed

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

    2015-09-01

    of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

  19. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells

    PubMed Central

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi

    2015-01-01

    expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells. PMID:26085159

  20. Exendin-4 promotes pancreatic β-cell proliferation via inhibiting the expression of Wnt5a.

    PubMed

    Wu, Xinger; Liang, Weiwei; Guan, Hongyu; Liu, Juan; Liu, Liehua; Li, Hai; He, Xiaoying; Zheng, Jing; Chen, Jie; Cao, Xiaopei; Li, Yanbing

    2017-02-01

    Exendin-4, a glucagon-like peptide-1 receptor agonist, is currently regarded as an effective therapeutic strategy for type-2 diabetes. Previous studies indicated that exendin-4 promoted β cell proliferation. However, the underlying mechanisms remain largely unknown. Recently it was reported that exendin-4 promoted pancreatic β cell proliferation by regulating the expression level of Wnt4. The present study was designed to investigate whether other Wnt isoforms take part in accommodation of β-cell proliferation. We found that exendin-4 promotes the proliferation and suppresses the expression of Wnt5a in INS-1 cell line and C57Bl/6 mouse pancreatic β-cells. Further mechanistic study demonstrated that exendin-4 promoted INS-1 cell proliferation partly through down-regulating the expression of Wnt5a. Furthermore, Wnt5a could induce the activation of calmodulin-dependent protein kinase II in INS-1 cells, thereby decreasing the cellular stable β-catenin and its nuclear translocation, and finally reduce the expression of cyclin D1. In addition, we also found that both of the receptors (Frz-2 and Ror-2) mediated the effect of Wnt5a on β cell line INS-1 proliferation. Taken together, this study suggests that Wnt5a plays a critical role in exendin-4-induced β-cell proliferation, indicating that Wnt5a might be a novel regulator in counterbalance of β cell mass.

  1. Promoter-Based Theranostics for Prostate Cancer

    DTIC Science & Technology

    2016-06-01

    for research contains sequences from bacterial origin. These bacterial sequences and mammalian promoters often rich in unmethylated 2’- deoxyribo...sequence enables plasmid DNA act as an artificial chromosome resulting in prolonged existence of the plasmid over multiple cell divisions. It is also

  2. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

    PubMed

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær; Rank, Julie; Hansen, Bjarne Gram; Andersen, Mikael Rørdam; Mortensen, Uffe Hasbro

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

  3. Lactate Metabolism is Associated with Mammalian Mitochondria

    PubMed Central

    Chen, Ying-Jr; Mahieu, Nathaniel G.; Huang, Xiaojing; Singh, Manmilan; Crawford, Peter A; Johnson, Stephen L.; Gross, Richard W.; Schaefer, Jacob

    2016-01-01

    It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. Within the fermenting cell itself, however, it is generally assumed that lactate is produced to replenish NAD+ and then is secreted. Here we explored the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. With high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells. PMID:27618187

  4. Mammalian lipoxygenases and their biological relevance

    PubMed Central

    Kuhn, Hartmut; Banthiya, Swathi; van Leyen, Klaus

    2015-01-01

    Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOX oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. PMID:25316652

  5. Circadian aspects of mammalian parturition: a review.

    PubMed

    Olcese, James

    2012-02-05

    The identification of circadian clocks in endocrine tissues has added considerable depth and complexity to our understanding of their physiology. A growing body of research reveals circadian clock gene expression in the uterus of non-pregnant and pregnant rodents. This review will focus on the mammalian uterus and its rhythmicity, particularly as it pertains to the circadian timing of parturition. This key event in the reproductive axis shows dramatic species-specific differences in its circadian phase. It is proposed here that these differences in the phasing of mammalian parturition are likely a function of opposite uterine cell responses to humoral cues. The argument will be made that melatonin fulfills many of the criteria to serve as a circadian signal in the initiation of human parturition, including specific actions on uterine smooth muscle cells that are consistent with a role for this hormone in the circadian timing of parturition.

  6. Avian and Mammalian Facilitative Glucose Transporters.

    PubMed

    Byers, Mary Shannon; Howard, Christianna; Wang, Xiaofei

    2017-04-05

    The GLUT members belong to a family of glucose transporter proteins that facilitate glucose transport across the cell membrane. The mammalian GLUT family consists of thirteen members (GLUTs 1-12 and H⁺-myo-inositol transporter (HMIT)). Humans have a recently duplicated GLUT member, GLUT14. Avians express the majority of GLUT members. The arrangement of multiple GLUTs across all somatic tissues signifies the important role of glucose across all organisms. Defects in glucose transport have been linked to metabolic disorders, insulin resistance and diabetes. Despite the essential importance of these transporters, our knowledge regarding GLUT members in avians is fragmented. It is clear that there are no chicken orthologs of mammalian GLUT4 and GLUT7. Our examination of GLUT members in the chicken revealed that some chicken GLUT members do not have corresponding orthologs in mammals. We review the information regarding GLUT orthologs and their function and expression in mammals and birds, with emphasis on chickens and humans.

  7. Potassium transport in the mammalian collecting duct.

    PubMed

    Muto, S

    2001-01-01

    The mammalian collecting duct plays a dominant role in regulating K(+) excretion by the nephron. The collecting duct exhibits axial and intrasegmental cell heterogeneity and is composed of at least two cell types: collecting duct cells (principal cells) and intercalated cells. Under normal circumstances, the collecting duct cell in the cortical collecting duct secretes K(+), whereas under K(+) depletion, the intercalated cell reabsorbs K(+). Assessment of the electrochemical driving forces and of membrane conductances for transcellular and paracellular electrolyte movement, the characterization of several ATPases, patch-clamp investigation, and cloning of the K(+) channel have provided important insights into the role of pumps and channels in those tubule cells that regulate K(+) secretion and reabsorption. This review summarizes K(+) transport properties in the mammalian collecting duct. Special emphasis is given to the mechanisms of how K(+) transport is regulated in the collecting duct.

  8. Mammalian Sperm Motility: Observation and Theory

    NASA Astrophysics Data System (ADS)

    Gaffney, E. A.; Gadêlha, H.; Smith, D. J.; Blake, J. R.; Kirkman-Brown, J. C.

    2011-01-01

    Mammalian spermatozoa motility is a subject of growing importance because of rising human infertility and the possibility of improving animal breeding. We highlight opportunities for fluid and continuum dynamics to provide novel insights concerning the mechanics of these specialized cells, especially during their remarkable journey to the egg. The biological structure of the motile sperm appendage, the flagellum, is described and placed in the context of the mechanics underlying the migration of mammalian sperm through the numerous environments of the female reproductive tract. This process demands certain specific changes to flagellar movement and motility for which further mechanical insight would be valuable, although this requires improved modeling capabilities, particularly to increase our understanding of sperm progression in vivo. We summarize current theoretical studies, highlighting the synergistic combination of imaging and theory in exploring sperm motility, and discuss the challenges for future observational and theoretical studies in understanding the underlying mechanics.

  9. Expanding the genetic code of mammalian cells.

    PubMed

    Italia, James S; Zheng, Yunan; Kelemen, Rachel E; Erickson, Sarah B; Addy, Partha S; Chatterjee, Abhishek

    2017-04-15

    In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Ricin trafficking in plant and mammalian cells.

    PubMed

    Lord, J Michael; Spooner, Robert A

    2011-07-01

    Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.

  11. Synaptic Release at Mammalian Bipolar Cell Terminals

    PubMed Central

    Wan, Qun-Fang; Heidelberger, Ruth

    2011-01-01

    Bipolar cells play a vital role in the transfer of visual information across the vertebrate retina. The synaptic output of these neurons is regulated by factors that are extrinsic and intrinsic. Relatively little is known about the intrinsic factors that regulate neurotransmitter exocytosis. Much of what we know about intrinsic presynaptic mechanisms that regulate glutamate release has come from the study of the unusually large and accessible synaptic terminal of the goldfish rod-dominant bipolar cell, the Mb1 bipolar cell. However, over the past several years, examination of presynaptic mechanisms governing neurotransmitter release has been extended to the mammalian rod bipolar cell. In this review, we discuss the recent advances in our understanding of synaptic vesicle dynamics and neurotransmitter release in rodent rod bipolar cells and consider how these properties help shape the synaptic output of the mammalian retina. PMID:21272392

  12. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  13. Isolation of genomic DNA from mammalian cells.

    PubMed

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Mammalian Evolution May not Be Strictly Bifurcating

    PubMed Central

    Hallström, Björn M.; Janke, Axel

    2010-01-01

    The massive amount of genomic sequence data that is now available for analyzing evolutionary relationships among 31 placental mammals reduces the stochastic error in phylogenetic analyses to virtually zero. One would expect that this would make it possible to finally resolve controversial branches in the placental mammalian tree. We analyzed a 2,863,797 nucleotide-long alignment (3,364 genes) from 31 placental mammals for reconstructing their evolution. Most placental mammalian relationships were resolved, and a consensus of their evolution is emerging. However, certain branches remain difficult or virtually impossible to resolve. These branches are characterized by short divergence times in the order of 1–4 million years. Computer simulations based on parameters from the real data show that as little as about 12,500 amino acid sites could be sufficient to confidently resolve short branches as old as about 90 million years ago (Ma). Thus, the amount of sequence data should no longer be a limiting factor in resolving the relationships among placental mammals. The timing of the early radiation of placental mammals coincides with a period of climate warming some 100–80 Ma and with continental fragmentation. These global processes may have triggered the rapid diversification of placental mammals. However, the rapid radiations of certain mammalian groups complicate phylogenetic analyses, possibly due to incomplete lineage sorting and introgression. These speciation-related processes led to a mosaic genome and conflicting phylogenetic signals. Split network methods are ideal for visualizing these problematic branches and can therefore depict data conflict and possibly the true evolutionary history better than strictly bifurcating trees. Given the timing of tectonics, of placental mammalian divergences, and the fossil record, a Laurasian rather than Gondwanan origin of placental mammals seems the most parsimonious explanation. PMID:20591845

  15. Structure and function of mammalian cilia.

    PubMed

    Satir, Peter; Christensen, Søren T

    2008-06-01

    In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease.

  16. Comparison of amphibian and mammalian thyroperoxidase ...

    EPA Pesticide Factsheets

    Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high con

  17. Epigenetic Regulation of the Mammalian Cell

    PubMed Central

    Baverstock, Keith; Rönkkö, Mauno

    2008-01-01

    Background Understanding how mammalian cells are regulated epigenetically to express phenotype is a priority. The cellular phenotypic transition, induced by ionising radiation, from a normal cell to the genomic instability phenotype, where the ability to replicate the genotype accurately is compromised, illustrates important features of epigenetic regulation. Based on this phenomenon and earlier work we propose a model to describe the mammalian cell as a self assembled open system operating in an environment that includes its genotype, neighbouring cells and beyond. Phenotype is represented by high dimensional attractors, evolutionarily conditioned for stability and robustness and contingent on rules of engagement between gene products encoded in the genetic network. Methodology/Findings We describe how this system functions and note the indeterminacy and fluidity of its internal workings which place it in the logical reasoning framework of predicative logic. We find that the hypothesis is supported by evidence from cell and molecular biology. Conclusions Epigenetic regulation and memory are fundamentally physical, as opposed to chemical, processes and the transition to genomic instability is an important feature of mammalian cells with probable fundamental relevance to speciation and carcinogenesis. A source of evolutionarily selectable variation, in terms of the rules of engagement between gene products, is seen as more likely to have greater prominence than genetic variation in an evolutionary context. As this epigenetic variation is based on attractor states phenotypic changes are not gradual; a phenotypic transition can involve the changed contribution of several gene products in a single step. PMID:18523589

  18. Mutation hot spots in mammalian mitochondrial DNA.

    PubMed

    Galtier, Nicolas; Enard, David; Radondy, Yoan; Bazin, Eric; Belkhir, Khalid

    2006-02-01

    Animal mitochondrial DNA is characterized by a remarkably high level of within-species homoplasy, that is, phylogenetic incongruence between sites of the molecule. Several investigators have invoked recombination to explain it, challenging the dogma of maternal, clonal mitochondrial inheritance in animals. Alternatively, a high level of homoplasy could be explained by the existence of mutation hot spots. By using an exhaustive mammalian data set, we test the hot spot hypothesis by comparing patterns of site-specific polymorphism and divergence in several groups of closely related species, including hominids. We detect significant co-occurrence of synonymous polymorphisms among closely related species in various mammalian groups, and a correlation between the site-specific levels of variability within humans (on one hand) and between Hominoidea species (on the other hand), indicating that mutation hot spots actually exist in mammalian mitochondrial coding regions. The whole data, however, cannot be explained by a simple mutation hot spots model. Rather, we show that the site-specific mutation rate quickly varies in time, so that the same sites are not hypermutable in distinct lineages. This study provides a plausible mutation model that potentially accounts for the peculiar distribution of mitochondrial sequence variation in mammals without the need for invoking recombination. It also gives hints about the proximal causes of mitochondrial site-specific hypermutability in humans.

  19. Mammalian Synthetic Biology: Engineering Biological Systems.

    PubMed

    Black, Joshua B; Perez-Pinera, Pablo; Gersbach, Charles A

    2017-06-21

    The programming of new functions into mammalian cells has tremendous application in research and medicine. Continued improvements in the capacity to sequence and synthesize DNA have rapidly increased our understanding of mechanisms of gene function and regulation on a genome-wide scale and have expanded the set of genetic components available for programming cell biology. The invention of new research tools, including targetable DNA-binding systems such as CRISPR/Cas9 and sensor-actuator devices that can recognize and respond to diverse chemical, mechanical, and optical inputs, has enabled precise control of complex cellular behaviors at unprecedented spatial and temporal resolution. These tools have been critical for the expansion of synthetic biology techniques from prokaryotic and lower eukaryotic hosts to mammalian systems. Recent progress in the development of genome and epigenome editing tools and in the engineering of designer cells with programmable genetic circuits is expanding approaches to prevent, diagnose, and treat disease and to establish personalized theranostic strategies for next-generation medicines. This review summarizes the development of these enabling technologies and their application to transforming mammalian synthetic biology into a distinct field in research and medicine.

  20. Aneuploidy in mammalian somatic cells in vivo.

    PubMed

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed.

  1. Mammalian masticatory muscles: homology, nomenclature, and diversification.

    PubMed

    Druzinsky, Robert E; Doherty, Alison H; De Vree, Frits L

    2011-08-01

    There is a deep and rich literature of comparative studies of jaw muscles in mammals but no recent analyses employ modern phylogenetic techniques to better understand evolutionary changes that have occurred in these muscles. In order to fully develop and utilize the Feeding Experiments End-user Database (FEED), we are constructing a comprehensive ontology of mammalian jaw muscles. This process has led to a careful consideration of nomenclature and homologies of the muscles and their constituent parts. Precise determinations of muscle attachments have shown that muscles with similar names are not necessarily homologous. Using new anatomical descriptions derived from the literature, we defined character states for the jaw muscles in diverse mammalian species. We then mapped those characters onto a recent phylogeny of mammals with the aid of the Mesquite software package. Our data further elucidate how muscle groups associated with the feeding apparatus differ and have become highly specialized in certain mammalian orders, such as Rodentia, while remaining conserved in other orders. We believe that careful naming of muscles and statistical analyses of their distributions among mammals, in association with the FEED database, will lead to new, significant insights into the functional, structural, and evolutionary morphology of the jaw muscles.

  2. Some principles of regeneration in mammalian systems.

    PubMed

    Carlson, Bruce M

    2005-11-01

    This article presents some general principles underlying regenerative phenomena in vertebrates, starting with the epimorphic regeneration of the amphibian limb and continuing with tissue and organ regeneration in mammals. Epimorphic regeneration following limb amputation involves wound healing, followed shortly by a phase of dedifferentiation that leads to the formation of a regeneration blastema. Up to the point of blastema formation, dedifferentiation is guided by unique regenerative pathways, but the overall developmental controls underlying limb formation from the blastema generally recapitulate those of embryonic limb development. Damaged mammalian tissues do not form a blastema. At the cellular level, differentiation follows a pattern close to that seen in the embryo, but at the level of the tissue and organ, regeneration is strongly influenced by conditions inherent in the local environment. In some mammalian systems, such as the liver, parenchymal cells contribute progeny to the regenerate. In others, e.g., skeletal muscle and bone, tissue-specific progenitor cells constitute the main source of regenerating cells. The substrate on which regeneration occurs plays a very important role in determining the course of regeneration. Epimorphic regeneration usually produces an exact replica of the structure that was lost, but in mammalian tissue regeneration the form of the regenerate is largely determined by the mechanical environment acting on the regenerating tissue, and it is normally an imperfect replica of the original. In organ hypertophy, such as that occurring after hepatic resection, the remaining liver mass enlarges, but there is no attempt to restore the original form.

  3. Comparison of amphibian and mammalian thyroperoxidase ...

    EPA Pesticide Factsheets

    Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high con

  4. MAMMALIAN CELLS CONTAIN A SECOND NUCLEOCYTOPLASMIC HEXOSAMINIDASE

    PubMed Central

    Gutternigg, Martin; Rendić, Dubravko; Voglauer, Regina; Iskratsch, Thomas; Wilson, Iain B. H.

    2010-01-01

    Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterised in recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterise for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability; furthermore, a myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localisation. Transcripts of the corresponding gene are expressed in a number of murine tissues. Based on its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be found from mammalian sources. PMID:19040401

  5. Entry of the bacterial pathogen Listeria monocytogenes into mammalian cells.

    PubMed

    Ireton, Keith

    2007-06-01

    The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to meningitis or abortion. Listeria provokes its internalization ('entry') into mammalian cells that are normally non-phagocytic, such as intestinal epithelial cells and hepatocytes. Entry provides access to a nutrient-rich cytosol and allows translocation across anatomical barriers. Here I discuss the two major internalization pathways used by Listeria. These pathways are initiated by binding of the bacterial surface proteins InlA or InlB to their respective host receptors, E-cadherin or Met. InlA mediates traversal of the intestinal barrier, whereas InlB promotes infection of the liver. At the cellular level, both InlA- and InlB-dependent entry require host signalling that promotes cytoskeletal rearrangements and pathogen engulfment. However, many of the specific signalling proteins in the two entry routes differ. InlA-mediated uptake uses components of adherens junctions that are coupled to F-actin and myosin, whereas InlB-dependent entry involves cytosolic adaptors that bridge Met to regulators of F-actin, including phosphoinositide 3-kinase and activators of the Arp2/3 complex. Unexpectedly, entry directed by InlB also involves endocytic components. Future work on InlA and InlB will lead to a better understanding of virulence, and may also provide novel insights into the normal biological functions of E-cadherin and Met.

  6. A Mammalian Circadian Clock Model Incorporating Daytime Expression Elements

    PubMed Central

    Jolley, Craig C.; Ukai-Tadenuma, Maki; Perrin, Dimitri; Ueda, Hiroki R.

    2014-01-01

    Models of the mammalian clock have traditionally been based around two feedback loops—the self-repression of Per/Cry by interfering with activation by BMAL/CLOCK, and the repression of Bmal/Clock by the REV-ERB proteins. Recent experimental evidence suggests that the D-box, a transcription factor binding site associated with daytime expression, plays a larger role in clock function than has previously been understood. We present a simplified clock model that highlights the role of the D-box and illustrate an approach for finding maximum-entropy ensembles of model parameters, given experimentally imposed constraints. Parameter variability can be mitigated using prior probability distributions derived from genome-wide studies of cellular kinetics. Our model reproduces predictions concerning the dual regulation of Cry1 by the D-box and Rev-ErbA/ROR response element (RRE) promoter elements and allows for ensemble-based predictions of phase response curves (PRCs). Nonphotic signals such as Neuropeptide Y (NPY) may act by promoting Cry1 expression, whereas photic signals likely act by stimulating expression from the E/E' box. Ensemble generation with parameter probability restraints reveals more about a model’s behavior than a single optimal parameter set. PMID:25229153

  7. Gene organization inside replication domains in mammalian genomes

    NASA Astrophysics Data System (ADS)

    Zaghloul, Lamia; Baker, Antoine; Audit, Benjamin; Arneodo, Alain

    2012-11-01

    We investigate the large-scale organization of human genes with respect to "master" replication origins that were previously identified as bordering nucleotide compositional skew domains. We separate genes in two categories depending on their CpG enrichment at the promoter which can be considered as a marker of germline DNA methylation. Using expression data in mouse, we confirm that CpG-rich genes are highly expressed in germline whereas CpG-poor genes are in a silent state. We further show that, whether tissue-specific or broadly expressed (housekeeping genes), the CpG-rich genes are over-represented close to the replication skew domain borders suggesting some coordination of replication and transcription. We also reveal that the transcription of the longest CpG-rich genes is co-oriented with replication fork progression so that the promoter of these transcriptionally active genes be located into the accessible open chromatin environment surrounding the master replication origins that border the replication skew domains. The observation of a similar gene organization in the mouse genome confirms the interplay of replication, transcription and chromatin structure as the cornerstone of mammalian genome architecture.

  8. Firefly luciferase gene: structure and expression in mammalian cells.

    PubMed Central

    de Wet, J R; Wood, K V; DeLuca, M; Helinski, D R; Subramani, S

    1987-01-01

    The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression. Images PMID:3821727

  9. Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian embryos.

    PubMed

    Blakewood, E G; Jaynes, J M; Johnson, W A; Godke, R A

    1989-12-01

    The fertile chicken egg may provide an effective, inexpensive method for promoting the development of early-stage embryos from other species. Presently, the loss of viability associated with the in vitro culture of mammalian embryos is hindering the use of in vitro fertilization with farm animals. Consequently, alternative in vitro laboratory methods are needed for the culture of mammalian embryos. A new method has been developed that involves the culture of mammalian embryos in the amniotic cavity of a developing chick embryo. Chick embryos were placed into shell-less incubation (37 C) at the 72-h developmental stage. After 24 h of shell-less incubation, agarose-embedded mammalian embryos were injected into the amniotic cavity of the chick embryo. The mammalian embryos were first placed into a drop of liquid agarose. One to four embryos were then aspirated into a beveled injection pipette and cooled, allowing the agarose to harden. Following penetration of the amnion with the beveled pipette, the agarose cylinder containing the embryos was expelled into the amniotic cavity. The shell-less culture system was then returned to incubation at 37 C for an additional 72 to 96 h. Following incubation, the amniotic cavity containing both chick and mammalian embryos was isolated and the agarose-embedded mammalian embryos were harvested. Significantly more embryos developed in the chick embryo amnion than in the control medium alone. Results obtained using this method on laboratory animals (mice) and on domestic mammals (goats and cattle) indicate that the chick-embryo amnion can support the development of early-stage, mammalian embryos to the blastocyst stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Engineering Escherichia coli into a protein delivery system for mammalian cells.

    PubMed

    Reeves, Analise Z; Spears, William E; Du, Juan; Tan, Kah Yong; Wagers, Amy J; Lesser, Cammie F

    2015-05-15

    Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

  11. Arctigenin from Fructus Arctii is a novel suppressor of heat shock response in mammalian cells

    PubMed Central

    Ishihara, Keiichi; Yamagishi, Nobuyuki; Saito, Youhei; Takasaki, Midori; Konoshima, Takao; Hatayama, Takumi

    2006-01-01

    Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoter-regulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy. PMID:16817321

  12. Life in the cold: links between mammalian hibernation and longevity.

    PubMed

    Wu, Cheng-Wei; Storey, Kenneth B

    2016-02-01

    The biological process of aging is the primary determinant of lifespan, but the factors that influence the rate of aging are not yet clearly understood and remain a challenging question. Mammals are characterized by >100-fold differences in maximal lifespan, influenced by relative variances in body mass and metabolic rate. Recent discoveries have identified long-lived mammalian species that deviate from the expected longevity quotient. A commonality among many long-lived species is the capacity to undergo metabolic rate depression, effectively re-programming normal metabolism in response to extreme environmental stress and enter states of torpor or hibernation. This stress tolerant phenotype often involves a reduction in overall metabolic rate to just 1-5% of the normal basal rate as well as activation of cytoprotective responses. At the cellular level, major energy savings are achieved via coordinated suppression of many ATP-expensive cell functions; e.g. global rates of protein synthesis are strongly reduced via inhibition of the insulin signaling axis. At the same time, various studies have shown activation of stress survival signaling during hibernation including up-regulation of protein chaperones, increased antioxidant defenses, and transcriptional activation of pro-survival signaling such as the FOXO and p53 pathways. Many similarities and parallels exist between hibernation phenotypes and different long-lived models, e.g. signal transduction pathways found to be commonly regulated during hibernation are also known to induce lifespan extension in animals such as Drosophila melanogaster and Caenorhabditis elegans. In this review, we highlight some of the molecular mechanisms that promote longevity in classic aging models C. elegans, Drosophila, and mice, while providing a comparative analysis to how they are regulated during mammalian hibernation.

  13. Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

    PubMed Central

    2015-01-01

    Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics. PMID:25360681

  14. Systematic transfer of prokaryotic sensors and circuits to mammalian cells.

    PubMed

    Stanton, Brynne C; Siciliano, Velia; Ghodasara, Amar; Wroblewska, Liliana; Clancy, Kevin; Trefzer, Axel C; Chesnut, Jonathan D; Weiss, Ron; Voigt, Christopher A

    2014-12-19

    Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10(5) members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.

  15. The calming effect of maternal carrying in different mammalian species.

    PubMed

    Esposito, Gianluca; Setoh, Peipei; Yoshida, Sachine; Kuroda, Kumi O

    2015-01-01

    Attachment theory postulates that mothers and their infants possess some basic physiological mechanisms that favor their dyadic interaction and bonding. Many studies have focused on the maternal physiological mechanisms that promote attachment (e.g., mothers' automatic responses to infant faces and/or cries), and relatively less have examined infant physiology. Thus, the physiological mechanisms regulating infant bonding behaviors remain largely undefined. This review elucidates some of the neurobiological mechanisms governing social bonding and cooperation in humans by focusing on maternal carrying and its beneficial effect on mother-infant interaction in mammalian species (e.g., in humans, big cats, and rodents). These studies show that infants have a specific calming response to maternal carrying. A human infant carried by his/her walking mother exhibits a rapid heart rate decrease, and immediately stops voluntary movement and crying compared to when he/she is held in a sitting position. Furthermore, strikingly similar responses were identified in mouse rodents, who exhibit immobility, diminished ultra-sonic vocalizations and heart rate. In general, the studies described in the current review demonstrate the calming effect of maternal carrying to be comprised of a complex set of behavioral and physiological components, each of which has a specific postnatal time window and is orchestrated in a well-matched manner with the maturation of the infants. Such reactions could have been evolutionarily adaptive in mammalian mother-infant interactions. The findings have implications for parenting practices in developmentally normal populations. In addition, we propose that infants' physiological response may be useful in clinical assessments as we discuss possible implications on early screening for child psychopathology (e.g., autism spectrum disorders and perinatal brain disorders).

  16. Functional Screening of Core Promoter Activity.

    PubMed

    Even, Dan Y; Kedmi, Adi; Ideses, Diana; Juven-Gershon, Tamar

    2017-01-01

    The core promoter is the DNA sequence that recruits the basal transcription machinery and directs accurate initiation of transcription. It is an active contributor to gene expression that can be rationally designed to manipulate the levels of expression. Core promoter function can be analyzed using different experimental approaches. Here, we describe the qualitative and quantitative analysis of engineered core promoter functions using the EGFP reporter gene that is driven by distinct core promoters. Expression plasmids are transfected into different mammalian cell lines, and the resulting fluorescence is monitored by live cell imaging , as well as by flow cytometry. In order to verify that the transcriptional activity of the examined core promoters is indeed a function of their activity, as opposed to differences in DNA uptake, real-time quantitative PCR analysis is performed. Importantly, the described methodology for functional screening of core promoter activity has enabled the analysis of engineered potent core promoters for extended time periods.

  17. ACE2 orthologues in non-mammalian vertebrates (Danio, Gallus, Fugu, Tetraodon and Xenopus).

    PubMed

    Chou, Chih-Fong; Loh, Chay Boon; Foo, Yik Khoon; Shen, Shuo; Fielding, Burtram C; Tan, Timothy H P; Khan, Sehaam; Wang, Yue; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo; Fu, Jianlin

    2006-08-01

    Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin-angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3'RACE. Analyses of the exon-intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.

  18. Protein and genome evolution in Mammalian cells for biotechnology applications.

    PubMed

    Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

    2009-06-01

    Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

  19. Mammalian Niche Conservation through Deep Time

    PubMed Central

    DeSantis, Larisa R. G.; Beavins Tracy, Rachel A.; Koontz, Cassandra S.; Roseberry, John C.; Velasco, Matthew C.

    2012-01-01

    Climate change alters species distributions, causing plants and animals to move north or to higher elevations with current warming. Bioclimatic models predict species distributions based on extant realized niches and assume niche conservation. Here, we evaluate if proxies for niches (i.e., range areas) are conserved at the family level through deep time, from the Eocene to the Pleistocene. We analyze the occurrence of all mammalian families in the continental USA, calculating range area, percent range area occupied, range area rank, and range polygon centroids during each epoch. Percent range area occupied significantly increases from the Oligocene to the Miocene and again from the Pliocene to the Pleistocene; however, mammalian families maintain statistical concordance between rank orders across time. Families with greater taxonomic diversity occupy a greater percent of available range area during each epoch and net changes in taxonomic diversity are significantly positively related to changes in percent range area occupied from the Eocene to the Pleistocene. Furthermore, gains and losses in generic and species diversity are remarkably consistent with ∼2.3 species gained per generic increase. Centroids demonstrate southeastern shifts from the Eocene through the Pleistocene that may correspond to major environmental events and/or climate changes during the Cenozoic. These results demonstrate range conservation at the family level and support the idea that niche conservation at higher taxonomic levels operates over deep time and may be controlled by life history traits. Furthermore, families containing megafauna and/or terminal Pleistocene extinction victims do not incur significantly greater declines in range area rank than families containing only smaller taxa and/or only survivors, from the Pliocene to Pleistocene. Collectively, these data evince the resilience of families to climate and/or environmental change in deep time, the absence of terminal Pleistocene

  20. Mammalian niche conservation through deep time.

    PubMed

    DeSantis, Larisa R G; Beavins Tracy, Rachel A; Koontz, Cassandra S; Roseberry, John C; Velasco, Matthew C

    2012-01-01

    Climate change alters species distributions, causing plants and animals to move north or to higher elevations with current warming. Bioclimatic models predict species distributions based on extant realized niches and assume niche conservation. Here, we evaluate if proxies for niches (i.e., range areas) are conserved at the family level through deep time, from the Eocene to the Pleistocene. We analyze the occurrence of all mammalian families in the continental USA, calculating range area, percent range area occupied, range area rank, and range polygon centroids during each epoch. Percent range area occupied significantly increases from the Oligocene to the Miocene and again from the Pliocene to the Pleistocene; however, mammalian families maintain statistical concordance between rank orders across time. Families with greater taxonomic diversity occupy a greater percent of available range area during each epoch and net changes in taxonomic diversity are significantly positively related to changes in percent range area occupied from the Eocene to the Pleistocene. Furthermore, gains and losses in generic and species diversity are remarkably consistent with ~2.3 species gained per generic increase. Centroids demonstrate southeastern shifts from the Eocene through the Pleistocene that may correspond to major environmental events and/or climate changes during the Cenozoic. These results demonstrate range conservation at the family level and support the idea that niche conservation at higher taxonomic levels operates over deep time and may be controlled by life history traits. Furthermore, families containing megafauna and/or terminal Pleistocene extinction victims do not incur significantly greater declines in range area rank than families containing only smaller taxa and/or only survivors, from the Pliocene to Pleistocene. Collectively, these data evince the resilience of families to climate and/or environmental change in deep time, the absence of terminal Pleistocene

  1. Mammalian developmental genetics in the twentieth century.

    PubMed

    Artzt, Karen

    2012-12-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas.

  2. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  3. Primer removal during mammalian mitochondrial DNA replication.

    PubMed

    Uhler, Jay P; Falkenberg, Maria

    2015-10-01

    The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  5. Derivation of the mammalian skull vault

    PubMed Central

    MORRISS-KAY, GILLIAN M.

    2001-01-01

    This review describes the evolutionary history of the mammalian skull vault as a basis for understanding its complex structure. Current information on the developmental tissue origins of the skull vault bones (mesoderm and neural crest) is assessed for mammals and other tetrapods. This information is discussed in the context of evolutionary changes in the proportions of the skull vault bones at the sarcopterygian-tetrapod transition. The dual tissue origin of the skull vault is considered in relation to the molecular mechanisms underlying osteogenic cell proliferation and differentiation in the sutural growth centres and in the proportionate contributions of different sutures to skull growth. PMID:11523816

  6. Mammalian odorant receptors: functional evolution and variation

    PubMed Central

    Jiang, Yue; Matsunami, Hiroaki

    2015-01-01

    In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. The mammalian OR repertoire has been subject to rapid evolution, and is highly diverse within the human population. Recent advances in the functional expression and ligand identification of ORs allow for functional analysis of OR evolution, and reveal that changes in OR protein sequences translate into high degrees of functional variations. Moreover, in several cases the functional variation of a single OR affects the perception of its cognate odor ligand, providing clues as to how an odor is coded at the receptor level. PMID:25660959

  7. Genome Editing Using Mammalian Haploid Cells

    PubMed Central

    Horii, Takuro; Hatada, Izuho

    2015-01-01

    Haploid cells are useful for studying gene functions because disruption of a single allele can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem cells (ESCs) in mice, rats, and monkeys provides a new platform for simple genetic manipulation of the mammalian genome. Use of haploid ESCs enhances the genome-editing potential of the CRISPR/Cas system. For example, CRISPR/Cas was used in haploid ESCs to generate multiple knockouts and large deletions at high efficiency. In addition, genome-wide screening is facilitated by haploid cell lines containing gene knockout libraries. PMID:26437403

  8. The virome in mammalian physiology and disease

    PubMed Central

    Virgin, Herbert W.

    2014-01-01

    The virome contains the most abundant and fastest-mutating genetic elements on Earth. The mammalian virome is constituted of viruses that infect host cells, virus-derived elements in our chromosomes, and viruses that infect the broad array of other types of organisms that inhabit us. Virome interactions with the host cannot be encompassed by a monotheistic view of viruses as pathogens. Instead, the genetic and transcriptional identity of mammals is defined in part by our co-evolved virome, a concept with profound implications for understanding health and disease. PMID:24679532

  9. Mammalian Kidney Development: Principles, Progress, and Projections

    PubMed Central

    Little, Melissa H.; McMahon, Andrew P.

    2012-01-01

    The mammalian kidney is a vital organ with considerable cellular complexity and functional diversity. Kidney development is notable for requiring distinct but coincident tubulogenic processes involving reciprocal inductive signals between mesenchymal and epithelial progenitor compartments. Key molecular pathways mediating these interactions have been identified. Further, advances in the analysis of gene expression and gene activity, coupled with a detailed knowledge of cell origins, are enhancing our understanding of kidney morphogenesis and unraveling the normal processes of postnatal repair and identifying disease-causing mechanisms. This article focuses on recent insights into central regulatory processes governing organ assembly and renal disease, and predicts future directions for the field. PMID:22550230

  10. AS52/GPT Mammalian Mutagenesis Assay

    DTIC Science & Technology

    1996-05-10

    dimethylnitrosamine (DMN) at 50 and 100 f.J.g/rnl was used as a 3 TLS Project Nn. A0ŗ-003: AS52/GPT Mammalian Mutagenesis Assay promutagen that requires metabolic...Chemical Source Lot No. air Air Products N/A calcium chloride Sigma 84F-0723 d imeth y !sulfoxide Fisher 933274 dimethylnitrosamine Sigma 82B0365...methanesulfonate (EMS) at 150 and 300 J.i-g/ml is used as a direct-acting mutagen for the nonactivated portion, and dimethylnitrosamine (DMN) at 150 and 300

  11. Structure and function of mammalian cilia

    PubMed Central

    Satir, Peter

    2008-01-01

    In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease. PMID:18365235

  12. Mammalian cell culture capacity for biopharmaceutical manufacturing.

    PubMed

    Ecker, Dawn M; Ransohoff, Thomas C

    2014-01-01

    : With worldwide sales of biopharmaceuticals increasing each year and continuing growth on the horizon, the manufacture of mammalian biopharmaceuticals has become a major global enterprise. We describe the current and future industry wide supply of manufacturing capacity with regard to capacity type, distribution, and geographic location. Bioreactor capacity and the use of single-use products for biomanufacturing are also profiled. An analysis of the use of this capacity is performed, including a discussion of current trends that will influence capacity growth, availability, and utilization in the coming years.

  13. Mammalian Developmental Genetics in the Twentieth Century

    PubMed Central

    Artzt, Karen

    2012-01-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas. PMID:23212897

  14. Molecular architecture of the mammalian circadian clock.

    PubMed

    Partch, Carrie L; Green, Carla B; Takahashi, Joseph S

    2014-02-01

    Circadian clocks coordinate physiology and behavior with the 24h solar day to provide temporal homeostasis with the external environment. The molecular clocks that drive these intrinsic rhythmic changes are based on interlocked transcription/translation feedback loops that integrate with diverse environmental and metabolic stimuli to generate internal 24h timing. In this review we highlight recent advances in our understanding of the core molecular clock and how it utilizes diverse transcriptional and post-transcriptional mechanisms to impart temporal control onto mammalian physiology. Understanding the way in which biological rhythms are generated throughout the body may provide avenues for temporally directed therapeutics to improve health and prevent disease.

  15. Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

    PubMed Central

    Labow, M A; Baim, S B; Shenk, T; Levine, A J

    1990-01-01

    A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture. Images PMID:2162473

  16. Design of a fluorescence-activated cell sorting-based Mammalian protein-protein interaction trap.

    PubMed

    Lievens, Sam; Van der Heyden, José; Vertenten, Els; Plum, Jean; Vandekerckhove, Joël; Tavernier, Jan

    2004-01-01

    The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), "positive" cells are selected by fluorescence-activated cell sorting (FACS).

  17. Lactate production by the mammalian blastocyst: manipulating the microenvironment for uterine implantation and invasion?

    PubMed

    Gardner, David K

    2015-04-01

    The mammalian blastocyst exhibits a high capacity for aerobic glycolysis, a metabolic characteristic of tumours. It has been considered that aerobic glycolysis is a means to ensure a high carbon flux to fulfil biosynthetic demands. Here, alternative explanations for this pattern of metabolism are considered. Lactate creates a microenvironment of low pH around the embryo to assist the disaggregation of uterine tissues to facilitate trophoblast invasion. Further it is proposed that lactate acts as a signalling molecule (especially at the reduced oxygen tension present at implantation) to elicit bioactive VEGF recruitment from uterine cells, to promote angiogenesis. Finally it is suggested that the region of high lactate/low pH created by the blastocyst modulates the activity of the local immune response, helping to create immune tolerance. Consequently, the mammalian blastocyst offers a model to study the role of microenvironments, and how metabolites and pH are used in signalling.

  18. Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells.

    PubMed

    Schröder, Sebastian; Herker, Eva; Itzen, Friederike; He, Daniel; Thomas, Sean; Gilchrist, Daniel A; Kaehlcke, Katrin; Cho, Sungyoo; Pollard, Katherine S; Capra, John A; Schnölzer, Martina; Cole, Philip A; Geyer, Matthias; Bruneau, Benoit G; Adelman, Karen; Ott, Melanie

    2013-11-07

    Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes.

  19. Circadian Gating of the Mammalian Cell Cycle Restriction Point: A Mathematical Analysis

    PubMed Central

    SU, JING; HENSON, MICHAEL A.

    2016-01-01

    A critical decision in the mammalian cell cycle is whether to pass through the restriction point (R-point) or enter the cell cycle. In this letter, we modeled the decision-making system of the mammalian cell cycle entry and the simulated circadian regulation of the R-point driven by external epithelial growth factor (EGF) patterns. Our conceptual model replicated key signaling behaviors observed experimentally, suggesting that the proposed network captured the essential system features. The model revealed the dramatic importance of the EGF dynamics on promoting cell proliferation, showed that the EGF signal duration was more important than the signal strength for driving cells past the R-point, and suggested that the loss of circadian control of the cell cycle entry could be associated with cancer development. PMID:28133623

  20. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature.

    PubMed

    Srinivasan, R Sathish; Dillard, Miriam E; Lagutin, Oleg V; Lin, Fu-Jung; Tsai, Sophia; Tsai, Ming-Jer; Samokhvalov, Igor M; Oliver, Guillermo

    2007-10-01

    The origin of the mammalian lymphatic vasculature has been debated for more than 100 years. Whether lymphatic endothelial cells have a single or dual, venous or mesenchymal origin remains controversial. To resolve this debate, we performed Cre/loxP-based lineage-tracing studies using mouse strains expressing Cre recombinase under the control of the Tie2, Runx1, or Prox1 promoter elements. These studies, together with the analysis of Runx1-mutant embryos lacking definitive hematopoiesis, conclusively determined that from venous-derived lymph sacs, lymphatic endothelial cells sprouted, proliferated, and migrated to give rise to the entire lymphatic vasculature, and that hematopoietic cells did not contribute to the developing lymph sacs. We conclude that the mammalian lymphatic system has a solely venous origin.

  1. Dynamic usage of transcription start sites within core promoters

    PubMed Central

    Kawaji, Hideya; Frith, Martin C; Katayama, Shintaro; Sandelin, Albin; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide

    2006-01-01

    Background Mammalian promoters do not initiate transcription at single, well defined base pairs, but rather at multiple, alternative start sites spread across a region. We previously characterized the static structures of transcription start site usage within promoters at the base pair level, based on large-scale sequencing of transcript 5' ends. Results In the present study we begin to explore the internal dynamics of mammalian promoters, and demonstrate that start site selection within many mouse core promoters varies among tissues. We also show that this dynamic usage of start sites is associated with CpG islands, broad and multimodal promoter structures, and imprinting. Conclusion Our results reveal a new level of biologic complexity within promoters - fine-scale regulation of transcription starting events at the base pair level. These events are likely to be related to epigenetic transcriptional regulation. PMID:17156492

  2. An Adaptive Threshold in Mammalian Neocortical Evolution

    PubMed Central

    Kalinka, Alex T.; Tomancak, Pavel; Huttner, Wieland B.

    2014-01-01

    Expansion of the neocortex is a hallmark of human evolution. However, determining which adaptive mechanisms facilitated its expansion remains an open question. Here we show, using the gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We find that variation in GI does not evolve linearly across species, but that mammals constitute two principal groups above and below a GI threshold value of 1.5, approximately equal to 109 neurons, which may be characterized by distinct constellations of physiological and life-history traits. By integrating data on neurogenic period, neuroepithelial founder pool size, cell-cycle length, progenitor-type abundances, and cortical neuron number into discrete mathematical models, we identify symmetric proliferative divisions of basal progenitors in the subventricular zone of the developing neocortex as evolutionarily necessary for generating a 14-fold increase in daily prenatal neuron production, traversal of the GI threshold, and thus establishment of two principal groups. We conclude that, despite considerable neuroanatomical differences, changes in the length of the neurogenic period alone, rather than any novel neurogenic progenitor lineage, are sufficient to explain differences in neuron number and neocortical size between species within the same principal group. PMID:25405475

  3. Ecological adaptation determines functional mammalian olfactory subgenomes

    PubMed Central

    Hayden, Sara; Bekaert, Michaël; Crider, Tess A.; Mariani, Stefano; Murphy, William J.; Teeling, Emma C.

    2010-01-01

    The ability to smell is governed by the largest gene family in mammalian genomes, the olfactory receptor (OR) genes. Although these genes are well annotated in the finished human and mouse genomes, we still do not understand which receptors bind specific odorants or how they fully function. Previous comparative studies have been taxonomically limited and mostly focused on the percentage of OR pseudogenes within species. No study has investigated the adaptive changes of functional OR gene families across phylogenetically and ecologically diverse mammals. To determine the extent to which OR gene repertoires have been influenced by habitat, sensory specialization, and other ecological traits, to better understand the functional importance of specific OR gene families and thus the odorants they bind, we compared the functional OR gene repertoires from 50 mammalian genomes. We amplified more than 2000 OR genes in aquatic, semi-aquatic, and flying mammals and coupled these data with 48,000 OR genes from mostly terrestrial mammals, extracted from genomic projects. Phylogenomic, Bayesian assignment, and principle component analyses partitioned species by ecotype (aquatic, semi-aquatic, terrestrial, flying) rather than phylogenetic relatedness, and identified OR families important for each habitat. Functional OR gene repertoires were reduced independently in the multiple origins of aquatic mammals and were significantly divergent in bats. We reject recent neutralist views of olfactory subgenome evolution and correlate specific OR gene families with physiological requirements, a preliminary step toward unraveling the relationship between specific odors and respective OR gene families. PMID:19952139

  4. Structure and function in mammalian societies

    PubMed Central

    Clutton-Brock, Tim

    2009-01-01

    Traditional interpretations of the evolution of animal societies have suggested that their structure is a consequence of attempts by individuals to maximize their inclusive fitness within constraints imposed by their social and physical environments. In contrast, some recent re-interpretations have argued that many aspects of social organization should be interpreted as group-level adaptations maintained by selection operating between groups or populations. Here, I review our current understanding of the evolution of mammalian societies, focusing, in particular, on the evolution of reproductive strategies in societies where one dominant female monopolizes reproduction in each group and her offspring are reared by other group members. Recent studies of the life histories of females in these species show that dispersing females often have little chance of establishing new breeding groups and so are likely to maximize their inclusive fitness by helping related dominants to rear their offspring. As in eusocial insects, increasing group size can lead to a progressive divergence in the selection pressures operating on breeders and helpers and to increasing specialization in their behaviour and life histories. As yet, there is little need to invoke group-level adaptations in order to account for the behaviour of individuals or the structure of mammalian groups. PMID:19805430

  5. Epidemiology of mammalian hepatitis E virus infection.

    PubMed

    Kaba, Mamadou; Moal, Valérie; Gérolami, René; Colson, Philippe

    2013-01-01

    Mammalian hepatitis E virus (HEV), the etiological agent of hepatitis E in humans, is a recently discovered infectious agent. It was identified for the first time in 1983 using electron microscopy on a faecal specimen of a person infected with non-A, non-B enterically-transmitted hepatitis. Based on retrospective and prospective studies, HEV was long described as one of the leading causes of acute viral hepatitis in tropical and subtropical countries, whereas in developed countries hepatitis E was considered an imported disease from HEV hyperendemic countries. Data from studies conducted during the past decade have greatly shifted our knowledge on the epidemiology and clinical spectrum of HEV. Recently, it has been shown that contrary to previous beliefs, hepatitis E is also an endemic disease in several developed countries, particularly in Japan and in Europe, as evidenced by reports of high anti-HEV immunoglobulin G prevalence in healthy individuals and an increasing number of non-travel-related acute hepatitis E cases. Moreover, a porcine reservoir and growing evidence of zoonotic transmission have been reported in these countries. This review summarizes the current knowledge on the epidemiology and prevention of transmission of mammalian HEV. Copyright © 2013 S. Karger AG, Basel.

  6. Ecology and evolution of mammalian biodiversity.

    PubMed

    Jones, Kate E; Safi, Kamran

    2011-09-12

    Mammals have incredible biological diversity, showing extreme flexibility in eco-morphology, physiology, life history and behaviour across their evolutionary history. Undoubtedly, mammals play an important role in ecosystems by providing essential services such as regulating insect populations, seed dispersal and pollination and act as indicators of general ecosystem health. However, the macroecological and macroevolutionary processes underpinning past and present biodiversity patterns are only beginning to be explored on a global scale. It is also particularly important, in the face of the global extinction crisis, to understand these processes in order to be able to use this knowledge to prevent future biodiversity loss and loss of ecosystem services. Unfortunately, efforts to understand mammalian biodiversity have been hampered by a lack of data. New data compilations on current species' distributions, ecologies and evolutionary histories now allow an integrated approach to understand this biodiversity. We review and synthesize these new studies, exploring the past and present ecology and evolution of mammalian biodiversity, and use these findings to speculate about the mammals of our future.

  7. Catabolic flexibility of mammalian-associated lactobacilli

    PubMed Central

    2013-01-01

    Metabolic flexibility may be generally defined as “the capacity for the organism to adapt fuel oxidation to fuel availability”. The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus. PMID:23680304

  8. Metabolic flux estimation in mammalian cell cultures.

    PubMed

    Goudar, Chetan T; Biener, Richard K; Piret, James M; Konstantinov, Konstantin B

    2014-01-01

    Metabolic flux analysis with its ability to quantify cellular metabolism is an attractive tool for accelerating cell line selection, medium optimization, and other bioprocess development activities. In the stoichiometric flux estimation approach, unknown fluxes are determined using intracellular metabolite mass balance expressions and measured extracellular rates. The simplicity of the stoichiometric approach extends its application to most cell culture systems, and the steps involved in metabolic flux estimation by the stoichiometric method are presented in detail in this chapter. Specifically, overdetermined systems are analyzed since the extra measurements can be used to check for gross measurement errors and system consistency. Cell-specific rates comprise the input data for flux estimation, and the logistic modeling approach is described for robust-specific rate estimation in batch and fed-batch systems. A simplified network of mammalian cell metabolism is used to illustrate the flux estimation procedure, and the steps leading up the consistency index determination are presented. If gross measurement errors are detected, a technique for determining the source of gross measurement error is also described. A computer program that performs most of the calculation described in this chapter is presented, and references to flux estimation software are provided. The procedure presented in this chapter should enable rapid metabolic flux estimation in any mammalian cell bioreaction network by the stoichiometric approach.

  9. The Mammalian Ovary from Genesis to Revelation

    PubMed Central

    Edson, Mark A.; Nagaraja, Ankur K.; Matzuk, Martin M.

    2009-01-01

    Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago. PMID:19776209

  10. Ecology and evolution of mammalian biodiversity

    PubMed Central

    Jones, Kate E.; Safi, Kamran

    2011-01-01

    Mammals have incredible biological diversity, showing extreme flexibility in eco-morphology, physiology, life history and behaviour across their evolutionary history. Undoubtedly, mammals play an important role in ecosystems by providing essential services such as regulating insect populations, seed dispersal and pollination and act as indicators of general ecosystem health. However, the macroecological and macroevolutionary processes underpinning past and present biodiversity patterns are only beginning to be explored on a global scale. It is also particularly important, in the face of the global extinction crisis, to understand these processes in order to be able to use this knowledge to prevent future biodiversity loss and loss of ecosystem services. Unfortunately, efforts to understand mammalian biodiversity have been hampered by a lack of data. New data compilations on current species' distributions, ecologies and evolutionary histories now allow an integrated approach to understand this biodiversity. We review and synthesize these new studies, exploring the past and present ecology and evolution of mammalian biodiversity, and use these findings to speculate about the mammals of our future. PMID:21807728

  11. Kinetic Analysis of a Mammalian Phospholipase D

    PubMed Central

    Henage, Lee G.; Exton, John H.; Brown, H. Alex

    2013-01-01

    In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Cα, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCα activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane. PMID:16339153

  12. The cellular code for mammalian thermosensation

    PubMed Central

    Pogorzala, Leah A.; Mishra, Santosh K.; Hoon, Mark A.

    2013-01-01

    Mammalian somatosenory neurons respond to thermal stimuli allowing animals to reliably discriminate hot from cold and select their preferred environments. We previously generated mice that are completely insensitive to temperatures from noxious cold to painful heat (−5 to 55 °C) by ablating several different classes of nociceptor early in development. Here we have adopted a selective ablation strategy in adult mice to dissect this phenotype and thereby demonstrated that separate populations of molecularly defined neurons respond to hot and cold. TRPV1-expressing neurons are responsible for all behavioral responses to temperatures between 40 and 50°C, while TRPM8-neurons are required for cold aversion. We also show that more extreme cold and heat activate additional populations of nociceptors including cells expressing Mrgprd. Thus, although eliminating Mrgprd-neurons alone does not affect behavioral responses to temperature, when combined with ablation of TRPV1- or TRPM8-cells, it significantly decreases responses to extreme heat and cold respectively. Notably, ablation of TRPM8-neurons distorts responses to preferred temperatures suggesting that the pleasant thermal sensation of warmth may in fact just reflect reduced aversive-input form TRPM8 and TRPV1-neurons. As predicted by this hypothesis, mice lacking both these classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50 °C. Taken together these results provide a simple cellular basis for mammalian thermosensation whereby two molecularly defined classes of sensory neurons detect and encode both attractive and aversive cues. PMID:23536068

  13. Power amplification in the mammalian cochlea.

    PubMed

    Lukashkin, Andrei N; Walling, Mark N; Russell, Ian J

    2007-08-07

    It was first suggested by Gold in 1948 [1] that the exquisite sensitivity and frequency selectivity of the mammalian cochlea is due to an active process referred to as the cochlear amplifier. It is thought that this process works by pumping energy to augment the otherwise damped sound-induced vibrations of the basilar membrane [2-4], a mechanism known as negative damping. The existence of the cochlear amplifier has been inferred from comparing responses of sensitive and compromised cochleae [5] and observations of acoustic emissions [6, 7] and through mathematical modeling [8, 9]. However, power amplification has yet to be demonstrated directly. Here, we prove that energy is indeed produced in the cochlea on a cycle-by-cycle basis. By using laser interferometry [10], we show that the nonlinear component of basilar-membrane responses to sound stimulation leads the forces acting on the membrane. This is possible only in active systems with negative damping [11]. Our finding provides the first direct evidence for power amplification in the mammalian cochlea. The finding also makes redundant current hypotheses of cochlear frequency sharpening and sensitization that are not based on negative damping.

  14. The terminal DNA structure of mammalian chromosomes.

    PubMed Central

    McElligott, R; Wellinger, R J

    1997-01-01

    In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated. PMID:9218811

  15. Genomic imprinting: a mammalian epigenetic discovery model.

    PubMed

    Barlow, Denise P

    2011-01-01

    Genomic imprinting is an epigenetic process leading to parental-specific expression of one to two percent of mammalian genes that offers one of the best model systems for a molecular analysis of epigenetic regulation in development and disease. In the twenty years since the first imprinted gene was identified, this model has had a significant impact on decoding epigenetic information in mammals. So far it has led to the discovery of long-range cis-acting control elements whose epigenetic state regulates small clusters of genes and of unusual macro noncoding RNAs (ncRNAs) that directly repress genes in cis, and critically, it has demonstrated that one biological role of DNA methylation is to allow expression of genes normally repressed by default. This review describes the progress in understanding how imprinted protein-coding genes are silenced; in particular, it focuses on the role of macro ncRNAs that have broad relevance as a potential new layer of regulatory information in the mammalian genome.

  16. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  17. Focusing on RISC assembly in mammalian cells

    SciTech Connect

    Hong Junmei; Wei Na; Chalk, Alistair; Wang Jue; Song, Yutong; Yi Fan; Qiao Renping; Sonnhammer, Erik L.L.; Wahlestedt, Claes; Liang Zicai Du, Quan

    2008-04-11

    RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.

  18. Expression of mammalian membrane proteins in mammalian cells using Semliki Forest virus vectors.

    PubMed

    Lundstrom, Kenneth

    2010-01-01

    One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation and bacterial systems to expression in animal cells. In contrast to soluble proteins, the expression levels have been relatively low due to the following reasons: The topology of membrane proteins requires special, posttranslational processing, folding, and insertion into membranes, which often are mammalian cell specific. Despite these strict demands, functional membrane proteins (G protein-coupled receptors, ion channels, and transporters) have been successfully expressed in bacterial, yeast, and insect cells. A general drawback observed in prokaryotic cells is that accumulation of foreign protein in membranes is toxic and results in growth arrest and therefore low yields of recombinant protein.In this chapter, the focus is on expression of recombinant mammalian membrane proteins in mammalian host cells, particularly applying Semliki Forest virus (SFV) vectors. Replication-deficient SFV vectors are rapidly generated at high titers in BHK-21 (Baby Hamster Kidney) cells, which then are applied for a broad range of mammalian and nonmammalian cells. The SFV system has provided high expression levels of topologically different proteins, especially for membrane proteins. Robust ligand-binding assays and functional coupling to G proteins and electrophysiological recordings have made the SFV system an attractive tool in drug discovery. Furthermore, the high susceptibility of SFV vectors to primary neurons has allowed various applications in neuroscience. Establishment of large-scale production in mammalian adherent and suspension cultures has allowed production of hundreds of milligrams of membrane proteins that has allowed their submission to serious structural biology approaches. In this

  19. Regulatory Divergence of Transcript Isoforms in a Mammalian Model System

    PubMed Central

    Thybert, David; Stefflova, Klara; Watt, Stephen; Flicek, Paul; Brazma, Alvis; Marioni, John C.; Odom, Duncan T.

    2015-01-01

    Phenotypic differences between species are driven by changes in gene expression and, by extension, by modifications in the regulation of the transcriptome. Investigation of mammalian transcriptome divergence has been restricted to analysis of bulk gene expression levels and gene-internal splicing. Using allele-specific expression analysis in inter-strain hybrids of Mus musculus, we determined the contribution of multiple cellular regulatory systems to transcriptome divergence, including: alternative promoter usage, transcription start site selection, cassette exon usage, alternative last exon usage, and alternative polyadenylation site choice. Between mouse strains, a fifth of genes have variations in isoform usage that contribute to transcriptomic changes, half of which alter encoded amino acid sequence. Virtually all divergence in isoform usage altered the post-transcriptional regulatory instructions in gene UTRs. Furthermore, most genes with isoform differences between strains contain changes originating from multiple regulatory systems. This result indicates widespread cross-talk and coordination exists among different regulatory systems. Overall, isoform usage diverges in parallel with and independently to gene expression evolution, and the cis and trans regulatory contribution to each differs significantly. PMID:26339903

  20. Radiofrequency exposure and mammalian cell toxicity, genotoxicity, and transformation.

    PubMed

    Meltz, Martin L

    2003-01-01

    The published in vitro literature relevant to the issue of the possible induction of toxicity, genotoxicity, and transformation of mammalian cells due to radiofrequency field (RF) exposure is examined. In some instances, information about related in vivo studies is presented. The review is from the perspective of technical merit and also biological consistency, especially with regard to those publications reporting a positive effect. The weight of evidence available indicates that, for a variety of frequencies and modulations with both short and long exposure times, at exposure levels that do not (or in some instances do) heat the biological sample such that there is a measurable increase in temperature, RF exposure does not induce (a). DNA strand breaks, (b). chromosome aberrations, (c). sister chromatid exchanges (SCEs), (d). DNA repair synthesis, (e). phenotypic mutation, or (f). transformation (cancer-like changes). While there is limited experimental evidence that RF exposure induces micronuclei formation, there is abundant evidence that it does not. There is some evidence that RF exposure does not induce DNA excision repair, suggesting the absence of base damage. There is also evidence that RF exposure does not inhibit excision repair after the induction of thymine dimers by UV exposure, as well as evidence that indicates that RF is not a co-carcinogen or a tumor promoter. The article is in part a tutorial, so that the reader can consider similarities and discrepancies between reports of RF-induced effects relative to one another.

  1. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    NASA Astrophysics Data System (ADS)

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-12-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

  2. Mammalian Cdh1/Fzr mediates its own degradation

    PubMed Central

    Listovsky, Tamar; Oren, Yifat S; Yudkovsky, Yana; Mahbubani, Hiro M; Weiss, Aryeh M; Lebendiker, Mario; Brandeis, Michael

    2004-01-01

    The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time. PMID:15029244

  3. Genetic studies of leucine transport in mammalian cells.

    PubMed

    Shotwell, M A; Lobatón, C D; Collarini, E J; Moreno, A; Giles, R E; Oxender, D L

    1984-05-15

    We have taken two approaches to the study of the genetics of leucine transport in mammalian cells. First, from a mutant Chinese hamster ovary cell line that has a temperature-sensitive leucyl-tRNA synthetase, we isolated temperature-resistant revertants with increased leucine transport activity. This transport elevation is reflected by increased Vmax values of leucine uptake and unchanged Km values of uptake. The temperature resistance in each revertant appears to result from the increased transport and not from any change in the leucyl-tRNA synthetase. We conclude that in each revertant there is a stable derepression of amino acid transport system L. In a second approach, we started with a Chinese hamster-human hybrid strain formed by the fusion of a temperature-sensitive leucyl-tRNA synthetase mutant hamster cell line and normal human leukocytes. From this temperature-sensitive hybrid strain we selected temperature-resistant hybrids, one class of which we found to have greatly elevated leucine transport activity. We have allowed human chromosomes to segregate from these high-transport hybrids, promoted by the presence of low concentrations of colcemid. The loss of the high-transport phenotype coincides with the loss of a single small human chromosome, which we are attempting to identify by using G-11 and G-banding staining techniques.

  4. Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

    PubMed

    Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

    2017-05-26

    Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Polybromo protein BAF180 functions in mammalian cardiac chamber maturation

    PubMed Central

    Wang, Zhong; Zhai, Weiguo; Richardson, James A.; Olson, Eric N.; Meneses, Juanito J.; Firpo, Meri T.; Kang, Chulho; Skarnes, William C.; Tjian, Robert

    2004-01-01

    BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRα, VDR, and PPARγ. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRα and PPARγ. Embryonic aggregation analyses reveal that in contrast to PPARγ-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARβ2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARβ2. These studies reveal unique functions of PBAF in cardiac chamber maturation. PMID:15601824

  6. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-01-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments. PMID:27924861

  7. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut

    PubMed Central

    Ghosh, Anil K.; Coppens, Isabelle; Gårdsvoll, Henrik; Ploug, Michael; Jacobs-Lorena, Marcelo

    2011-01-01

    Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process. PMID:21949403

  8. Epigenetic Control of Mammalian LINE-1 Retrotransposon by Retinoblastoma Proteins

    PubMed Central

    Montoya-Durango, Diego E.; Liu, Yongqing; Teneng, Ivo; Kalbfleisch, Ted; Lacy, Mary E.; Steffen, Marlene C.; Ramos, Kenneth S.

    2009-01-01

    Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. ChIP experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of co-repressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation. PMID:19427507

  9. Mammalian social odours: attraction and individual recognition

    PubMed Central

    Brennan, Peter A; Kendrick, Keith M

    2006-01-01

    Mammalian social systems rely on signals passed between individuals conveying information including sex, reproductive status, individual identity, ownership, competitive ability and health status. Many of these signals take the form of complex mixtures of molecules sensed by chemosensory systems and have important influences on a variety of behaviours that are vital for reproductive success, such as parent–offspring attachment, mate choice and territorial marking. This article aims to review the nature of these chemosensory cues and the neural pathways mediating their physiological and behavioural effects. Despite the complexities of mammalian societies, there are instances where single molecules can act as classical pheromones attracting interest and approach behaviour. Chemosignals with relatively high volatility can be used to signal at a distance and are sensed by the main olfactory system. Most mammals also possess a vomeronasal system, which is specialized to detect relatively non-volatile chemosensory cues following direct contact. Single attractant molecules are sensed by highly specific receptors using a labelled line pathway. These act alongside more complex mixtures of signals that are required to signal individual identity. There are multiple sources of such individuality chemosignals, based on the highly polymorphic genes of the major histocompatibility complex (MHC) or lipocalins such as the mouse major urinary proteins. The individual profile of volatile components that make up an individual odour signature can be sensed by the main olfactory system, as the pattern of activity across an array of broadly tuned receptor types. In addition, the vomeronasal system can respond highly selectively to non-volatile peptide ligands associated with the MHC, acting at the V2r class of vomeronasal receptor. The ability to recognize individuals or their genetic relatedness plays an important role in mammalian social behaviour. Thus robust systems for olfactory

  10. Fibroblast growth factor-9 in marsupial testicular development.

    PubMed

    Chung, J W; Pask, A J; Yu, H; Renfree, M B

    2011-01-01

    FGF9 is a member of the fibroblast growth factor (FGF) family and is critical for early testicular development and germ cell survival in the mouse. Fgf9 reinforces the testis determinant Sox9 and antagonizes Wnt4, an ovarian factor. To determine whether FGF9 has a conserved role in the mammalian gonad, we examined its expression in the gonads of a marsupial, the tammar wallaby Macropus eugenii, and compared it to WNT4 expression. Marsupial FGF9 is highly conserved with orthologues from eutherian mammals, including humans. FGF9 protein was detected in both the testis and ovary before sexual differentiation, but it subsequently became sexually dimorphic during the period of testicular differentiation. The protein was specifically enriched in the seminiferous cords of the developing testis in the Sertoli and germ cells. FGF9 mRNA expression was upregulated in the tammar testis at the time of seminiferous cord formation and downregulated in the developing ovary in an opposite profile to that of marsupial WNT4. These observations suggest that FGF9 promotes male fate in the early gonad of marsupials through an antagonistic relationship with WNT4 as it does in eutherian mammals.

  11. Genetic redundancy of GATA factors in the extraembryonic trophoblast lineage ensures the progression of preimplantation and postimplantation mammalian development

    PubMed Central

    Home, Pratik; Kumar, Ram Parikshan; Ganguly, Avishek; Saha, Biswarup; Milano-Foster, Jessica; Bhattacharya, Bhaswati; Ray, Soma; Gunewardena, Sumedha; Paul, Arindam; Camper, Sally A.; Fields, Patrick E.

    2017-01-01

    GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure. PMID:28232602

  12. The Evolution of Mammalian Olfactory Receptor Genes

    PubMed Central

    Issel-Tarver, L.; Rine, J.

    1997-01-01

    We performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans. The human subfamilies were localized to chromosomes 7, 11, and 19. The two subfamilies that were tightly linked in the dog genome were also tightly linked in the human genome. The four subfamilies were compared in human (primate), horse (perissodactyl), and a variety of artiodactyls and carnivores. Some changes in gene number were detected, but overall subfamily size appeared to have been established before the divergence of these mammals 60-100 million years ago. PMID:9017400

  13. Mechanism of Protein Biosynthesis in Mammalian Mitochondria

    PubMed Central

    Christian, Brooke E.; Spremulli, Linda L.

    2011-01-01

    Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. PMID:22172991

  14. Cenozoic climate change influences mammalian evolutionary dynamics

    PubMed Central

    Figueirido, Borja; Janis, Christine M.; Pérez-Claros, Juan A.; De Renzi, Miquel; Palmqvist, Paul

    2012-01-01

    Global climate change is having profound impacts on the natural world. However, climate influence on faunal dynamics at macroevolutionary scales remains poorly understood. In this paper we investigate the influence of climate over deep time on the diversity patterns of Cenozoic North American mammals. We use factor analysis to identify temporally correlated assemblages of taxa, or major evolutionary faunas that we can then study in relation to climatic change over the past 65 million years. These taxa can be grouped into six consecutive faunal associations that show some correspondence with the qualitative mammalian chronofaunas of previous workers. We also show that the diversity pattern of most of these chronofaunas can be correlated with the stacked deep-sea benthic foraminiferal oxygen isotope (δ18O) curve, which strongly suggests climatic forcing of faunal dynamics over a large macroevolutionary timescale. This study demonstrates the profound influence of climate on the diversity patterns of North American terrestrial mammals over the Cenozoic. PMID:22203974

  15. Regulation of Rap GTPases in mammalian neurons.

    PubMed

    Shah, Bhavin; Püschel, Andreas W

    2016-10-01

    Small GTPases are central regulators of many cellular processes. The highly conserved Rap GTPases perform essential functions in the mammalian nervous system during development and in mature neurons. During neocortical development, Rap1 is required to regulate cadherin- and integrin-mediated adhesion. In the adult nervous system Rap1 and Rap2 regulate the maturation and plasticity of dendritic spine and synapses. Although genetic studies have revealed important roles of Rap GTPases in neurons, their regulation by guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them by stimulating their intrinsic GTPase activity is just beginning to be explored in vivo. Here we review how GEFs and GAPs regulate Rap GTPases in the nervous system with a focus on their in vivo function.

  16. KN-93 inhibits IKr in mammalian cardiomyocytes

    PubMed Central

    Hegyi, Bence; Chen-Izu, Ye; Jian, Zhong; Shimkunas, Rafael; Izu, Leighton T.; Banyasz, Tamas

    2015-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function. PMID:26463508

  17. KN-93 inhibits IKr in mammalian cardiomyocytes.

    PubMed

    Hegyi, Bence; Chen-Izu, Ye; Jian, Zhong; Shimkunas, Rafael; Izu, Leighton T; Banyasz, Tamas

    2015-12-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 is widely used in multiple fields of cardiac research especially for studying the mechanisms of cardiomyopathy and cardiac arrhythmias. Whereas KN-93 is a potent inhibitor of CaMKII, several off-target effects have also been found in expression cell systems and smooth muscle cells, but there is no information on the KN93 side effects in mammalian ventricular myocytes. In this study we explore the effect of KN-93 on the rapid component of delayed rectifier potassium current (IKr) in the ventricular myocytes from rabbit and guinea pig hearts. Our data indicate that KN-93 exerts direct inhibitory effect on IKr that is not mediated via CaMKII. This off-target effect of KN93 should be taken into account when interpreting the data from using KN93 to investigate the role of CaMKII in cardiac function.

  18. Studies on the mammalian toxicity of fenthion*

    PubMed Central

    Francis, Jean I.; Barnes, J. M.

    1963-01-01

    This paper constitutes a report on mammalian toxicological investigations of fen hion, carried out as part of the WHO malaria eradication programme, and on the conclusions drawn from them. Fenthion is found to be of intermediate toxicity to the four rodent species studied. In rats the signs of poisoning develop rather slowly but persist for several days, male rats being more susceptible than females, whereas for most phosphorothionates the converse is true. The results suggest that fenthion is not simply oxidized from the P=S compound to the P=O. It has been stated that the sulfoxide and sulfone are produced before the P=S→P=O oxidation takes place, but experiments suggest that further changes are involved. The findings are discussed in relation to the possible health hazard that might be encountered by those who have to apply fenthion as a residual spray. PMID:14056272

  19. Crystal structure of mammalian acid sphingomyelinase

    PubMed Central

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X.; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  20. Mechanism of protein biosynthesis in mammalian mitochondria.

    PubMed

    Christian, Brooke E; Spremulli, Linda L

    2012-01-01

    Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Mammalian synthetic biology for studying the cell

    PubMed Central

    Mathur, Melina; Xiang, Joy S.

    2017-01-01

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

  2. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  3. Peromyscus as a Mammalian Epigenetic Model

    PubMed Central

    Shorter, Kimberly R.; Crossland, Janet P.; Webb, Denessia; Szalai, Gabor; Felder, Michael R.; Vrana, Paul B.

    2012-01-01

    Deer mice (Peromyscus) offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential. PMID:22567379

  4. Mammalian Autophagy: How Does It Work?

    PubMed

    Bento, Carla F; Renna, Maurizio; Ghislat, Ghita; Puri, Claudia; Ashkenazi, Avraham; Vicinanza, Mariella; Menzies, Fiona M; Rubinsztein, David C

    2016-06-02

    Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.

  5. Fundamentals of Expression in Mammalian Cells.

    PubMed

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions.

  6. Signaling Mechanisms in Mammalian Myoblast Fusion

    PubMed Central

    Hindi, Sajedah M.; Tajrishi, Marjan M.; Kumar, Ashok

    2013-01-01

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion. PMID:23612709

  7. Signaling mechanisms in mammalian myoblast fusion.

    PubMed

    Hindi, Sajedah M; Tajrishi, Marjan M; Kumar, Ashok

    2013-04-23

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.

  8. New consensus nomenclature for mammalian keratins

    PubMed Central

    Schweizer, Jürgen; Bowden, Paul E.; Coulombe, Pierre A.; Langbein, Lutz; Lane, E. Birgitte; Magin, Thomas M.; Maltais, Lois; Omary, M. Bishr; Parry, David A.D.; Rogers, Michael A.; Wright, Mathew W.

    2006-01-01

    Keratins are intermediate filament–forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species. PMID:16831889

  9. Mammalian synthetic biology for studying the cell.

    PubMed

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  10. [Thiamine triphosphatase activity in mammalian mitochondria].

    PubMed

    Rusina, I M; Makarchikov, A F

    2003-01-01

    Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.

  11. Mammalian Metallothionein-2A and Oxidative Stress

    PubMed Central

    Ling, Xue-Bin; Wei, Hong-Wei; Wang, Jun; Kong, Yue-Qiong; Wu, Yu-You; Guo, Jun-Li; Li, Tian-Fa; Li, Ji-Ke

    2016-01-01

    Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy. PMID:27608012

  12. Cellular and Chemical Neuroscience of Mammalian Sleep

    PubMed Central

    Datta, Subimal

    2010-01-01

    Extraordinary strides have been made toward understanding the complexities and regulatory mechanisms of sleep over the past two decades, thanks to the help of rapidly evolving technologies. At its most basic level, mammalian sleep is a restorative process of the brain and body. Beyond its primary restorative purpose, sleep is essential for a number of vital functions. Our primary research interest is to understand the cellular and molecular mechanisms underlying the regulation of sleep and its cognitive functions. Here I will reflect on our own research contributions to fifty years of extraordinary advances in the neurobiology of slow-wave sleep (SWS) and rapid eye movement (REM) sleep regulation. I conclude this review by suggesting some potential future directions to further our understanding of the neurobiology of sleep. PMID:20359944

  13. Predictive chromatin signatures in the mammalian genome

    PubMed Central

    Hon, Gary C.; Hawkins, R. David; Ren, Bing

    2009-01-01

    The DNA sequence of an organism is a blueprint of life: it harbors not only the information about proteins and other molecules produced in each cell, but also instructions on when and where such molecules are made. Chromatin, the structure of histone and DNA that has co-evolved with eukaryotic genome, also contains information that indicates the function and activity of the underlying DNA sequences. Such information exists in the form of covalent modifications to the histone proteins that comprise the nucleosome. Thanks to the development of high throughput technologies such as DNA microarrays and next generation DNA sequencing, we have begun to associate the various combinations of chromatin modification patterns with functional sequences in the human genome. Here, we review the rapid progress from descriptive observations of histone modification profiles to highly predictive models enabling use of chromatin signatures to enumerate novel functional sequences in mammalian genomes that have escaped previous detection. PMID:19808796

  14. Mammalian protein glycosylation--structure versus function.

    PubMed

    Defaus, S; Gupta, P; Andreu, D; Gutiérrez-Gallego, R

    2014-06-21

    Carbohydrates fulfil many common as well as extremely important functions in nature. They show a variety of molecular displays--e.g., free mono-, oligo-, and polysaccharides, glycolipids, proteoglycans, glycoproteins, etc.--with particular roles and localizations in living organisms. Structure-specific peculiarities are so many and diverse that it becomes virtually impossible to cover them all from an analytical perspective. Hence this manuscript, focused on mammalian glycosylation, rather than a complete list of analytical descriptors or recognized functions for carbohydrate structures, comprehensively reviews three central issues in current glycoscience, namely (i) structural analysis of glycoprotein glycans, covering both classical and novel approaches for teasing out the structural puzzle as well as potential pitfalls of these processes; (ii) an overview of functions attributed to carbohydrates, covering from monosaccharide to complex, well-defined epitopes and full glycans, including post-glycosylational modifications, and (iii) recent technical advances allowing structural identification of glycoprotein glycans with simultaneous assignation of biological functions.

  15. Cenozoic climate change influences mammalian evolutionary dynamics.

    PubMed

    Figueirido, Borja; Janis, Christine M; Pérez-Claros, Juan A; De Renzi, Miquel; Palmqvist, Paul

    2012-01-17

    Global climate change is having profound impacts on the natural world. However, climate influence on faunal dynamics at macroevolutionary scales remains poorly understood. In this paper we investigate the influence of climate over deep time on the diversity patterns of Cenozoic North American mammals. We use factor analysis to identify temporally correlated assemblages of taxa, or major evolutionary faunas that we can then study in relation to climatic change over the past 65 million years. These taxa can be grouped into six consecutive faunal associations that show some correspondence with the qualitative mammalian chronofaunas of previous workers. We also show that the diversity pattern of most of these chronofaunas can be correlated with the stacked deep-sea benthic foraminiferal oxygen isotope (δ(18)O) curve, which strongly suggests climatic forcing of faunal dynamics over a large macroevolutionary timescale. This study demonstrates the profound influence of climate on the diversity patterns of North American terrestrial mammals over the Cenozoic.

  16. Genetic reassortment of mammalian reoviruses in mice.

    PubMed Central

    Wenske, E A; Chanock, S J; Krata, L; Fields, B N

    1985-01-01

    Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo. PMID:4057359

  17. Clues to the functions of mammalian sleep.

    PubMed

    Siegel, Jerome M

    2005-10-27

    The functions of mammalian sleep remain unclear. Most theories suggest a role for non-rapid eye movement (NREM) sleep in energy conservation and in nervous system recuperation. Theories of REM sleep have suggested a role for this state in periodic brain activation during sleep, in localized recuperative processes and in emotional regulation. Across mammals, the amount and nature of sleep are correlated with age, body size and ecological variables, such as whether the animals live in a terrestrial or an aquatic environment, their diet and the safety of their sleeping site. Sleep may be an efficient time for the completion of a number of functions, but variations in sleep expression indicate that these functions may differ across species.

  18. Mammalian telomeres and their partnership with lamins

    PubMed Central

    Burla, Romina; La Torre, Mattia; Saggio, Isabella

    2016-01-01

    ABSTRACT Chromosome ends are complex structures, which require a panel of factors for their elongation, replication, and protection. We describe here the mechanics of mammalian telomeres, dynamics and maintainance in relation to lamins. Multiple biochemical connections, including association of telomeres to the nuclear envelope and matrix, of telomeric proteins to lamins, and of lamin-associated proteins to chromosome ends, underline the interplay between lamins and telomeres. Paths toward senescence, such as defective telomere replication, altered heterochromatin organization, and impaired DNA repair, are common to lamins' and telomeres' dysfunction. The convergence of phenotypes can be interpreted through a model of dynamic, lamin-controlled functional platforms dedicated to the function of telomeres as fragile sites. The features of telomeropathies and laminopathies, and of animal models underline further overlapping aspects, including the alteration of stem cell compartments. We expect that future studies of basic biology and on aging will benefit from the analysis of this telomere-lamina interplay. PMID:27116558

  19. Differential Light Scattering from Spherical Mammalian Cells

    PubMed Central

    Brunsting, Albert; Mullaney, Paul F.

    1974-01-01

    The differential scattered light intensity patterns of spherical mammalian cells were measured with a new photometer which uses high-speed film as the light detector. The scattering objects, interphase and mitotic Chinese hamster ovary cells and HeLa cells, were modeled as (a) a coated sphere, accounting for nucleus and cytoplasm, and (b) a homogeneous sphere when no cellular nucleus was present. The refractive indices and size distribution of the cells were measured for an accurate comparison of the theoretical model with the light-scattering measurements. The light scattered beyond the forward direction is found to contain information about internal cellular morphology, provided the size distribution of the cells is not too broad. ImagesFIGURE 1 PMID:4134589

  20. Cooperative breeding and monogamy in mammalian societies

    PubMed Central

    Lukas, Dieter; Clutton-Brock, Tim

    2012-01-01

    Comparative studies of social insects and birds show that the evolution of cooperative and eusocial breeding systems has been confined to species where females mate completely or almost exclusively with a single male, indicating that high levels of average kinship between group members are necessary for the evolution of reproductive altruism. In this paper, we show that in mammals, the evolution of cooperative breeding has been restricted to socially monogamous species which currently represent 5 per cent of all mammalian species. Since extra-pair paternity is relatively uncommon in socially monogamous and cooperatively breeding mammals, our analyses support the suggestion that high levels of average kinship between group members have played an important role in the evolution of cooperative breeding in non-human mammals, as well as in birds and insects. PMID:22279167

  1. Chemical analysis of individual mammalian cells

    SciTech Connect

    Tan, W.; Yeung, E.S.

    1994-12-31

    The extremely small size of mammalian cells creates an unusual challenge for the analytical chemist, both in terms of separation and detection. Under a microscope, it is possible to confirm the injection of individual cells such as erythrocyte into capillaries with 10-{mu}m i.d. by hydrostatic pressure. The ionic contents can then be separated by capillary electrophoresis after the cell lyses. Enzymes at the zeptomole level can be monitored by on-column fluorescence enzyme assay. On-column particle-counting immunoassay can be applied to a broad range of analytes (antigens), also at the zeptomole level. The authors report here the simultaneous determination of the amounts of glucose-6-phosphate dehydrogenase (G6PDH) and their activities in individual erythrocytes by using a combination of the two detection schemes. Insights into the degradation of proteins as a function of cell age can be derived.

  2. Feeling the pressure in mammalian somatosensation

    PubMed Central

    Lumpkin, Ellen A; Bautista, Diana M

    2014-01-01

    Mechanoreceptor cells of the somatosensory system initiate the perception of touch and pain. Molecules required for mechanosensation have been identified from invertebrate neurons, and recent functional studies indicate that ion channels of the transient receptor potential and degenerin/epithelial Na+ channel families are likely to be transduction channels. The expression of related channels in mammalian somatosensory neurons has fueled the notion that these channels mediate mechanotransduction in vertebrates; however, genetic disruption and heterologous expression have not yet revealed a direct role for any of these candidates in somatosensory mechanotransduction. Thus, new systems are needed to define the function of these ion channels in somatosensation and to pinpoint molecules or signaling pathways that underlie mechanotransduction in vertebrates. PMID:19683913

  3. Cellular and chemical neuroscience of mammalian sleep.

    PubMed

    Datta, Subimal

    2010-05-01

    Extraordinary strides have been made toward understanding the complexities and regulatory mechanisms of sleep over the past two decades thanks to the help of rapidly evolving technologies. At its most basic level, mammalian sleep is a restorative process of the brain and body. Beyond its primary restorative purpose, sleep is essential for a number of vital functions. Our primary research interest is to understand the cellular and molecular mechanisms underlying the regulation of sleep and its cognitive functions. Here I will reflect on our own research contributions to 50 years of extraordinary advances in the neurobiology of slow-wave sleep (SWS) and rapid eye movement (REM) sleep regulation. I conclude this review by suggesting some potential future directions to further our understanding of the neurobiology of sleep.

  4. Mammalian sperm nuclear organization: resiliencies and vulnerabilities.

    PubMed

    Champroux, A; Torres-Carreira, J; Gharagozloo, P; Drevet, J R; Kocer, A

    2016-01-01

    Sperm cells are remarkably complex and highly specialized compared to somatic cells. Their function is to deliver to the oocyte the paternal genomic blueprint along with a pool of proteins and RNAs so a new generation can begin. Reproductive success, including optimal embryonic development and healthy offspring, greatly depends on the integrity of the sperm chromatin structure. It is now well documented that DNA damage in sperm is linked to reproductive failures both in natural and assisted conception (Assisted Reproductive Technologies [ART]). This manuscript reviews recent important findings concerning - the unusual organization of mammalian sperm chromatin and its impact on reproductive success when modified. This review is focused on sperm chromatin damage and their impact on embryonic development and transgenerational inheritance.

  5. Global Epigenomic Reconfiguration During Mammalian Brain Development

    PubMed Central

    Nery, Joseph R.; Urich, Mark; Puddifoot, Clare A.; Johnson, Nicholas D.; Lucero, Jacinta; Huang, Yun; Dwork, Andrew J.; Schultz, Matthew D.; Yu, Miao; Tonti-Filippini, Julian; Heyn, Holger; Hu, Shijun; Wu, Joseph C.; Rao, Anjana; Esteller, Manel; He, Chuan; Haghighi, Fatemeh G.; Sejnowski, Terrence J.; Behrens, M. Margarita; Ecker, Joseph R.

    2013-01-01

    DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity. PMID:23828890

  6. Redox signaling during hypoxia in mammalian cells.

    PubMed

    Smith, Kimberly A; Waypa, Gregory B; Schumacker, Paul T

    2017-10-01

    Hypoxia triggers a wide range of protective responses in mammalian cells, which are mediated through transcriptional and post-translational mechanisms. Redox signaling in cells by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) occurs through the reversible oxidation of cysteine thiol groups, resulting in structural modifications that can change protein function profoundly. Mitochondria are an important source of ROS generation, and studies reveal that superoxide generation by the electron transport chain increases during hypoxia. Other sources of ROS, such as the NAD(P)H oxidases, may also generate oxidant signals in hypoxia. This review considers the growing body of work indicating that increased ROS signals during hypoxia are responsible for regulating the activation of protective mechanisms in diverse cell types. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Engineered Trehalose Permeable to Mammalian Cells.

    PubMed

    Abazari, Alireza; Meimetis, Labros G; Budin, Ghyslain; Bale, Shyam Sundhar; Weissleder, Ralph; Toner, Mehmet

    2015-01-01

    Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

  8. Scaling of the mammalian middle ear.

    PubMed

    Nummela, S

    1995-05-01

    This study considers the general question how animal size limits the size and information receiving capacity of sense organs. To clarify this in the case of the mammalian middle ear, I studied 63 mammalian species, ranging from a small bat to the Indian elephant. I determined the skull mass and the masses of the ossicles malleus, incus and stapes (M, I and S), and measured the tympanic membrane area, A1. The ossicular mass (in mg) is generally negatively allometric to skull mass (in g), the regression equation for the whole material (excluding true seals) being y = 1.373 x(0.513). However, for very small mammals the allometry approaches isometry. Within a group of large mammals no distinct allometry can be discerned. The true seals (Phocidae) are exceptional by having massive ossicles. The size relations within the middle ear are generally rather constant. However, the I/M relation is slightly positively allometric, y = 0.554 x(1.162). Two particularly isometric relations were found; the S/(M + I) relation for the ossicles characterized by the regression equation y = 0.054 x(0.993), and the relation between a two-dimensional measure of the ossicles and the tympanic membrane ares, (M + I)2/3 /A1. As in isometric ears the sound energy collected by the tympanic membrane is linearly related to its area, the latter isometry suggests that, regardless of animal size, a given ossicular cross-sectional area is exposed to a similar sound-induced stress. Possible morphological middle ear adaptations to particular acoustic environments are discussed.

  9. Cortical pathways to the mammalian amygdala.

    PubMed

    McDonald, A J

    1998-06-01

    The amygdaloid nuclear complex is critical for producing appropriate emotional and behavioral responses to biologically relevant sensory stimuli. It constitutes an essential link between sensory and limbic areas of the cerebral cortex and subcortical brain regions, such as the hypothalamus, brainstem, and striatum, that are responsible for eliciting emotional and motivational responses. This review summarizes the anatomy and physiology of the cortical pathways to the amygdala in the rat, cat and monkey. Although the basic anatomy of these systems in the cat and monkey was largely delineated in studies conducted during the 1970s and 1980s, detailed information regarding the cortico-amygdalar pathways in the rat was only obtained in the past several years. The purpose of this review is to describe the results of recent studies in the rat and to compare the organization of cortico-amygdalar projections in this species with that seen in the cat and monkey. In all three species visual, auditory, and somatosensory information is transmitted to the amygdala by a series of modality-specific cortico-cortical pathways ("cascades") that originate in the primary sensory cortices and flow toward higher order association areas. The cortical areas in the more distal portions of these cascades have stronger and more extensive projections to the amygdala than the more proximal areas. In all three species olfactory and gustatory/visceral information has access to the amygdala at an earlier stage of cortical processing than visual, auditory and somatosensory information. There are also important polysensory cortical inputs to the mammalian amygdala from the prefrontal and hippocampal regions. Whereas the overall organization of cortical pathways is basically similar in all mammalian species, there is anatomical evidence which suggests that there are important differences in the extent of convergence of cortical projections in the primate versus the nonprimate amygdala.

  10. Identification of mammalian orthologs using local synteny

    PubMed Central

    2009-01-01

    Background Accurate determination of orthology is central to comparative genomics. For vertebrates in particular, very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make inference of orthology between genes difficult. Many methods have been developed to identify orthologous genes, mostly based upon analysis of the inferred protein sequence of the genes. More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment in vertebrates. Such methods have been most successfully implemented in fungal genomes and have long been used in prokaryotic genomes, where gene order is far less variable than in vertebrates. However, to our knowledge, no explicit comparison of synteny and sequence based definitions of orthology has been reported in vertebrates, or, more specifically, in mammals. Results We test a simple method for the measurement and utilization of gene order (local synteny) in the identification of mammalian orthologs by investigating the agreement between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny is a robust substitute to coding sequence for identifying orthologs. However, 7% of pairs were found to be discordant between local synteny and Inparanoid. These cases of discordance result from evolutionary events including retrotransposition and genome rearrangements. Conclusions By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and might be used to identify cases of non-orthologous gene

  11. Defining the mammalian CArGome

    PubMed Central

    Sun, Qiang; Chen, Guang; Streb, Jeffrey W.; Long, Xiaochun; Yang, Yumei; Stoeckert, Christian J.; Miano, Joseph M.

    2006-01-01

    Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton. PMID:16365378

  12. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

    PubMed Central

    Condreay, J. Patrick; Witherspoon, Sam M.; Clay, William C.; Kost, Thomas A.

    1999-01-01

    Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression. PMID:9874783

  13. Dynamic Shuttling of Tia-1 Accompanies the Recruitment of mRNA to Mammalian Stress Granules

    PubMed Central

    Kedersha, Nancy; Cho, Michael R.; Li, Wei; Yacono, Patrick W.; Chen, Samantha; Gilks, Natalie; Golan, David E.; Anderson, Paul

    2000-01-01

    Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein–tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1ΔRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression. PMID:11121440

  14. An Analytical Study of Mammalian Bite Wounds Requiring Inpatient Management

    PubMed Central

    Lee, Young-Geun; Kim, Woo-Kyung

    2013-01-01

    Background Mammalian bite injuries create a public health problem because of their frequency, potential severity, and increasing number. Some researchers have performed fragmentary analyses of bite wounds caused by certain mammalian species. However, little practical information is available concerning serious mammalian bite wounds that require hospitalization and intensive wound management. Therefore, the purpose of this study was to perform a general review of serious mammalian bite wounds. Methods We performed a retrospective review of the medical charts of 68 patients who were referred to our plastic surgery department for the treatment of bite wounds between January 2003 and October 2012. The cases were analyzed according to the species, patient demographics, environmental factors, injury characteristics, and clinical course. Results Among the 68 cases of mammalian bite injury, 58 (85%) were caused by dogs, 8 by humans, and 2 by cats. Most of those bitten by a human and both of those bitten by cats were male. Only one-third of all the patients were children or adolescents. The most frequent site of injury was the face, with 40 cases, followed by the hand, with 16 cases. Of the 68 patients, 7 were treated with secondary intention healing. Sixty-one patients underwent delayed procedures, including delayed direct closure, skin graft, composite graft, and local flap. Conclusions Based on overall findings from our review of the 68 cases of mammalian bites, we suggest practical guidelines for the management of mammalian bite injuries, which could be useful in the treatment of serious mammalian bite wounds. PMID:24286042

  15. Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

    ERIC Educational Resources Information Center

    Drew, Liam J.; Fusi, Stefano; Hen, René

    2013-01-01

    In the adult mammalian brain, newly generated neurons are continuously incorporated into two networks: interneurons born in the subventricular zone migrate to the olfactory bulb, whereas the dentate gyrus (DG) of the hippocampus integrates locally born principal neurons. That the rest of the mammalian brain loses significant neurogenic capacity…

  16. Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

    ERIC Educational Resources Information Center

    Drew, Liam J.; Fusi, Stefano; Hen, René

    2013-01-01

    In the adult mammalian brain, newly generated neurons are continuously incorporated into two networks: interneurons born in the subventricular zone migrate to the olfactory bulb, whereas the dentate gyrus (DG) of the hippocampus integrates locally born principal neurons. That the rest of the mammalian brain loses significant neurogenic capacity…

  17. Rabconnectin-3 is a functional regulator of mammalian Notch signaling.

    PubMed

    Sethi, Nilay; Yan, Yan; Quek, Debra; Schupbach, Trudi; Kang, Yibin

    2010-11-05

    The Notch signaling pathway is important for cell fate decisions in embryonic development and adult life. Defining the functional importance of the Notch pathway in these contexts requires the elucidation of essential signal transduction components that have not been fully characterized. Here, we show that Rabconnectin-3B is required for the Notch pathway in mammalian cells. siRNA-mediated silencing of Rabconnectin-3B in mammalian cells attenuated Notch signaling and disrupted the activation and nuclear accumulation of the Notch target Hes1. Rabconnectin-3B knockdown also disrupted V-ATPase activity in mammalian cells, consistent with previous observations in Drosophila. Pharmacological inhibition of the V-ATPase complex significantly reduced Notch signaling in mammalian cells. Finally, Rabconnectin-3B knockdown phenocopied functional disruption of Notch signaling during osteoclast differentiation. Collectively, these findings define an important role for Rabconnectin-3 and V-ATPase activity in the Notch signaling pathway in mammalian cells.

  18. USE OF NON-MAMMALIAN ALTERNATIVE MODELS FOR NEUROTOXICOLOGICAL STUDY

    PubMed Central

    Peterson, Randall T.; Nass, Richard; Boyd, Windy A.; Freedman, Jonathan H.; Dong, Ke; Narahashi, Toshio

    2009-01-01

    The field of neurotoxicology needs to satisfy two opposing demands: the testing of a growing list of chemicals, and resource limitations and ethical concerns associated with testing using traditional mammalian species. National and international government agencies have defined a need to reduce, refine or replace mammalian species in toxicological testing with alternative testing methods and non-mammalian models. Toxicological assays using alternative animal models may relieve some of this pressure by allowing testing of more compounds while reducing expense and using fewer mammals. Recent advances in genetic technologies and the strong conservation between human and non-mammalian genomes allows for the dissection of the molecular pathways involved in neurotoxicological responses and neurological diseases using genetically tractable organisms. In this review, applications of four non-mammalian species, Zebrafish, cockroach, Drosophila, and Caenorhabditis elegans, in the investigation of neurotoxicology and neurological diseases are presented. PMID:18538410

  19. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

    PubMed

    Muir, Elizabeth M; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W; Keynes, Roger J; Rogers, John H

    2010-01-15

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

  20. The differentiation of mammalian ovarian granulosa cells – living in the shadow of cellular developmental capacity.

    PubMed

    Chachuła, A; Kranc, W; Budna, J; Bryja, A; Ciesiólka, S; Wojtanowicz-Markiewicz, K; Piotrowska, H; Bukowska, D; Krajecki, M; Antosik, P; Brüssow, K P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The mammalian cumulus-oocyte complex (COCs) promotes oocyte growth and development during long stages of folliculogenesis and oogenesis. Before ovulation, the follicle is formed by a variety of fully differentiated cell populations; cumulus cells (CCs) that tightly surround the female gamete, granulosa cells (GCs) and theca cells (TCs) which build the internal and external mass of the follicular wall. It is well documented that CCs surrounding the oocyte are necessary for resumption of meiosis and full maturation of the gamete. However, the role of the granulosa cells in acquisition of MII stage and/or full fertilization ability is not yet entirely known. In this article, we present an overview of mammalian oocytes and their relationship to the surrounding cumulus and granulosa cells. We also describe the processes of GCs differentiation and developmental capacity. Finally, we describe several markers of mammalian GCs, which could be used for positive identification of isolated cells. The developmental capacity of oocytes and surrounding somatic cells – a “fingerprint” of folliculogenesis and oogenesis.

  1. Enhanced Neurite Growth from Mammalian Neurons in Three-Dimensional Salmon Fibrin Gels

    PubMed Central

    Ju, Yo-El; Janmey, Paul A.; McCormick, Margaret; Sawyer, Evelyn S.; Flanagan, Lisa A.

    2007-01-01

    Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels or to any copurifying plasminogen. Co-purified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury. PMID:17258313

  2. Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Shrivastav, Tulsidas G; Chaube, Shail K

    2017-05-01

    Calcium (Ca(++) ) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest, and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca(++) in meiotic cell cycle resumption, arrest, and apoptosis in mammalian oocytes. Release of Ca(++) from internal stores and/or Ca(++) influx from extracellular medium causes moderate increase of intracellular Ca(++) ([Ca(++) ]i) level and reactive oxygen species (ROS). Increase of Ca(++) as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization, thereby meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in oocytes. A sustained increase of [Ca(++) ]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca(++) ]i triggers Fas ligand-mediated oocyte apoptosis. Further, OS triggers mitochondria-mediated oocyte apoptosis in several mammalian species. Thus, Ca(++) exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca(++) ]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M-II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria- as well as Fas ligand-mediated apoptotic pathways. Indeed, Ca(++) regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. J. Cell. Physiol. 232: 976-981, 2017. © 2016 Wiley Periodicals, Inc.

  3. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells.

    PubMed

    Weinberg, Benjamin H; Pham, N T Hang; Caraballo, Leidy D; Lozanoski, Thomas; Engel, Adrien; Bhatia, Swapnil; Wong, Wilson W

    2017-05-01

    Engineered genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. We present a robust, general, scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE), to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on recombinases under the control of a single promoter, which integrates circuit signals on a single transcriptional layer. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells and devised a quantitative, vector-proximity metric to evaluate their performance. Of 113 circuits analyzed, 109 functioned (96.5%) as intended without optimization. The circuits, which are available through Addgene, include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; circuits with small-molecule-inducible control; and circuits that incorporate CRISPR-Cas9 to regulate endogenous genes. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.

  4. Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.

    PubMed

    Mao, Junhao; Kim, Byeong-Moo; Rajurkar, Mihir; Shivdasani, Ramesh A; McMahon, Andrew P

    2010-05-01

    Homeostasis of the vertebrate digestive tract requires interactions between an endodermal epithelium and mesenchymal cells derived from the splanchnic mesoderm. Signaling between these two tissue layers is also crucial for patterning and growth of the developing gut. From early developmental stages, sonic hedgehog (Shh) and indian hedgehog (Ihh) are secreted by the endoderm of the mammalian gut, indicative of a developmental role. Further, misregulated hedgehog (Hh) signaling is implicated in both congenital defects and cancers arising from the gastrointestinal tract. In the mouse, only limited gastrointestinal anomalies arise following removal of either Shh or Ihh. However, given the considerable overlap in their endodermal expression domains, a functional redundancy between these signals might mask a more extensive role for Hh signaling in development of the mammalian gut. To address this possibility, we adopted a conditional approach to remove both Shh and Ihh functions from early mouse gut endoderm. Analysis of compound mutants indicates that continuous Hh signaling is dispensable for regional patterning of the gut tube, but is essential for growth of the underlying mesenchyme. Additional in vitro analysis, together with genetic gain-of-function studies, further demonstrate that Hh proteins act as paracrine mitogens to promote the expansion of adjacent mesenchymal progenitors, including those of the smooth muscle compartment. Together, these studies provide new insights into tissue interactions underlying mammalian gastrointestinal organogenesis and disease.

  5. Evaluation of Cre Recombinase Delivery in Mammalian Cells using Baculovirus Infection

    PubMed Central

    Erbs, Eric; Pradhan, Amynah A.; Matifas, Audrey; Kieffer, Brigitte L.; Massotte, Dominique

    2013-01-01

    In vivo conditional knock-out of a protein is a method of choice to decipher its biological function. It can be achieved by encoding the cre-recombinase on a recombinant virus to exert spatio-temporal control of its expression and enzymatic activity and, subsequently, of the target gene deletion. Recombinant baculoviruses have been successfully used to express a wide range of proteins in insect cells. More recently, their potential to infect mammalian cells has been addressed but, so far, their ability to yield a conditional knock-out as a result of efficient in vivo cre-recombinase gene delivery has not been examined. Cre-recombinase fused to the green fluorescent protein was cloned under the control of the CAG promoter in a recombinant Autographa californica baculovirus expressing the vesicular stomatitis virus envelope G protein for increased mammalian cell infection. Gene delivery was evaluated in vitro in mammalian cells, neuroblastoma and mouse primary neuronal cultures as well as in vivo in the mouse brain. Infection with adeno-associated viruses encoding the cre-recombinase fused to the green fluorescent protein was performed as a positive control. Our results indicate that baculovirus infection leads to functional cre-recombinase expression in non-neuronal and neuroblastoma cell lines but not in mouse primary neuronal cultures or brain. PMID:23732834

  6. The Dual Lives of Bidirectional Promoters

    PubMed Central

    Wakano, Clay; Byun, Jung S.; Di, Li-Jun; Gardner, Kevin

    2012-01-01

    The sequencing of the human genome led to many insights into gene organization and structure. One interesting observation was the high frequency of bidirectional promoters characterized by two protein encoding genes whose promoters are arranged in a divergent or “head-to-head” configuration with less than 2000 base pairs of intervening sequence. Computational estimates published by various groups indicate that nearly 10% of the coding gene promoters are arranged in such a manner and the extent of this bias is a unique feature of mammalian genomes. Moreover, as a class, head-to-head promoters appear to be enriched in specific categories of gene function. Here we review the structure, composition, genomic properties and functional classifications of genes controlled by bidirectional promoters and explore the biological implication of these features. PMID:22366276

  7. Retinal research using the perfused mammalian eye.

    PubMed

    Niemeyer, G

    2001-05-01

    The effort to isolate and maintain alive in vitro an intact mammalian eye is rewarded by the full control provided over the arterial input and exclusion of systemic regulatory or compensatory mechanisms. Electrical recording of typical light-evoked field potentials from retina and optic nerve can be complemented by single-cell recording. Thus, light-induced electrical activity reflects the function of the retinal pigment epithelium, of the layers of the retina and of the ganglion cells or their axons. Retinal function in vitro is documented by electrophysiological and morphological methods revealing subtle features of retinal information processing as well as optic nerve signals that approach-at threshold stimulus intensity-the human psychophysical threshold. Such sensitivity of third-order retinal neurons is described for the first time. This well controlled in vitro preparation has been used successfully for biophysical, metabolic and pharmacological studies. Examples are provided that demonstrate the marked sensibility of the rod system to changes in glucose supply. Moreover, histochemical identification of glycogen stores revealed labeling of the second- and third-order neurons subserving the rod system, in addition to labeling of Müller (glial) cells in the cat retina. The glycogen content of the cat retina is augmented by prolonged anesthesia, largely depleted by ischemia after enucleation and enhanced by insulin. Pharmacological experiments using agonists and antagonists of putative retinal neurotransmitters are summarized and outlined using the muscarinic cholinergic agonist QNB as an example. Actions and uptake of the neuromodulator adenosine are presented in detail, including inhibitory effects on physiologically characterized ganglion cells. Neuronal effects of adenosine are distinguished from those resulting from vasodilatation and from glycogenolysis induced by the neuromodulator. To open the blood-retina barrier, a hyperosmotic challenge can be

  8. Health Promotion

    PubMed Central

    Wilson, Ron

    1992-01-01

    How physicians address issues of disease prevention and health promotion is discussed and current standards of screening for disease and counseling practices are reviewed. Collaboration among all health professionals is necessary if preventive medicine is to be effective. PMID:21221259

  9. Mammalian prion amyloid formation in bacteria

    PubMed Central

    Macedo, Bruno; Cordeiro, Yraima; Ventura, Salvador

    2016-01-01

    ABSTRACT Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrPSc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrPSc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility. PMID:26910379

  10. Angiogenesis is inhibitory for mammalian digit regeneration

    PubMed Central

    Yu, Ling; Yan, Mingquan; Simkin, Jennifer; Ketcham, Paulina D.; Leininger, Eric; Han, Manjong

    2014-01-01

    Abstract The regenerating mouse digit tip is a unique model for investigating blastema formation and epimorphic regeneration in mammals. The blastema is characteristically avascular and we previously reported that blastema expression of a known anti‐angiogenic factor gene, Pedf, correlated with a successful regenerative response (Yu, L., Han, M., Yan, M., Lee, E. C., Lee, J. & Muneoka, K. (2010). BMP signaling induces digit regeneration in neonatal mice. Development, 137, 551–559). Here we show that during regeneration Vegfa transcripts are not detected in the blastema but are expressed at the onset of differentiation. Treating the amputation wound with vascular endothelial growth factor enhances angiogenesis but inhibits regeneration. We next tested bone morphogenetic protein 9 (BMP9), another known mediator of angiogenesis, and found that BMP9 is also a potent inhibitor of digit tip regeneration. BMP9 induces Vegfa expression in the digit stump suggesting that regenerative failure is mediated by enhanced angiogenesis. Finally, we show that BMP9 inhibition of regeneration is completely rescued by treatment with pigment epithelium‐derived factor. These studies show that precocious angiogenesis is inhibitory for regeneration, and provide compelling evidence that the regulation of angiogenesis is a critical factor in designing therapies aimed at stimulating mammalian regeneration. PMID:27499862

  11. Myocardial ischemic protection in natural mammalian hibernation.

    PubMed

    Yan, Lin; Kudej, Raymond K; Vatner, Dorothy E; Vatner, Stephen F

    2015-03-01

    Hibernating myocardium is an important clinical syndrome protecting the heart with chronic myocardial ischemia, named for its assumed resemblance to hibernating mammals in winter. However, the effects of myocardial ischemic protection have never been studied in true mammalian hibernation, which is a unique strategy for surviving extreme winter environmental stress. The goal of this investigation was to test the hypothesis that ischemic stress may also be protected in woodchucks as they hibernate in winter. Myocardial infarction was induced by coronary occlusion followed by reperfusion in naturally hibernating woodchucks in winter with and without hibernation and in summer, when not hibernating. The ischemic area at risk was similar among groups. Myocardial infarction was significantly less in woodchucks in winter, whether hibernating or not, compared with summer, and was similar to that resulting after ischemic preconditioning. Whereas several genes were up or downregulated in both hibernating woodchuck and with ischemic preconditioning, one mechanism was unique to hibernation, i.e., activation of cAMP-response element binding protein (CREB). When CREB was upregulated in summer, it induced protection similar to that observed in the woodchuck heart in winter. The cardioprotection in hibernation was also mediated by endothelial nitric oxide synthase, rather than inducible nitric oxide synthase. Thus, the hibernating woodchuck heart is a novel model to study cardioprotection for two major reasons: (1) powerful cardioprotection occurs naturally in winter months in the absence of any preconditioning stimuli, and (2) it resembles ischemic preconditioning, but with novel mechanisms, making this model potentially useful for clinical translation.

  12. Nitric oxide negatively regulates mammalian adult neurogenesis

    NASA Astrophysics Data System (ADS)

    Packer, Michael A.; Stasiv, Yuri; Benraiss, Abdellatif; Chmielnicki, Eva; Grinberg, Alexander; Westphal, Heiner; Goldman, Steven A.; Enikolopov, Grigori

    2003-08-01

    Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

  13. A synthetic low-frequency mammalian oscillator

    PubMed Central

    Tigges, Marcel; Dénervaud, Nicolas; Greber, David; Stelling, Joerg; Fussenegger, Martin

    2010-01-01

    Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives. PMID:20197318

  14. Ventricular Fibrillation in Mammalian Hearts: Simulation Results

    NASA Astrophysics Data System (ADS)

    Fenton, Flavio H.

    2002-03-01

    The computational approach to understanding the initiation and evolution of cardiac arrhythmias forms a necessary link between experiment and theory. Numerical simulations combine useful mathematical models and complex geometry while offering clean and comprehensive data acquisition, reproducible results that can be compared to experiments, and the flexibility of exploring parameter space systematically. However, because cardiac dynamics occurs on many scales (on the order of 10^9 cells of size 10-100 microns with more than 40 ionic currents and time scales as fast as 0.01ms), roughly 10^17 operations are required to simulate just one second of real time. These intense computational requirements lead to significant implementation challenges even on existing supercomputers. Nevertheless, progress over the last decade in understanding the effects of some spatial scales and spatio-temporal dynamics on cardiac cell and tissue behavior justifies the use of certain simplifications which, along with improved models for cellular dynamics and detailed digital models of cardiac anatomy, are allowing simulation studies of full-size ventricles and atria. We describe this simulation problem from a combined numerical, physical and biological point of view, with an emphasis on the dynamics and stability of scroll waves of electrical activity in mammalian hearts and their relation to tachycardia, fibrillation and sudden death. Detailed simulations of electrical activity in ventricles including complex anatomy, anisotropic fiber structure, and electrophysiological effects of two drugs (DAM and CytoD) are presented and compared with experimental results.

  15. Movement Symmetries and the Mammalian Vestibular System

    NASA Astrophysics Data System (ADS)

    McCollum, Gin; Boyle, Richard

    2000-03-01

    Unity of movement requires vertebrates to have an ability to symmetrize along the midline. For example, human erect stance involves symmetry with respect to gravity. The mammalian vestibular system provides a mechanism for maintaining symmetries, which is also open to influence and adaptation by the rest of the organism. The vestibular system includes the inner ear endorgans and central nuclei, along with projections to oculomotor, cerebellar, thalamic, and spinal motor centers. The vestibular endorgans - the semicircular canals and the otoliths - use sensory hairs to register inertia. The vestibular endorgans are right-left symmetric and the semicircular canals form an approximately orthogonal coordinate system for angular motion. Primary afferent axons project from the endorgans to the vestibular nuclei (and a few other places). The vestibular nuclei integrate vestibular, visual, and somatosensory signals, along with a proposed copy of the voluntary motor command and signals from other central structures. The relationship between the canals and the otoliths gives rise to symmetries among neurons, in the organization among the several vestibular nuclei, and in the projections from the vestibular nuclei. These symmetries organize the space of body movements so that functional relationships are maintained in spite of the many free variables of body movement. They also provide a foundation for adaptive reinterpretation of the relationship between canal and otolith signals, for example in freefall.

  16. Multiple Functions and Regulation of Mammalian Peroxiredoxins.

    PubMed

    Rhee, Sue Goo; Kil, In Sup

    2017-06-20

    Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

  17. Tubulin dynamics in cultured mammalian cells

    PubMed Central

    1984-01-01

    Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells. PMID:6501419

  18. Activation of mammalian tyrosinase by ferrous ions.

    PubMed

    Palumbo, A; d'Ischia, M; Misuraca, G; Carratú, L; Prota, G

    1990-03-26

    Kinetic experiments are reported showing that mammalian tyrosinase from B16 mouse melanoma is significantly activated by catalytic amounts of ferrous ions. Monitoring of tyrosine oxidation by both dopachrome formation and oxygen consumption showed that ferrous ions at micromolar concentrations induce a marked enzymatic activity with 0.01 U/ml of highly purified tyrosinase, whereas no detectable reaction occurs in the absence of metal over a sufficiently prolonged period of time. The extent of the activating effect, which is specific for the reduced form of iron, is proportional to the concentration of the added metal with a typical saturation profile, no further effect being observed beyond a threshold value. Changing the buffer system from phosphate to hepes or tris results in a marked decrease of the Fe2(+)-induced activation. Scavengers of active oxygen species, such as superoxide dismutase, catalase, formate and mannitol have no detectable effect on the tyrosinase activity. These results are accounted for in terms of an activation mechanism involving reduction of the cupric ions at the active site of the resting enzyme.

  19. Melanopsin signalling in mammalian iris and retina.

    PubMed

    Xue, T; Do, M T H; Riccio, A; Jiang, Z; Hsieh, J; Wang, H C; Merbs, S L; Welsbie, D S; Yoshioka, T; Weissgerber, P; Stolz, S; Flockerzi, V; Freichel, M; Simon, M I; Clapham, D E; Yau, K-W

    2011-11-02

    Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

  20. Mammalian cyanide detoxification with sulphane sulphur.

    PubMed

    Westley, J

    1988-01-01

    It has been understood for nearly a century that most of the cyanide entering mammals by either ingestion or inhalation of sublethal quantities is detoxified by combination with cyanide-reactive sulphur. The detoxifying reaction is primarily enzymic, and the enzymes that catalyse it, as well as those that catalyse reactions which generate the cyanide-reactive sulphane sulphur, have been isolated and studied extensively. However, analytical methods suitable for quantitative characterization of the physiological pool of sulphur available for this detoxification have only recently come under development. A new method is based on the evaluation of cyanide depletion under controlled, mild reaction conditions in the presence of a sulphurtransferase and cofactor. The use of differential pulse polarography with nested internal standards makes this a practical procedure for samples containing as little as a few nanomoles of cyanide-reactive material. As measured by this method, the sulphane sulphur concentration in mammalian liver tissue is in the fractional millimolar range, with even less in the bloodstream. Nevertheless, the specific binding of sulphane sulphur by serum albumin, and the rapid cyanolysis of the albumin-bound sulphur, may yet indicate a significant role for the sulphurtransferase capability of serum albumin in cyanide detoxification.

  1. Cholesterol, the central lipid of mammalian cells

    PubMed Central

    Maxfield, Frederick R.; van Meer, Gerrit

    2010-01-01

    Summary of recent advances Despite its importance for mammalian cell biology and human health, there are many basic aspects of cholesterol homeostasis that are not well understood. Even for the well-characterized delivery of cholesterol to cells via lipoproteins, a novel regulatory mechanism has been discovered recently, involving a serum protein called PCSK9, which profoundly affects lipoproteins and their receptors. Cells can export cholesterol by processes that require the activity of ABC transporters, but the molecular mechanisms for cholesterol transport remain unclear. Cholesterol levels in different organelles vary by 5–10 fold, and the mechanisms for maintaining these differences are now partially understood. Several proteins have been proposed to play a role in the inter-organelle movement of cholesterol, but many aspects of the mechanisms for regulating intracellular transport and distribution of cholesterol remain to be worked out. The endoplasmic reticulum is the main organelle responsible for regulation of cholesterol synthesis, and careful measurements have shown that the proteins responsible for sterol sensing respond over a very narrow range of cholesterol concentrations to provide very precise, switch-like control over cholesterol synthesis. PMID:20627678

  2. Cell fate regulation in early mammalian development

    NASA Astrophysics Data System (ADS)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  3. Mitochondrial dynamics in mammalian health and disease.

    PubMed

    Liesa, Marc; Palacín, Manuel; Zorzano, Antonio

    2009-07-01

    The meaning of the word mitochondrion (from the Greek mitos, meaning thread, and chondros, grain) illustrates that the heterogeneity of mitochondrial morphology has been known since the first descriptions of this organelle. Such a heterogeneous morphology is explained by the dynamic nature of mitochondria. Mitochondrial dynamics is a concept that includes the movement of mitochondria along the cytoskeleton, the regulation of mitochondrial architecture (morphology and distribution), and connectivity mediated by tethering and fusion/fission events. The relevance of these events in mitochondrial and cell physiology has been partially unraveled after the identification of the genes responsible for mitochondrial fusion and fission. Furthermore, during the last decade, it has been identified that mutations in two mitochondrial fusion genes (MFN2 and OPA1) cause prevalent neurodegenerative diseases (Charcot-Marie Tooth type 2A and Kjer disease/autosomal dominant optic atrophy). In addition, other diseases such as type 2 diabetes or vascular proliferative disorders show impaired MFN2 expression. Altogether, these findings have established mitochondrial dynamics as a consolidated area in cellular physiology. Here we review the most significant findings in the field of mitochondrial dynamics in mammalian cells and their implication in human pathologies.

  4. Roles of the oviduct in mammalian fertilization

    PubMed Central

    Coy, P; García-Vázquez, FA; Visconti, PE; Avilés, M

    2014-01-01

    The oviduct or Fallopian tube is the anatomical region where every new life begins in mammalian species. After a long journey, the spermatozoa meet the oocyte in the specific site of the oviduct named ampulla, and fertilization takes place. The successful fertilization depends on several biological processes which occur in the oviduct some hours before this rendezvous and affect both gametes. Estrogen and progesterone, released from the ovary, orchestrate a series of changes by genomic- and non-genomic pathways in the oviductal epithelium affecting gene expression, proteome and secretion of its cells into the fluid bathing the oviductal lumen. In addition, new regulatory molecules are being discovered playing important roles in oviductal physiology and fertilization. The present review tries to describe these processes, building a comprehensive map of the physiology of the oviduct, to better understand the importance of this organ in reproduction. With this purpose, gamete transport, sperm and oocyte changes in the oviductal environment and other interactions between gametes and oviduct are discussed in light of recent publications in the field. PMID:23028122

  5. Characterization of hibernating ribosomes in mammalian cells

    PubMed Central

    Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A

    2011-01-01

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions. PMID:21768774

  6. Functional amyloid formation within mammalian tissue.

    PubMed

    Fowler, Douglas M; Koulov, Atanas V; Alory-Jost, Christelle; Marks, Michael S; Balch, William E; Kelly, Jeffery W

    2006-01-01

    Amyloid is a generally insoluble, fibrous cross-beta sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin-a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.

  7. Historical Perspectives: plasticity of mammalian skeletal muscle.

    PubMed

    Pette, D

    2001-03-01

    More than 40 years ago, the nerve cross-union experiment of Buller, Eccles, and Eccles provided compelling evidence for the essential role of innervation in determining the properties of mammalian skeletal muscle fibers. Moreover, this experiment revealed that terminally differentiated muscle fibers are not inalterable but are highly versatile entities capable of changing their phenotype from fast to slow or slow to fast. With the use of various experimental models, numerous studies have since confirmed and extended the notion of muscle plasticity. Together, these studies demonstrated that motoneuron-specific impulse patterns, neuromuscular activity, and mechanical loading play important roles in both the maintenance and transition of muscle fiber phenotypes. Depending on the type, intensity, and duration of changes in any of these factors, muscle fibers adjust their phenotype to meet the altered functional demands. Fiber-type transitions resulting from multiple qualitative and quantitative changes in gene expression occur sequentially in a regular order within a spectrum of pure and hybrid fiber types.

  8. Angiogenesis is inhibitory for mammalian digit regeneration.

    PubMed

    Yu, Ling; Yan, Mingquan; Simkin, Jennifer; Ketcham, Paulina D; Leininger, Eric; Han, Manjong; Muneoka, Ken

    2014-06-01

    The regenerating mouse digit tip is a unique model for investigating blastema formation and epimorphic regeneration in mammals. The blastema is characteristically avascular and we previously reported that blastema expression of a known anti-angiogenic factor gene, Pedf, correlated with a successful regenerative response (Yu, L., Han, M., Yan, M., Lee, E. C., Lee, J. & Muneoka, K. (2010). BMP signaling induces digit regeneration in neonatal mice. Development, 137, 551-559). Here we show that during regeneration Vegfa transcripts are not detected in the blastema but are expressed at the onset of differentiation. Treating the amputation wound with vascular endothelial growth factor enhances angiogenesis but inhibits regeneration. We next tested bone morphogenetic protein 9 (BMP9), another known mediator of angiogenesis, and found that BMP9 is also a potent inhibitor of digit tip regeneration. BMP9 induces Vegfa expression in the digit stump suggesting that regenerative failure is mediated by enhanced angiogenesis. Finally, we show that BMP9 inhibition of regeneration is completely rescued by treatment with pigment epithelium-derived factor. These studies show that precocious angiogenesis is inhibitory for regeneration, and provide compelling evidence that the regulation of angiogenesis is a critical factor in designing therapies aimed at stimulating mammalian regeneration.

  9. Genetically programmed superparamagnetic behavior of mammalian cells.

    PubMed

    Kim, Taeuk; Moore, David; Fussenegger, Martin

    2012-12-31

    Although magnetic fields and paramagnetic inorganic materials were abundant on planet earth during the entire evolution of living species the interaction of organisms with these physical forces remains a little-understood phenomenon. Interestingly, rather than being genetically encoded, organisms seem to accumulate and take advantage of inorganic nanoparticles to sense or react to magnetic fields. Using a synthetic biology-inspired approach we have genetically programmed mammalian cells to show superparamagnetic behavior. The combination of ectopic production of the human ferritin heavy chain 1 (hFTH1), engineering the cells for expression of an iron importer, the divalent metal ion transferase 1 (DMT1) and the design of an iron-loading culture medium to maximize cellular iron uptake enabled efficient iron mineralization in intracellular ferritin particles and conferred superparamagnetic behavior to the entire cell. When captured by a magnetic field the superparamagnetic cells reached attraction velocities of up to 30 μm/s and could be efficiently separated from complex cell mixtures using standard magnetic cell separation equipment. Technology that enables magnetic separation of genetically programmed superparamagnetic cells in the absence of inorganic particles could foster novel opportunities in diagnostics and cell-based therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Myocardial ischemic protection in natural mammalian hibernation

    PubMed Central

    Yan, Lin; Kudej, Raymond K.; Vatner, Dorothy E.

    2015-01-01

    Hibernating myocardium is an important clinical syndrome protecting the heart with chronic myocardial ischemia, named for its assumed resemblance to hibernating mammals in winter. However, the effects of myocardial ischemic protection have never been studied in true mammalian hibernation, which is a unique strategy for surviving extreme winter environmental stress. The goal of this investigation was to test the hypothesis that ischemic stress may also be protected in woodchucks as they hibernate in winter. Myocardial infarction was induced by coronary occlusion followed by reperfusion in naturally hibernating woodchucks in winter with and without hibernation and in summer, when not hibernating. The ischemic area at risk was similar among groups. Myocardial infarction was significantly less in woodchucks in winter, whether hibernating or not, compared with summer, and was similar to that resulting after ischemic preconditioning. Whereas several genes were up or downregulated in both hibernating woodchuck and with ischemic preconditioning, one mechanism was unique to hibernation, i.e., activation of cAMP-response element binding protein (CREB). When CREB was upregulated in summer, it induced protection similar to that observed in the woodchuck heart in winter. The cardioprotection in hibernation was also mediated by endothelial nitric oxide synthase, rather than inducible nitric oxide synthase. Thus, the hibernating woodchuck heart is a novel model to study cardioprotection for two major reasons: (1) powerful cardioprotection occurs naturally in winter months in the absence of any preconditioning stimuli, and (2) it resembles ischemic preconditioning, but with novel mechanisms, making this model potentially useful for clinical translation. PMID:25613166

  11. Late Quaternary mammalian zoogeography of eastern Washington

    NASA Astrophysics Data System (ADS)

    Lyman, R. Lee; Livingston, Stephanie D.

    1983-11-01

    The late Quaternary mammalian zoogeographic history of eastern Washington as revealed by archaeological and paleontological research conforms to a set of past environmental conditions inferred from botanical data. During the relatively cool and moist late Pleistocene and early Holocene, Cervus cf. elaphus, Ovis canadensis, Vulpes vulpes, Martes americana, Alopex lagopus, and perhaps Rangifer sp., taxa with ecological preferences for mesic steppe habitats, were present in the now xeric Columbia Basin. As the climate became progressively warmer and drier during the late Pleistocene and early Holocene, Antilocapra americana, Onychomys leucogaster, Spermophilus townsendii, and Neotoma cinerea, taxa with ecological preferences for xeric steppe habitats, appear in the Columbia Basin. Bison sp. and Taxidea taxus may have been present in eastern Washington for the last 20,000 yr. Middle and late Holocene records for Oreamnos americanus, Spermophilus columbianus, S. townsendii, Lagurus curtatus, and Urocyon cinereoargenteus in central eastern Washington suggest fluctuations in the ranges of these taxa that conform to a middle Holocene period of less effective precipitation and a ca. 3500-yr-old period of more effective precipitation before essentially modern environmental conditions prevailed.

  12. Repair of radiation damage in mammalian cells

    SciTech Connect

    Setlow, R.B.

    1981-01-01

    The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis.

  13. ORC proteins in the mammalian zygote.

    PubMed

    Ortega, Michael A; Nguyen, Hieu; Ward, W Steven

    2016-01-01

    The origin recognition complex (ORC) proteins, ORC1-6, are the first known proteins that bind DNA replication origins to mark the competency for the initiation of DNA synthesis. These proteins have complex mechanisms of assembly into the ORC complex and unexpected localizations in the mitotic chromosomes, cytoplasm, and nuclear structures. The mammalian zygote is a potentially important model that may contribute to our understanding of the mechanisms and features influencing origin establishment and in the identification of other functions of the ORC proteins. Together with expected localizations to the chromatin during G1, we found an unexpected distribution in the cytoplasm that appeared to accumulate ORC proteins suggesting potential roles for ORC subunits in mitosis and chromatin segregation. ORC1, 2, 3, and 5 all localize to the area between the separating maternal chromosomes shortly after fertilization. ORC4 forms a cage around the set of chromosomes that will be extruded during polar body formation before it binds to the chromatin shortly before zygotic DNA replication. These data suggest that the ORC proteins may also play roles in preparing the cell for DNA replication in addition to their direct role in establishing functional replication origins.

  14. ORC Proteins in the Mammalian Zygote

    PubMed Central

    Ortega, Michael A.; Nguyen, Hieu; Ward, W. Steven

    2015-01-01

    Summary The origin recognition complex (ORC) proteins, ORC1-6, are the first proteins that bind DNA replication origins to mark the competency for the initiation of DNA synthesis. These proteins have complex mechanisms of assembly into the ORC complex, and unexpected localizations in the mitotic chromosomes, cytoplasm, and nuclear structures. The mammalian zygote is a potentially important model that may contribute to our understanding of the mechanisms and features influencing origin establishment, and in identifying other functions of the ORC proteins. Along with expected localizations to the chromatin during G1, we found unexpected distribution in the cytoplasm that appeared to accumulate ORC proteins, which suggest potential roles for ORC subunits in mitosis and chromatin segregation. ORC1, 2, 3 and 5 all localize to the area between the separating maternal chromosomes shortly after fertilization. ORC4 forms a cage around the set of chromosomes that will be extruded during polar body formation before it binds to the chromatin shortly before zygotic DNA replication. These data suggest that the ORC proteins may also play roles in preparing the cell for DNA replication in addition to their direct role in establishing functional replication origins. PMID:26453397

  15. Timing of circadian genes in mammalian tissues

    PubMed Central

    Korenčič, Anja; Košir, Rok; Bordyugov, Grigory; Lehmann, Robert; Rozman, Damjana; Herzel, Hanspeter

    2014-01-01

    Circadian clocks are endogenous oscillators driving daily rhythms in physiology. The cell-autonomous clock is governed by an interlocked network of transcriptional feedback loops. Hundreds of clock-controlled genes (CCGs) regulate tissue specific functions. Transcriptome studies reveal that different organs (e.g. liver, heart, adrenal gland) feature substantially varying sets of CCGs with different peak phase distributions. To study the phase variability of CCGs in mammalian peripheral tissues, we develop a core clock model for mouse liver and adrenal gland based on expression profiles and known cis-regulatory sites. ‘Modulation factors’ associated with E-boxes, ROR-elements, and D-boxes can explain variable rhythms of CCGs, which is demonstrated for differential regulation of cytochromes P450 and 12 h harmonics. By varying model parameters we explore how tissue-specific peak phase distributions can be generated. The central role of E-boxes and ROR-elements is confirmed by analysing ChIP-seq data of BMAL1 and REV-ERB transcription factors. PMID:25048020

  16. Desiccation response of mammalian cells: anhydrosignaling.

    PubMed

    Huang, Zebo; Tunnacliffe, Alan

    2007-01-01

    Dehydration through evaporation, or air drying, is expected to have both similarities and differences to osmostress. Both stresses involve water loss, but the degree of dehydration will ultimately be more severe during desiccation. Despite the severity of desiccation stress, there are examples of organisms that can survive almost complete water loss, including resurrection plants and plant seeds, certain invertebrates among the nematodes, brine shrimps, tardigrades and bdelloid rotifers, and many microorganisms, including bakers' yeast. During desiccation, these organisms enter a state of suspended animation, a process known as anhydrobiosis ("life without water"). For other organisms, desiccation is lethal, but there is considerable interest in using what is known about anhydrobiosis to confer desiccation tolerance on sensitive cell types, such as mammalian cells. Success with this approach, which we have termed anhydrobiotic engineering, will require a more complete knowledge of the mechanisms of desiccation tolerance and the sensing and response of nontolerant organisms to extreme dehydration. With this goal in mind, we have attempted to characterize the response of human tissue culture cells to desiccation and to compare this response with osmotic upshift. This chapter describes some of the methods used to begin to uncover the response to evaporative water loss in human cell cultures.

  17. The First Mammalian Aldehyde Oxidase Crystal Structure

    PubMed Central

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-01-01

    Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

  18. Ion channels of the mammalian urethra

    PubMed Central

    Kyle, Barry D

    2014-01-01

    The mammalian urethra is a muscular tube responsible for ensuring that urine remains in the urinary bladder until urination. In order to prevent involuntary urine leakage, the urethral musculature must be capable of constricting the urethral lumen to an extent that exceeds bladder intravesicular pressure during the urine-filling phase. The main challenge in anti-incontinence treatments involves selectively-controlling the excitability of the smooth muscles in the lower urinary tract. Almost all strategies to battle urinary incontinence involve targeting the bladder and as a result, this tissue has been the focus for the majority of research and development efforts. There is now increasing recognition of the value of targeting the urethral musculature in the treatment and management of urinary incontinence. Newly-identified and characterized ion channels and pathways in the smooth muscle of the urethra provides a range of potential therapeutic targets for the treatment of urinary incontinence. This review provides a summary of the current state of knowledge of the ion channels discovered in urethral smooth muscle cells that regulate their excitability. PMID:25483582

  19. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  20. Evolution of the mammalian dentate gyrus.

    PubMed

    Hevner, Robert F

    2016-02-15

    The dentate gyrus (DG), a part of the hippocampal formation, has important functions in learning, memory, and adult neurogenesis. Compared with homologous areas in sauropsids (birds and reptiles), the mammalian DG is larger and exhibits qualitatively different phenotypes: 1) folded (C- or V-shaped) granule neuron layer, concave toward the hilus and delimited by a hippocampal fissure; 2) nonperiventricular adult neurogenesis; and 3) prolonged ontogeny, involving extensive abventricular (basal) migration and proliferation of neural stem and progenitor cells (NSPCs). Although gaps remain, available data indicate that these DG traits are present in all orders of mammals, including monotremes and marsupials. The exception is Cetacea (whales, dolphins, and porpoises), in which DG size, convolution, and adult neurogenesis have undergone evolutionary regression. Parsimony suggests that increased growth and convolution of the DG arose in stem mammals concurrently with nonperiventricular adult hippocampal neurogenesis and basal migration of NSPCs during development. These traits could all result from an evolutionary change that enhanced radial migration of NSPCs out of the periventricular zones, possibly by epithelial-mesenchymal transition, to colonize and maintain nonperiventricular proliferative niches. In turn, increased NSPC migration and clonal expansion might be a consequence of growth in the cortical hem (medial patterning center), which produces morphogens such as Wnt3a, generates Cajal-Retzius neurons, and is regulated by Lhx2. Finally, correlations between DG convolution and neocortical gyrification (or capacity for gyrification) suggest that enhanced abventricular migration and proliferation of NSPCs played a transformative role in growth and folding of neocortex as well as archicortex.

  1. A mammalian acetate switch regulates stress erythropoiesis

    PubMed Central

    Xu, Min; Nagati, Jason S.; Xie, Jian; Li, Jiwen; Walters, Holly; Moon, Young-Ah; Gerard, Robert D.; Huang, Chou-Long; Comerford, Sarah A.; Hammer, Robert E.; Horton, Jay D.; Chen, Rui; Garcia, Joseph A.

    2014-01-01

    Endocrine erythropoietin (Epo), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor Hypoxia Inducible Factor 2 (HIF-2). We previously reported that the lysine acetyltransferase Cbp is required for HIF-2α acetylation and efficient HIF-2 dependent Epo induction during hypoxia. We now show these processes require acetate-dependent acetyl CoA synthetase 2 (Acss2). In Hep3B hepatoma cells and in Epo-generating organs of hypoxic or acutely anemic mice, acetate levels increase and Acss2 is required for HIF-2α acetylation, Cbp/HIF-2α complex formation and recruitment to the Epo enhancer, and efficient Epo induction. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an Acss2-dependent manner. In acquired and genetic chronic anemia mouse models, acetate supplementation also increases Epo expression and resting hematocrits. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and Epo induction during pathophysiological states marked by tissue hypoxia. PMID:25108527

  2. From Immunity and Vaccines to Mammalian Regeneration

    PubMed Central

    Heber-Katz, Ellen

    2015-01-01

    Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21cip1/waf1. The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution. PMID:26116734

  3. Characterization of hibernating ribosomes in mammalian cells.

    PubMed

    Krokowski, Dawid; Gaccioli, Francesca; Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A; Taylor, Derek; Hatzoglou, Maria

    2011-08-15

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.

  4. Network features of the mammalian circadian clock.

    PubMed

    Baggs, Julie E; Price, Tom S; DiTacchio, Luciano; Panda, Satchidananda; Fitzgerald, Garret A; Hogenesch, John B

    2009-03-10

    The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.

  5. Sources of Error in Mammalian Genetic Screens.

    PubMed

    Sack, Laura Magill; Davoli, Teresa; Xu, Qikai; Li, Mamie Z; Elledge, Stephen J

    2016-09-08

    Genetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias. Copyright © 2016 Sack et al.

  6. Sources of Error in Mammalian Genetic Screens

    PubMed Central

    Sack, Laura Magill; Davoli, Teresa; Xu, Qikai; Li, Mamie Z.; Elledge, Stephen J.

    2016-01-01

    Genetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc. This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias. PMID:27402361

  7. Mitochondrial inheritance is mediated by microtubules in mammalian cell division.

    PubMed

    Lawrence, Elizabeth; Mandato, Craig

    2013-11-01

    The mitochondrial network fragments and becomes uniformly dispersed within the cytoplasm when mammalian cells enter mitosis. Such morphology and distribution of mitochondria was previously thought to facilitate the stochastic inheritance of mitochondria by daughter cells. In contrast, we recently reported that mitochondria in dividing mammalian cells are inherited by an ordered mechanism of inheritance mediated by microtubules. We showed that mitochondria are progressively enriched at the cell equator and depleted at the poles throughout division. Furthermore, the mitochondrial distribution during division is dependent on microtubules, indicating an ordered inheritance strategy. The microtubule-mediated positioning of mitochondria in dividing mammalian cells may have functional consequences for cell division and/or mitochondrial inheritance.

  8. Fishing for mammalian paradigms in the teleost immune system.

    PubMed

    Sunyer, J Oriol

    2013-04-01

    Recent years have witnessed a renaissance in the study of fish immune systems. Such studies have greatly expanded the knowledge of the evolution and diversification of vertebrate immune systems. Several findings in those studies have overturned old paradigms about the immune system and led to the discovery of novel aspects of mammalian immunity. Here I focus on how findings pertaining to immunity in teleost (bony) fish have led to major new insights about mammalian B cell function in innate and adaptive immunity. Additionally, I illustrate how the discovery of the most ancient mucosal immunoglobulin described thus far will help resolve unsettled paradigms of mammalian mucosal immunity.

  9. Fishing for mammalian paradigms in the teleost immune system

    PubMed Central

    Sunyer, J Oriol

    2013-01-01

    Recent years have witnessed a renaissance in the study of fish immune systems. Such studies have greatly expanded the knowledge of the evolution and diversification of vertebrate immune systems. Several findings in those studies have overturned old paradigms about the immune system and led to the discovery of novel aspects of mammalian immunity. Here I focus on how findings pertaining to immunity in teleost (bony) fish have led to major new insights about mammalian B cell function in innate and adaptive immunity. Additionally, I illustrate how the discovery of the most ancient mucosal immunoglobulin described thus far will help resolve unsettled paradigms of mammalian mucosal immunity. PMID:23507645

  10. Vitamin H-regulated transgene expression in mammalian cells

    PubMed Central

    Weber, Wilfried; Bacchus, William; Daoud-El Baba, Marie; Fussenegger, Martin

    2007-01-01

    Although adjustable transgene expression systems are considered essential for future therapeutic and biopharmaceutical manufacturing applications, the currently available transcription control modalities all require side-effect-prone inducers such as immunosupressants, hormones and antibiotics for fine-tuning. We have designed a novel mammalian transcription-control system, which is reversibly fine-tuned by non-toxic vitamin H (also referred to as biotin). Ligation of vitamin H, by engineered Escherichia coli biotin ligase (BirA), to a synthetic biotinylation signal fused to the tetracycline-dependent transactivator (tTA), enables heterodimerization of tTA to a streptavidin-linked transrepressor domain (KRAB), thereby abolishing tTA-mediated transactivation of specific target promoters. As heterodimerization of tTA to KRAB is ultimately conditional upon the presence of vitamin H, the system is vitamin H responsive. Transgenic Chinese hamster ovary cells, engineered for vitamin H-responsive gene expression, showed high-level, adjustable and reversible production of a human model glycoprotein in bench-scale culture systems, bioreactor-based biopharmaceutical manufacturing scenarios, and after implantation into mice. The vitamin H-responsive expression systems showed unique band pass filter-like regulation features characterized by high-level expression at low (0–2 nM biotin), maximum repression at intermediate (100–1000 nM biotin), and high-level expression at increased (>100 000 nM biotin) biotin concentrations. Sequential ON-to-OFF-to-ON, ON-to-OFF and OFF-to-ON expression profiles with graded expression transitions can all be achieved by simply increasing the level of a single inducer molecule without exchanging the culture medium. These novel expression characteristics mediated by an FDA-licensed inducer may foster advances in therapeutic cell engineering and manufacturing of difficult-to-produce protein therapeutics. PMID:17827215

  11. Mechanisms regulating melatonin synthesis in the mammalian pineal organ.

    PubMed

    Schomerus, Christof; Korf, Horst-Werner

    2005-12-01

    The day/night rhythm in melatonin production is a characteristic feature in vertebrate physiology. This hormonal signal reliably reflects the environmental light conditions and is independent of behavioral aspects. In all mammalian species, melatonin production is regulated by norepinephrine, which is released from sympathetic nerve fibers exclusively at night. Norepinephrine elevates the intracellular cAMP concentration via beta-adrenergic receptors and activates the cAMP-dependent protein kinase A. This pathway is crucial for regulation of the penultimate enzyme in melatonin biosynthesis, the arylalkylamine N-acetyltransferase (AANAT); cAMP/protein kinase A may, however, act in different ways. In ungulates and primates, pinealocytes constantly synthesize AANAT protein from continually available Aanat mRNA. During the day-in the absence of noradrenergic stimulation-the protein is immediately destroyed by proteasomal proteolysis. At nighttime, elevated cAMP levels cause phosphorylation of AANAT by protein kinase A. This posttranslational modification leads to interaction of phosphorylated AANAT with regulatory 14-3-3 proteins, which protect AANAT from degradation. Increases in AANAT protein are paralleled by increases in enzyme activity. Stimulation of the cAMP/protein kinase A pathway may also activate pineal gene expression. In rodents, transcriptional activation of the Aanat gene is the primary mechanism for the induction of melatonin biosynthesis and results in marked day/night fluctuations in Aanat mRNA. It involves protein kinase A-dependent phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) and binding of phosphorylated CREB in the promoter region of the Aanat gene. In conclusion, a common neuroendocrine principle, the nocturnal rise in melatonin, is controlled by strikingly diverse regulatory mechanisms. This diversity has emerged in the course of evolution and reflects the high adaptive plasticity of the

  12. Control of mammalian gene expression by amino acids, especially glutamine.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Husson, Annie

    2009-04-01

    Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic-leucine zipper factors, activating transcription factors and CCAAT/enhancer-binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient-sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient-sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine-responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor-kappaB in the anti-inflammatory action of glutamine, the stimulatory role of activating protein-1 and the inhibitory role of C/EBP homology binding protein in growth-promotion, and the role of c-myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen-activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene

  13. Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters.

    PubMed

    Chen, Yun; Pai, Athma A; Herudek, Jan; Lubas, Michal; Meola, Nicola; Järvelin, Aino I; Andersson, Robin; Pelechano, Vicent; Steinmetz, Lars M; Jensen, Torben Heick; Sandelin, Albin

    2016-09-01

    Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.

  14. Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

    PubMed Central

    Chen, Yun; Pai, Athma A.; Herudek, Jan; Lubas, Michal; Meola, Nicola; Järvelin, Aino I.; Andersson, Robin; Pelechano, Vicent; Steinmetz, Lars M.; Heick Jensen, Torben; Sandelin, Albin

    2016-01-01

    Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes. PMID:27455346

  15. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    SciTech Connect

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  16. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  17. From quiescence to proliferation: Cdk oscillations drive the mammalian cell cycle.

    PubMed

    Gérard, Claude; Goldbeter, Albert

    2012-01-01

    We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks) driving the mammalian cell cycle (Gérard and Goldbeter, 2009). The model contains four modules, each centered around one cyclin/Cdk complex. Cyclin D/Cdk4-6 and cyclin E/Cdk2 promote progression in G1 and elicit the G1/S transition, respectively; cyclin A/Cdk2 ensures progression in S and the transition S/G2, while the activity of cyclin B/Cdk1 brings about the G2/M transition. This model shows that in the presence of sufficient amounts of growth factor the Cdk network is capable of temporal self-organization in the form of sustained oscillations, which correspond to the ordered, sequential activation of the various cyclin/Cdk complexes that control the successive phases of the cell cycle. The results suggest that the switch from cellular quiescence to cell proliferation corresponds to the transition from a stable steady state to sustained oscillations in the Cdk network. The transition depends on a finely tuned balance between factors that promote or hinder progression in the cell cycle. We show that the transition from quiescence to proliferation can occur in multiple ways that alter this balance. By resorting to bifurcation diagrams, we analyze the mechanism of oscillations in the Cdk network. Finally, we show that the complexity of the detailed model can be greatly reduced, without losing its key dynamical properties, by considering a skeleton model for the Cdk network. Using such a skeleton model for the mammalian cell cycle we show that positive feedback (PF) loops enhance the amplitude and the robustness of Cdk oscillations with respect to molecular noise. We compare the relative merits of the detailed and skeleton versions of the model for the Cdk network driving the mammalian cell cycle.

  18. The integrin-binding domain of invasin is sufficient to allow bacterial entry into mammalian cells.

    PubMed Central

    Rankin, S; Isberg, R R; Leong, J M

    1992-01-01

    Yersinia pseudotuberculosis is able to enter normally nonphagocytic host cells by multiple pathways, the most efficient of which is mediated by invasin, a 986-amino-acid bacterial outer membrane protein. It has previously been shown that the C-terminal 192 amino acids of invasin are sufficient to bind mammalian cells. To determine if additional regions of the invasin protein are necessary to promote entry, we developed a novel assay that tests the ability of various invasin derivatives to confer on Staphylococcus aureus the ability to enter animal cells. We determined that the 192-amino-acid cell-binding region of invasin, when used to coat the bacterial cell surface, was also sufficient to promote cellular penetration. These results suggest that the simple binding of invasin to its receptors is sufficient to mediate entry and that the bacterium plays a largely passive role in the entry process. Images PMID:1500198

  19. Apparent microRNA-Target-specific Histone Modification in Mammalian Spermatogenesis

    PubMed Central

    Taguchi, Y-H

    2015-01-01

    BACKGROUND Epigenetics is an important mRNA expression regulator. However, how distinct epigenetic factors, such as microRNAs (miRNAs) and promoter methylation, cooperatively regulate mRNA expression is rarely discussed. Recently, apparent miRNA regulation of promoter methylation was identified by bioinformatic analysis; however, it has not yet been experimentally confirmed. If miRNA regulation of other epigenetic factors were identified, it would reveal another layer of epigenetic regulation. In this paper, histone modifications (H3K4me1, H3K4me3, H3K27me3, H3K27ac, H3K9ac, and H2AZ) during mammalian spermatogenesis were studied and the apparent miRNA-target-specific histone modification was investigated by bioinformatic analyses of publicly available datasets. RESULTS We identified several miRNAs’ target genes that are significantly associated with histone modification during mammalian spermatogenesis. MiRNAs that target genes associated with the most significant histone modifications are expressed before or during spermatogenesis; thus the results were convincing. CONCLUSIONS In this paper, we identified apparent miRNA regulation of histone modifications using a bioinformatics approach. The biological mechanisms of this effect should be further experimentally investigated. PMID:25780334

  20. R-loops induce repressive chromatin marks over mammalian gene terminators.

    PubMed

    Skourti-Stathaki, Konstantina; Kamieniarz-Gdula, Kinga; Proudfoot, Nicholas J

    2014-12-18

    The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potentially harmful effects on genome integrity owing to the fragility of the displaced DNA coding strand. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes. Furthermore, we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing before efficient termination. Here we reveal an unanticipated link between R-loops and RNA-interference-dependent H3K9me2 formation over pause-site termination regions in mammalian protein-coding genes. We show that R-loops induce antisense transcription over these pause elements, which in turn leads to the generation of double-stranded RNA and the recruitment of DICER, AGO1, AGO2 and the G9a histone lysine methyltransferase. Consequently, an H3K9me2 repressive mark is formed and heterochromatin protein 1γ (HP1γ) is recruited, which reinforces Pol II pausing before efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.

  1. Mammalian agmatinases constitute unusual members in the family of Mn(2+)-dependent ureahydrolases.

    PubMed

    Romero, Nicol; Benítez, José; Garcia, David; González, Arlette; Bennun, Leonardo; García-Robles, María A; López, Vasthi; Wilson, Liam A; Schenk, Gerhard; Carvajal, Nelson; Uribe, Elena

    2017-01-01

    Agmatine (1-amino-4-guanidinobutane) plays an important role in a range of metabolic functions, in particular in the brain. Agmatinases (AGMs) are enzymes capable of converting agmatine to the polyamine putrescine and urea. AGMs belong to the family of Mn(2+)-dependent ureahydrolases. However, no AGM from a mammalian source has yet been extracted in catalytically active form. While in human AGM the six amino acid ligands that coordinate the two Mn(2+) ions in the active site are conserved, four mutations are observed in the murine enzyme. Here, we demonstrate that similar to its human counterpart murine AGM does not appear to have in vitro catalytic activity, independent of the presence of Mn(2+). However, in presence of agmatine both enzymes are very efficient in promoting cell growth of a yeast strain that is deficient in polyamine biosynthesis (Saccharomyces cerevisiae strain TRY104Δspe1). Furthermore, mutations among the putative Mn(2+) binding residues had no effect on the ability of murine AGM to promote growth of the yeast culture. It thus appears that mammalian AGMs form a distinct group within the family of ureahydrolases that (i) either fold in a manner distinct from other members in this family, or (ii) require accessory proteins to bind Mn(2+) in a mechanism related to that observed for the Ni(2+)-dependent urease.

  2. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina

    PubMed Central

    Ooto, Sotaro; Akagi, Tadamichi; Kageyama, Ryoichiro; Akita, Joe; Mandai, Michiko; Honda, Yoshihito; Takahashi, Masayo

    2004-01-01

    It has long been believed that the retina of mature mammals is incapable of regeneration. In this study, using the N-methyl-d-aspartate neurotoxicity model of adult rat retina, we observed that some Müller glial cells were stimulated to proliferate in response to a toxic injury and produce bipolar cells and rod photoreceptors. Although these newly produced neurons were limited in number, retinoic acid treatment promoted the number of regenerated bipolar cells. Moreover, misexpression of basic helix–loop–helix and homeobox genes promoted the induction of amacrine, horizontal, and rod photoreceptor specific phenotypes. These findings demonstrated that retinal neurons regenerated even in adult mammalian retina after toxic injury. Furthermore, we could partially control the fate of the regenerated neurons with extrinsic factors or intrinsic genes. The Müller glial cells constitute a potential source for the regeneration of adult mammalian retina and can be a target for drug delivery and gene therapy in retinal degenerative diseases. PMID:15353594

  3. Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development.

    PubMed

    Li, Wei; Xiong, Yiqin; Shang, Ching; Twu, Karen Y; Hang, Calvin T; Yang, Jin; Han, Pei; Lin, Chieh-Yu; Lin, Chien-Jung; Tsai, Feng-Chiao; Stankunas, Kryn; Meyer, Tobias; Bernstein, Daniel; Pan, Minggui; Chang, Ching-Pin

    2013-01-29

    Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.

  4. Identification of a mammalian silicon transporter.

    PubMed

    Ratcliffe, Sarah; Jugdaohsingh, Ravin; Vivancos, Julien; Marron, Alan; Deshmukh, Rupesh; Ma, Jian Feng; Mitani-Ueno, Namiki; Robertson, Jack; Wills, John; Boekschoten, Mark V; Müller, Michael; Mawhinney, Robert C; Kinrade, Stephen D; Isenring, Paul; Bélanger, Richard R; Powell, Jonathan J

    2017-05-01

    Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na(+) concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon. Copyright © 2017 the American Physiological Society.

  5. Assays for mammalian tyrosinase: a comparative study

    SciTech Connect

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.

  6. Repair of furocoumarin adducts in mammalian cells

    SciTech Connect

    Zolan, M.E.; Smith, C.A.; Hanawalt, P.C.

    1984-12-01

    DNA repair was studied in cultured mammalian cells treated with the furocoumarins 8-methoxypsoralen (8-MOP), aminomethyl trioxsalen, or angelicin and irradiated with near UV light. The amount of DNA cross-linked by 8-MOP in normal human cells decreased by about one-half in 24 hours after treatment; no decrease was observed in xeroderma pigmentosum cells, group A. At present, it is not known to what extent this decrease represents complete repair events at the sites of cross-links. Furocoumarin adducts elicited excision repair in normal human and monkey cells but not in xeroderma pigmentosum group A cells. This excision repair resembled in several aspects that elicited by pyrimidine dimers, formed in DNA by irradiation with 254-nm UV light; however, it appeared that for at least 8-MOP and aminomethyl trioxsalen, removal of adducts was not as efficient as was the removal of pyrimidine dimers. A comparison was also made of repair in the 172-base-pair repetitive alpha-DNA component of monkey cells to repair in the bulk of the genome. Although repair elicited by pyrimidine dimers in alpha-DNA was the same as in the bulk DNA, that following treatment of cells with either aminomethyl trioxsalen or angelicin and near UV was markedly deficient in alpha-DNA. This deficiency reflected the removal of fewer adducts from alpha-DNA after the same initial adduct frequencies. These results could mean that each furocoumarin may produce several structurally distinct adducts to DNA in cells and that the capacity of cellular repair systems to remove these various adducts may vary greatly.

  7. Ontogenetic development of the mammalian circadian system.

    PubMed

    Weinert, Dietmar

    2005-01-01

    This review summarizes the current knowledge about the ontogenetic development of the circadian system in mammals. The developmental changes of overt rhythms are discussed, although the main focus of the review is the underlying neuronal and molecular mechanisms. In addition, the review describes ontogenetic development, not only as a process of morpho-functional maturation. The need of repeated adaptations and readaptations due to changing developmental stage and environmental conditions is also considered. The review analyzes mainly rodent data, obtained from the literature and from the author's own studies. Results from other species, including humans, are presented to demonstrate common features and species-dependent differences. The review first describes the development of the suprachiasmatic nuclei as the central pacemaker system and shows that intrinsic circadian rhythms are already generated in the mammalian fetus. As in adult organisms, the period length is different from 24 h and needs continuous correction by environmental periodicities, or zeitgebers. The investigation of the ontogenetic development of the mechanisms of entrainment reveals that, at prenatal and early postnatal stages, non-photic cues deriving from the mother are effective. Light-dark entrainment develops later. At a certain age, both photic and non-photic zeitgebers may act in parallel, even though the respective time information is 12 h out of phase. That leads to a temporary internal desynchronization. Because rhythmic information needs to be transferred to effector organs, the corresponding neural and humoral signalling pathways are also briefly described. Finally, to be able to transform a rhythmic signal into an overt rhythm, the corresponding effector organs must be functionally mature. As many of these organs are able to generate their own intrinsic rhythms, another aspect of the review is dedicated to the development of peripheral oscillators and mechanisms of their entrainment

  8. Functional Evolution of Mammalian Odorant Receptors

    PubMed Central

    Adipietro, Kaylin A.; Mainland, Joel D.; Matsunami, Hiroaki

    2012-01-01

    The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands. PMID:22807691

  9. High-Frequency Power Gain in the Mammalian Cochlea

    NASA Astrophysics Data System (ADS)

    Maoiléidigh, Dáibhid Ó.; Hudspeth, A. J.

    2011-11-01

    Amplification in the mammalian inner ear is thought to