Nutrient acquisition strategies of mammalian cells.

Science.gov (United States)

Palm, Wilhelm; Thompson, Craig B

2017-06-07

Mammalian cells are surrounded by diverse nutrients, such as glucose, amino acids, various macromolecules and micronutrients, which they can import through transmembrane transporters and endolysosomal pathways. By using different nutrient sources, cells gain metabolic flexibility to survive periods of starvation. Quiescent cells take up sufficient nutrients to sustain homeostasis. However, proliferating cells depend on growth-factor-induced increases in nutrient uptake to support biomass formation. Here, we review cellular nutrient acquisition strategies and their regulation by growth factors and cell-intrinsic nutrient sensors. We also discuss how oncogenes and tumour suppressors promote nutrient uptake and thereby support the survival and growth of cancer cells.

Liv. 52 protection against radiation induced lesions in mammalian liver

International Nuclear Information System (INIS)

Saini, M.R.; Saini, N.

1985-01-01

Effect of Liv. 52 on mammalian liver was studied after whole-body exposure to 5.5 Gy of 60 Co gamma radiation. It was found that the drug protected the organ against radiation-induced changes. The protective effect was manifested in the form of early recovery as indicated by the absence of pathological changes like cytoplasmic degranulation, loss of nulei from many cells and abnormal architecture at 10 days and restoration of normal structure by 4 weeks. Liv. 52 may neutralize the peroxides formed from water molecules after irradiation which are toxic and cause the damage to the organ. Thus it seems that the drug may act as detoxicating agent. (author)

Transcription Factor Zbtb20 Controls Regional Specification of Mammalian Archicortex

DEFF Research Database (Denmark)

Rosenthal, Eva Helga

2010-01-01

Combinatorial expression of sets of transcription factors (TFs) along the mammalian cortex controls its subdivision into functional areas. Unlike neocortex, only few recent data suggest genetic mechanisms controlling the regionalization of the archicortex. TF Emx2 plays a crucial role in patterning...... later on becoming restricted exclusively to postmitotic neurons of hippocampus (Hi) proper, dentate gyrus (DG), and two transitory zones, subiculum (S) and retrosplenial cortex (Rsp). Analysis of Zbtb20-/- mice revealed altered cortical patterning at the border between neocortex and archicortex...

The impact of locally multiply damaged sites (LMDS) induced by ionizing radiation in mammalian cells

International Nuclear Information System (INIS)

Averbeck, D.; Boucher, D.

2006-01-01

Monte Carlo calculations have shown that ionising radiations produce a specific type of clustered cell damage called locally multiply damaged sites or LMDS. These lesions consist of closely positioned single-strand breaks, (oxidative) base damage and DNA double-strand breaks (DSB) in between one helical turn of DNA. As specific markers of radiation-induced damage these lesions are likely to condition biological responses and are thus of great interest for radiation protection. Calculations indicate that there should be more LMDS induced by high than by low LET radiation, and they should be absent in un-irradiated cells. Processes like K-shell activation and local Auger electron emission can be expected to add complex DSB or LMDS, producing significant chromosomal damage. In the discussion of the specificity of ionising radiation in comparison to other genotoxic agents, many arguments have been put forward that these lesions should be particularly deleterious for living cells. Complex lesions of that type should represent big obstacles for DNA repair and give rise to high lethality. Moreover, cellular attempts to repair them could accentuate harm, leading to mutations, genetic instability and cancer. In vitro experiments with oligonucleotides containing an artificially introduced set of base damage and SSB in different combinations have shown that depending on the close positioning of the damage on DNA, repair enzymes, and even whole cell extracts, are unable to repair properly and may stimulate mis-repair. Pulsed field gel electrophoresis (PFGE) in conjunction with enzymatic treatments has been used to detect LMDS in mammalian cells after high and low LET radiation. In order to further define the importance of LMDS for radiation induced cellular responses, we studied the induction of LMDS as a function of radiation dose and dose rate in mammalian cells (CHO and MRC5) using 137 Cs gamma-radiation. Using PFGE and specific glycosylases to convert oxidative damage into

The impact of locally multiply damaged sites (LMDS) induced by ionizing radiation in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Averbeck, D.; Boucher, D. [Institut Curie-Section de Recherche, UMR2027 CNRS, LCR-V28 du CEA, Centre Universitaire, 91405 Orsay Cedex (France)

2006-07-01

Monte Carlo calculations have shown that ionising radiations produce a specific type of clustered cell damage called locally multiply damaged sites or LMDS. These lesions consist of closely positioned single-strand breaks, (oxidative) base damage and DNA double-strand breaks (DSB) in between one helical turn of DNA. As specific markers of radiation-induced damage these lesions are likely to condition biological responses and are thus of great interest for radiation protection. Calculations indicate that there should be more LMDS induced by high than by low LET radiation, and they should be absent in un-irradiated cells. Processes like K-shell activation and local Auger electron emission can be expected to add complex DSB or LMDS, producing significant chromosomal damage. In the discussion of the specificity of ionising radiation in comparison to other genotoxic agents, many arguments have been put forward that these lesions should be particularly deleterious for living cells. Complex lesions of that type should represent big obstacles for DNA repair and give rise to high lethality. Moreover, cellular attempts to repair them could accentuate harm, leading to mutations, genetic instability and cancer. In vitro experiments with oligonucleotides containing an artificially introduced set of base damage and SSB in different combinations have shown that depending on the close positioning of the damage on DNA, repair enzymes, and even whole cell extracts, are unable to repair properly and may stimulate mis-repair. Pulsed field gel electrophoresis (PFGE) in conjunction with enzymatic treatments has been used to detect LMDS in mammalian cells after high and low LET radiation. In order to further define the importance of LMDS for radiation induced cellular responses, we studied the induction of LMDS as a function of radiation dose and dose rate in mammalian cells (CHO and MRC5) using {sup 137}Cs gamma-radiation. Using PFGE and specific glycosylases to convert oxidative damage

Role of somatostatin receptor-2 in gentamicin-induced auditory hair cell loss in the Mammalian inner ear.

Directory of Open Access Journals (Sweden)

Yves Brand

Full Text Available Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin-a peptide with hormone/neurotransmitter properties-can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.

Chromosomal damages and mutagenesis in mammalian and human cells induced by ionizing radiations with different LET

International Nuclear Information System (INIS)

Govorun, R.D.

1997-01-01

On the basis of literature and proper data the inference was made about essential role of structural chromosomal (and gene) damages in spontaneous and radiation-induced mutagenesis of mammalian and human cells on HPRT-loci. The evidences of increasing role of these damages in the mutagenesis after the influence of ionizing radiations with high LET are adduced. The consequences of HPRT-gene damages have been examined hypothetically. The geterogeneity of mutant subclones on their cytogenetical properties were revealed experimentally. The data reflect a phenomenon of the reproductive chromosomal instability in many generations of mutant cell. The mutagenesis of mammalian cells is also accompanied by the impairment of chromosome integrity with high probability as a stage of appropriate genome reorganization because of changed vital conditions

Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

International Nuclear Information System (INIS)

Pedersen, R.A.; Brandriff, B.

1979-01-01

A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos

DNA repair and radiation sensitivity in mammalian cells

International Nuclear Information System (INIS)

Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

1993-01-01

Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

Angiogenesis is inhibitory for mammalian digit regeneration

Science.gov (United States)

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

C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1.

Science.gov (United States)

Meng, Xiangzhi; Schoggins, John; Rose, Lloyd; Cao, Jingxin; Ploss, Alexander; Rice, Charles M; Xiang, Yan

2012-04-01

Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.

Redundancy and molecular evolution: the rapid Induction of bone formation by the mammalian transforming growth factor-β3 isoform

Directory of Open Access Journals (Sweden)

Ugo Ripamonti

2016-09-01

Full Text Available The soluble osteogenic molecular signals of the transforming growth factor-β (TGF-β supergene family are the molecular bases of the induction of bone formation and postnatal bone tissue morphogenesis with translation into clinical contexts. The mammalian TGF-β3 isoform, a pleiotropic member of the family, controls a vast array of biological processes including the induction of bone formation. Recombinant hTGF-β3 induces substantial bone formation when implanted with either collagenous bone matrices or coral-derived macroporous bioreactors in the rectus abdominis muscle of the non-human primate Papio ursinus. In marked contrast, the three mammalian TGF-βs do not initiate the induction of bone formation in rodents and lagomorphs. The induction of bone by hTGF-β3/preloaded bioreactors is orchestrated by inducing fibrin-fibronectin rings that structurally organize tissue patterning and morphogenesis within the macroporous spaces. Induced advancing extracellular matrix rings provide the structural anchorage for hyper chromatic cells, interpreted as differentiating osteoblasts re-programmed by hTGF-β3 from invading myoblastic and/or pericytic differentiated cells. Runx2 and Osteocalcin expression are significantly up-regulated correlating to multiple invading cells differentiating into the osteoblastic phenotype. Bioreactors pre-loaded with recombinant human Noggin (hNoggin, a BMPs antagonist, show down-regulation of BMP-2 and other profiled osteogenic proteins’ genes resulting in minimal bone formation. Coral-derived macroporous constructs preloaded with binary applications of hTGF-β3 and hNoggin also show down-regulation of BMP-2 with the induction of limited bone formation. The induction of bone formation by hTGF-β3 is via the BMPs pathway and it is thus blocked by hNoggin. Our systematic studies in Papio ursinus with translational hTGF-β3 in large cranio-mandibulo-facial defects in humans are now requesting the re-evaluation of Bone

Evolutionary changes of Hox genes and relevant regulatory factors provide novel insights into mammalian morphological modifications.

Science.gov (United States)

Li, Kui; Sun, Xiaohui; Chen, Meixiu; Sun, Yingying; Tian, Ran; Wang, Zhengfei; Xu, Shixia; Yang, Guang

2018-01-01

The diversity of body plans of mammals accelerates the innovation of lifestyles and the extensive adaptation to different habitats, including terrestrial, aerial and aquatic habitats. However, the genetic basis of those phenotypic modifications, which have occurred during mammalian evolution, remains poorly explored. In the present study, we synthetically surveyed the evolutionary pattern of Hox clusters that played a powerful role in the morphogenesis along the head-tail axis of animal embryos and the main regulatory factors (Mll, Bmi1 and E2f6) that control the expression of Hox genes. A deflected density of repetitive elements and lineage-specific radical mutations of Mll have been determined in marine mammals with morphological changes, suggesting that evolutionary changes may alter Hox gene expression in these lineages, leading to the morphological modification of these lineages. Although no positive selection was detected at certain ancestor nodes of lineages, the increased ω values of Hox genes implied the relaxation of functional constraints of these genes during the mammalian evolutionary process. More importantly, 49 positively-selected sites were identified in mammalian lineages with phenotypic modifications, indicating adaptive evolution acting on Hox genes and regulatory factors. In addition, 3 parallel amino acid substitutions in some Hox genes were examined in marine mammals, which might be responsible for their streamlined body. © 2017 The Authors. Integrative Zoology published by International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

International Nuclear Information System (INIS)

Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

2015-01-01

Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs

Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

Energy Technology Data Exchange (ETDEWEB)

Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Song, Sang Heon [Department of Internal Medicine, Pusan National University Hospital, Busan (Korea, Republic of); Shin, Hwa Kyoung [Department of Anatomy, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [Medical Research Institute, Department of Pharmacology, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

2015-08-14

Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

Energy Technology Data Exchange (ETDEWEB)

Chung, Eun Joo [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Sowers, Anastasia; Thetford, Angela [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); McKay-Corkum, Grace; Chung, Su I. [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Mitchell, James B. [Radiation Biology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States); Citrin, Deborah E., E-mail: citrind@mail.nih.gov [Radiation Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland (United States)

2016-11-15

Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

Science.gov (United States)

Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A; Harwalkar, Jyoti; Shoskes, Aaron C; Elson, Paul; Beach, Jordan R; Hussey, George S; Schiemann, William P; Egelhoff, Thomas T; Howe, Philip H; Hansel, Donna E

2016-05-01

Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers. Copyright © 2016. Published by Elsevier Inc.

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

Directory of Open Access Journals (Sweden)

Liliana Costa

2012-06-01

Full Text Available 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.

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

Science.gov (United States)

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

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

Science.gov (United States)

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

2016-12-15

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The fungicide mancozeb induces toxic effects on mammalian granulosa cells

International Nuclear Information System (INIS)

Paro, Rita; Tiboni, Gian Mario; Buccione, Roberto; Rossi, Gianna; Cellini, Valerio; Canipari, Rita; Cecconi, Sandra

2012-01-01

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant. Highlights: ► The fungicide mancozeb affects oocyte spindle morphology and fertilization rate. ► We investigated the toxic effects of mancozeb on mouse and human granulosa cells. ► Granulosa cells modify their morphology and expression level of p53. ► Mancozeb induces a premalignant-like status in exposed cells.

The fungicide mancozeb induces toxic effects on mammalian granulosa cells

Energy Technology Data Exchange (ETDEWEB)

Paro, Rita [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Tiboni, Gian Mario [Department of Medicine and Aging, Section of Reproductive Sciences, University “G. D' Annunzio”, Chieti-Pescara (Italy); Buccione, Roberto [Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti (Italy); Rossi, Gianna; Cellini, Valerio [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Canipari, Rita [Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Embryology, School of Pharmacy and Medicine, “Sapienza” University of Rome, Rome (Italy); Cecconi, Sandra, E-mail: sandra.cecconi@cc.univaq.it [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy)

2012-04-15

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant. Highlights: ► The fungicide mancozeb affects oocyte spindle morphology and fertilization rate. ► We investigated the toxic effects of mancozeb on mouse and human granulosa cells. ► Granulosa cells modify their morphology and expression level of p53. ► Mancozeb induces a premalignant-like status in exposed cells.

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

Science.gov (United States)

Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

2004-07-16

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

International Nuclear Information System (INIS)

Campos-Nebel, Marcelo de; Larripa, Irene; Gonzalez-Cid, Marcela

2008-01-01

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by γH2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced DSB

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Campos-Nebel, Marcelo de [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)], E-mail: mnebel@hematologia.anm.edu.ar; Larripa, Irene; Gonzalez-Cid, Marcela [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)

2008-11-10

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by {gamma}H2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced

Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

International Nuclear Information System (INIS)

Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

2015-01-01

Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway

Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

Energy Technology Data Exchange (ETDEWEB)

Li, Kuiqing; Chen, Xu [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Liu, Cheng [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Gu, Peng; Li, Zhuohang; Wu, Shaoxu [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Xu, Kewei [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Lin, Tianxin, E-mail: tianxinl@sina.com [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China); Huang, Jian, E-mail: urolhj@sina.com [Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120 (China)

2015-05-01

Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway.

MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin

International Nuclear Information System (INIS)

Zhang, Zi-wei; Guo, Rui-wei; Lv, Jin-lin; Wang, Xian-mei; Ye, Jin-shan; Lu, Ni-hong; Liang, Xing; Yang, Li-xia

2017-01-01

Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis. - Highlights: • Suggesting a new mechanism of insulin-triggered VSMC functions. • Providing a new therapeutic strategies that target atherosclerosis in T2DM patients. • Providing a new strategies that target in-stent restenosis in T2DM patients.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Science.gov (United States)

Kashir, Junaid; Deguchi, Ryusaku; Jones, Celine; Coward, Kevin; Stricker, Stephen A

2013-10-01

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca²⁺) in all animals that have been examined, and such Ca²⁺ elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²⁺ transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca²⁺ dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca²⁺ signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca²⁺ signals are typically propagated as global waves that depend on Ca²⁺ release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP₃). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca²⁺ transients are also examined. In addition, the importance of fertilization-induced Ca²⁺ signals for activating development is underscored by noting some major downstream effects of these signals in various animals. © 2013 Wiley Periodicals, Inc.

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

Science.gov (United States)

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.

Regulation of Autophagy by Glucose in Mammalian Cells

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Erwin Knecht

2012-07-01

Full Text Available Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

International Nuclear Information System (INIS)

Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

2003-01-01

Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

Comparative studies of radiation-induced chromosome aberrations in several mammalian species

International Nuclear Information System (INIS)

Muramatsu, S.; Matsuoka, O.

1976-01-01

The dose-response relationship for inducing chromosome aberrations in peripheral lymphocytes of five mammalian species - man, cynomolgus monkey, rabbit, domestic cat and beagle dog - were studied comparatively by whole-blood microculture technique following in-vitro exposures at various doses with 200-kVp X rays. The yields of induced chromosome aberrations were dependent on exposure doses between 48 and 480 rads in all the species examined. The relationship between exposure dose (D in rads) and frequency of induced dicentrics per cell (Y) was expressed by: Ysub((man)) = 14.38x10 -6 Dsup(1.94); Ysub((monkey)) = 18.12x10 -6 Dsup(1.86); Ysub((rabbit)) = 1.88x10 -6 Dsup(2.06); Ysub((cat)) = 78.66x10 -6 Dsup(1.35); Ysub((dog)) = 46.13x10 -6 Dsup(1.37). Taking the frequency of dicentrics in man as 1.00, the relative frequency in each species was estimated as 0.79, 0.24, 0.22 and 0.16 in monkey, rabbit, cat and dog, respectively. From these results the consistent relationship could not be discovered between X-ray doses and the dicentric yield based on the arm number effect proposed by Brewen et al., whereas the nuclear DNA contents and the arm number in all the species used are roughly similar to those in man. The authors considered that such interspecies differences may be derived from the cellular and/or physiological features of PHA-responsible lymphocytes (T-cells) in each species, and that may be due to the level of development of each species on the phylogenetic or evolutionary scale. (author)

A promoter-level mammalian expression atlas

KAUST Repository

Forest, Alistair R R

2014-03-26

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.

A promoter-level mammalian expression atlas

KAUST Repository

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

2014-01-01

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.

The cancer paradigms of mammalian regeneration: can mammals regenerate as amphibians?

Science.gov (United States)

Sarig, Rachel; Tzahor, Eldad

2017-04-01

Regeneration in mammals is restricted to distinct tissues and occurs mainly by expansion and maturation of resident stem cells. During regeneration, even subtle mutations in the proliferating cells may cause a detrimental effect by eliciting abnormal differentiation or malignant transformation. Indeed, cancer in mammals has been shown to arise through deregulation of stem cells maturation, which often leads to a differentiation block and cell transformation. In contrast, lower organisms such as amphibians retain a remarkable regenerative capacity in various organs, which occurs via de- and re-differentiation of mature cells. Interestingly, regenerating amphibian cells are highly resistant to oncogenic transformation. Therapeutic approaches to improve mammalian regeneration mainly include stem-cell transplantations; but, these have proved unsuccessful in non-regenerating organs such as the heart. A recently developed approach is to induce de-differentiation of mature cardiomyocytes using factors that trigger their re-entry into the cell cycle. This novel approach raises numerous questions regarding the balance between transformation and regeneration induced by de-differentiation of mature mammalian somatic cells. Can this balance be controlled artificially? Do de-differentiated cells acquire the protection mechanisms seen in regenerating cells of lower organisms? Is this model unique to the cardiac tissue, which rarely develops tumors? This review describes regeneration processes in both mammals and lower organisms and, particularly, the ability of regenerating cells to avoid transformation. By comparing the characteristics of mammalian embryonic and somatic cells, we discuss therapeutic strategies of using various cell populations for regeneration. Finally, we describe a novel cardiac regeneration approach and its implications for regenerative medicine. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email

Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells

International Nuclear Information System (INIS)

Grosovsky, A.J.; de Boer, J.G.; de Jong, P.J.; Drobetsky, E.A.; Glickman, B.W.

1988-01-01

The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage

Involvement of TRPV3 and TRPM8 ion channel proteins in induction of mammalian cold-inducible proteins.

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Fujita, Takanori; Liu, Yu; Higashitsuji, Hiroaki; Itoh, Katsuhiko; Shibasaki, Koji; Fujita, Jun; Nishiyama, Hiroyuki

2018-01-01

Cold-inducible RNA-binding protein (CIRP), RNA-binding motif protein 3 (RBM3) and serine and arginine rich splicing factor 5 (SRSF5) are RNA-binding proteins that are transcriptionally upregulated in response to moderately low temperatures and a variety of cellular stresses in mammalian cells. Induction of these cold-inducible proteins (CIPs) is dependent on transient receptor potential (TRP) V4 channel protein, but seems independent of its ion channel activity. We herein report that in addition to TRPV4, TRPV3 and TRPM8 are necessary for the induction of CIPs. We established cell lines from the lung of TRPV4-knockout (KO) mouse, and observed induction of CIPs in them by western blot analysis. A TRPV4 antagonist RN1734 suppressed the induction in wild-type mouse cells, but not in TRPV4-KO cells. A TRPV3 channel blocker S408271 and a TRPM8 channel blocker AMTB as well as siRNAs against TRPV3 and TRPM8 suppressed the CIP induction in mouse TRPV4-KO cells and human U-2 OS cells. A TRPV3 channel agonist 2-APB induced CIP expression, but camphor did not. Neither did a TRPM8 channel agonist WS-12. These results suggest that TRPV4, TRPV3 and TRPM8 proteins, but not their ion channel activities are necessary for the induction of CIPs at 32 °C. Identification of proteins that differentially interact with these TRP channels at 37 °C and 32 °C would help elucidate the underlying mechanisms of CIP induction by hypothermia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Viral and Host Factors Required for Avian H5N1 Influenza A Virus Replication in Mammalian Cells

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Hong Zhang

2013-06-01

Full Text Available Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions.

Role of Notch signaling in the mammalian heart

Energy Technology Data Exchange (ETDEWEB)

Zhou, X.L.; Liu, J.C. [Department of Cardiac Surgery, The First Affiliated Hospital, Nanchang University, Donghu District, Nanchang, Jiangxi (China)

2013-12-12

Notch signaling is an evolutionarily ancient, highly conserved pathway important for deciding cell fate, cellular development, differentiation, proliferation, apoptosis, adhesion, and epithelial-to-mesenchymal transition. Notch signaling is also critical in mammalian cardiogenesis, as mutations in this signaling pathway are linked to human congenital heart disease. Furthermore, Notch signaling can repair myocardial injury by promoting myocardial regeneration, protecting ischemic myocardium, inducing angiogenesis, and negatively regulating cardiac fibroblast-myofibroblast transformation. This review provides an update on the known roles of Notch signaling in the mammalian heart. The goal is to assist in developing strategies to influence Notch signaling and optimize myocardial injury repair.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism

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Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-01-01

Background Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. Methods We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and rel...

Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

International Nuclear Information System (INIS)

Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

1988-01-01

Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

Effect of an aminothiol (WR-1065) on radiation-induced mutagenesis and cytotoxicity in two repair-deficient mammalian cell lines

International Nuclear Information System (INIS)

Grdina, D.J.; Nagy, B.; Meechan, P.J.

1991-01-01

WR-2721 and its free thiol WR-1065 have been found to effectively protect against radiation- and/or chemotherapy-induced mutagenesis, transformation and carcinogenesis. With respect to the antimutagenic effect, WR-1065 significantly reduced the frequency of HGPRT mutants even when it was administered up to three hours following exposure of cells to radiation. The mechanisms of action most often attributed to these agents include their ability to scavenge free radicals, enter into chemical repair processes through the donation of hydrogen atoms, and induce intracellular hypoxia by means of auto-oxidative processes. Although evidence exists for each of these processes, none is sufficiently satisfactory to account for the post-irradiation protection of WR-1065 against mutation induction in mammalian cells. The most elegant work describing the role of aminothiols on cellular enzymatic repair processes has focused on well-characterized repair-proficient and -deficient bacterial and yeast cell systems. Protection against radiation-induced cytotoxicity by the aminothiol cysteamine was absent in E. coli cell lines that were characterized as having genetically defective repair systems. Until recently, such studies could not be effectively performed with mammalian cells. However, with the isolation and characterization of rodent cell lines deficient in their ability to repair DNA damage, it is now possible to investigate the role of cell-mediated repair systems on aminothiol radioprotection. Specifically, the authors have investigated the effects of WR-1065 on radiation-induced mutagenesis and cytotoxicity in cell lines EM9 and xrs-5, which are defective in DNA single-strand break (SSB) and double-strand break (DSB) rejoining, respectively. Corresponding parental repair-proficient cell lines, AA8 and K1, were also studied for comparative purposes. 26 refs., 5 figs., 2 tabs

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

Science.gov (United States)

Elmroth, Kecke; Stenerlöw, Bo

2005-04-01

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

Science.gov (United States)

Baer, Matthew L; Henderson, Scott C; Colello, Raymond J

2015-01-01

Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.

Elucidating the Role of Injury-Induced Electric Fields (EFs in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

Directory of Open Access Journals (Sweden)

Matthew L Baer

Full Text Available Injury to the vertebrate central nervous system (CNS induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.

Cinnamic aldehyde suppresses hypoxia-induced angiogenesis via inhibition of hypoxia-inducible factor-1α expression during tumor progression.

Science.gov (United States)

Bae, Woom-Yee; Choi, Jae-Sun; Kim, Ja-Eun; Jeong, Joo-Won

2015-11-01

During tumor progression, hypoxia-inducible factor 1 (HIF-1) plays a critical role in tumor angiogenesis and tumor growth by regulating the transcription of several genes in response to a hypoxic environment and changes in growth factors. This study was designed to investigate the effects of cinnamic aldehyde (CA) on tumor growth and angiogenesis and the mechanisms underlying CA's anti-angiogenic activities. We found that CA administration inhibits tumor growth and blocks tumor angiogenesis in BALB/c mice. In addition, CA treatment decreased HIF-1α protein expression and vascular endothelial growth factor (VEGF) expression in mouse tumors and Renca cells exposed to hypoxia in vitro. Interestingly, CA treatment did not affect the stability of von Hippel-Lindau protein (pVHL)-associated HIF-1α and CA attenuated the activation of mammalian target of rapamycin (mTOR) pathway. Collectively, these findings strongly indicate that the anti-angiogenic activity of CA is, at least in part, regulated by the mTOR pathway-mediated suppression of HIF-1α protein expression and these findings suggest that CA may be a potential drug for human cancer therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

Science.gov (United States)

Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian hepatic stellate cells

International Nuclear Information System (INIS)

Chen, Ming-Feng; Huang, S. Joseph; Huang, Chao-Cheng; Liu, Pei-Shan; Lin, Kun-I; Liu, Ching-Wen; Hsieh, Wen-Chuan; Shiu, Li-Yen; Chen, Chang-Han

2016-01-01

Saikosaponin d (SSd) is one of the main active triterpene saponins in Bupleurum falcatum. It has a steroid-like structure, and is reported to have pharmacological activities, including liver protection in rat, cell cycle arrest and apoptosis induction in several cancer cell lines. However, the biological functions and molecular mechanisms of mammalian cells under SSd treatment are still unclear. The cytotoxicity and apoptosis of hepatic stellate cells (HSCs) upon SSd treatment were discovered by MTT assay, colony formation assay and flow cytometry. The collage I/III, caspase activity and apoptotic related genes were examined by quantitative PCR, Western blotting, immunofluorescence and ELISA. The mitochondrial functions were monitored by flow cytometry, MitoTracker staining, ATP production and XF24 bioenergetic assay. This study found that SSd triggers cell death via an apoptosis path. An example of this path might be typical apoptotic morphology, increased sub-G1 phase cell population, inhibition of cell proliferation and activation of caspase-3 and caspase-9. However, the apoptotic effects induced by SSd are partially blocked by the caspase-3 inhibitor, Z-DEVD-FMK, suggesting that SSd may trigger both HSC-T6 and LX-2 cell apoptosis through caspase-3-dependent and independent pathways. We also found that SSd can trigger BAX and BAK translocation from the cytosol to the mitochondria, resulting in mitochondrial function inhibition, membrane potential disruption. Finally, SSd also increases the release of apoptotic factors. The overall analytical data indicate that SSd-elicited cell death may occur through caspase-3-dependent, caspase-3-independent and mitochondrial pathways in mammalian HSCs, and thus can delay the formation of liver fibrosis by reducing the level of HSCs

DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

1988-08-01

The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after ..gamma..-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation.

DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

International Nuclear Information System (INIS)

Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

1988-01-01

The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after γ-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation. (author)

Heavy ion induced genetic effects in mammalian cells. Final report

International Nuclear Information System (INIS)

Kiefer, J.; Brend'amour, M.; Casares, A.; Egenolf, R.; Gutermuth, F.; Ikpeme, S.E.; Koch, S.; Kost, M.; Loebrich, M.; Pross, H.D.; Russmann, C.; Schmidt, P.; Schneider, E.; Stoll, U.; Weber, K.J.

2001-01-01

DNA double-strand breaks (DSBs) are generally assumed to be the most relevant initial event producing radiation-induced cellular lethality, as well as mutations and transformations. The dependence of their formation on radiation quality has been recently reviewed. Contrary to earlier observations there seems to be now agreement that the RBE does not increase above unity with increasing LET in mammalian cells when conventional techniques are applied which are not able to resolve smaller fragments. If they are, however, included in the analysis maximum RBE values around 2 are obtained. The situation is different with yeast: An increased effectiveness for DSB induction has been reported with alpha particles, as well as for heavy ions. This may be due to differences in methods or to chromosomal structure, as discussed in more detail in this paper. DSB induction was measured for a LET range of 100 to 11500 keV/? m in yeast cells using pulsed field gel electrophoresis. Under the conditions applied the chromosomes of the yeast cells could be separated according to size allowing the direct quantification of the DSB yield by measuring the intensity of the largest chromosomes. The results demonstrate clearly that DSB induction in yeast depends on radiation quality. The derived cross-sections for DSB induction were also compared to those for cell inactivation determined in parallel experiments under identical irradiation conditions. (orig.)

Is ultraviolet enhanced reactivation of mammalian virus mutagenic

International Nuclear Information System (INIS)

Bockstahler, L.E.; Hellman, K.B.; Cantwell, J.M.; Strickland, A.

1981-01-01

Ultraviolet enhanced reactivation consists of an increase in the survival of certain uv-irradiated mammalian viruses when assayed for infectivity in uv-irradiated host mammalian cells, as compared with unirradiated cells. In this report ultraviolet enhanced reactivation is described, and a review is presented of investigations from this and other laboratories to establish whether or not this process is mutagenic. The answer to this question may help establish if error-prone DNA repair is induced in irradiated mammalian cells. We approached the mutagenesis question by examining the phenotypic reversion of a uv-irradiated temperature sensitive mutant of Herpes simplex virus to wild type growth in uv-irradiated monkey kidney cells. Apparent reversion was observed in both irradiated and unirradiated cells. No correlation could be found between the extent of reversion and uv exposure to the cells. The conclusions from studies reported by other investigators using various mammalian virus mutagenesis systems are conflicting. It was generally agreed that viral mutagenesis occurs when irradiated virus is passaged through either irradiated or unexposed cells. However, in some studies it was found that the frequency of mutagenesis in irradiated cells was greater than that in unirradiated cells, while in other studies increased mutagenesis in irradiated cells was not observed

The nonenzymatic subunit of pseutarin C, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V.

Science.gov (United States)

Rao, Veena S; Swarup, Sanjay; Kini, R Manjunatha

2003-08-15

Pseutarin C is a group C prothrombin activator from the venom of the eastern brown snake Pseudonaja textilis. It is a multi-subunit protein complex consisting of catalytic and nonenzymatic subunits similar to coagulation factor Xa and factor Va, respectively. Here we describe the complete sequence of the nonenzymatic subunit. Based on the partial amino acid sequence of the nonenzymatic subunit, degenerate primers were designed. Using a "walking" strategy based on sequentially designed primers, we determined the complete cDNA sequence of the nonenzymatic subunit. The cDNA encodes a protein of 1461 amino acid residues, which includes a 30-residue signal peptide, a mature protein of 1430 amino acid residues, and a stop codon. cDNA blot analysis showed a single transcript of approximately 4.6 kb. The deduced amino acid sequence shows approximately 50% identity to mammalian factor V and by homology has a similar domain structure consisting of domains A1-A2-B-A3-C1-C2. Interestingly, the B domain of pseutarin C is shorter than that of mammalian factor V (FV). Although most of the proteolytic activation sites are conserved, 2 of 3 proteolytic sites cleaved by activated protein C are mutated, and thus activated protein C is not able to inactivate this procoagulant toxin. The predicted posttranslational modifications, including disulfide bonds, N-glycosylation, phosphorylation, and sulfation, in pseutarin C are significantly different compared with bovine factor V. Thus, our data demonstrate that the nonenzymatic subunit of group C prothrombin activators is structurally similar to mammalian FV.

Structural differences between yeast and mammalian microtubules revealed by cryo-EM

Energy Technology Data Exchange (ETDEWEB)

Howes, Stuart C. [Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; Geyer, Elisabeth A. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; LaFrance, Benjamin [Univ. of California, Berkeley, CA (United States). Molecular and Cell Biology Graduate Program; Zhang, Rui [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Kellogg, Elizabeth H. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Westermann, Stefan [Univ. of Duisburg-Essen, Essen (Germany). Dept. of Molecular Genetics, Center for Medical Biotechnology; Rice, Luke M. [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biochemistry; Nogales, Eva [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Molecular Biology and California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

2017-06-26

Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis.

Science.gov (United States)

Das, Ranjan; Xu, Shanhua; Nguyen, Tuyet Thi; Quan, Xianglan; Choi, Seong-Kyung; Kim, Soo-Jin; Lee, Eun Young; Cha, Seung-Kuy; Park, Kyu-Sang

2015-12-25

TGF-β is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-β1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-β receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-β1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-β1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTORactivation. Our data suggest that TGF-β1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic

Plateau properties in mammalian spinal interneurons during transmitter-induced locomotor activity

DEFF Research Database (Denmark)

Kiehn, O.; Johnson, B. R.; Raastad, M.

1996-01-01

We examined the organization of spinal networks controlling locomotion in the isolated spinal cord of the neonatal rat, and in this study we provide the first demonstration of plateau and bursting mechanisms in mammalian interneurons that show locomotor-related activity. Using tight-seal whole...

X-rays sensitive mammalian cell mutant

International Nuclear Information System (INIS)

Utsumi, Hiroshi

1982-01-01

A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation

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Atilgan Yilmaz

2015-11-01

Full Text Available In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e., reversal of differentiated state, as a means to produce progenitor cells to eventually replace damaged tissues. Therefore, induced activation of dedifferentiation responses in mammalian tissues holds an immense promise for regenerative medicine. Here we demonstrate that ectopic expression of Msx2 in cultured mouse myotubes recapitulates several aspects of amphibian muscle dedifferentiation. We found that MSX2, but not MSX1, leads to cellularization of myotubes and downregulates the expression of myotube markers, such as MHC, MRF4 and myogenin. RNA sequencing of myotubes ectopically expressing Msx2 showed downregulation of over 500 myotube-enriched transcripts and upregulation of over 300 myoblast-enriched transcripts. MSX2 selectively downregulated expression of Ptgs2 and Ptger4, two members of the prostaglandin pathway with important roles in myoblast fusion during muscle differentiation. Ectopic expression of Msx2, as well as Msx1, induced partial cell cycle re-entry of myotubes by upregulating CyclinD1 expression but failed to initiate S-phase. Finally, MSX2-induced dedifferentiation in mouse myotubes could be recapitulated by a pharmacological treatment with trichostatin A (TSA, bone morphogenetic protein 4 (BMP4 and fibroblast growth factor 1 (FGF1. Together, these observations indicate that MSX2 is a major driver of dedifferentiation in mammalian muscle cells.

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

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Mori, Jun; Takahashi-Yanaga, Fumi; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

2005-01-01

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G 0 /G 1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3β (GSK-3β). Depletion of endogenous GSK-3β by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3β and found that DIF-1 dephosphorylated GSK-3β on Ser 9 and induced the nuclear translocation of GSK-3β, suggesting that DIF-1 activated GSK-3β. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr 286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3β-mediated phosphorylation of Thr 286

An Analytical Study of Mammalian Bite Wounds Requiring Inpatient Management

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Young-Geun Lee

2013-11-01

Full Text Available BackgroundMammalian 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.MethodsWe 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.ResultsAmong 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.ConclusionsBased 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.

Cell physiology regulation by hypoxia inducible factor-1: Targeting oxygen-related nanomachineries of hypoxic cells.

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Eskandani, Morteza; Vandghanooni, Somayeh; Barar, Jaleh; Nazemiyeh, Hossein; Omidi, Yadollah

2017-06-01

Any dysfunctionality in maintaining the oxygen homeostasis by mammalian cells may elicit hypoxia/anoxia, which results in inescapable oxidative stress and possible subsequent detrimental impacts on certain cells/tissues with high demands to oxygen molecules. The ischemic damage in turn can trigger initiation of a number of diseases including organs ischemia, metabolic disorders, inflammatory diseases, different types of malignancies, and alteration in wound healing process. Thus, full comprehension of molecular mechanism(s) and cellular physiology of the oxygen homeostasis is the cornerstone of the mammalian cells metabolism, energetic pathways and health and disease conditions. An imbalance in oxygen content within the cellular microenvironment activates a cascade of molecular events that are often compensated, otherwise pathologic condition occurs through a complexed network of biomolecules. Hypoxia inducible factor-1 (HIF-1) plays a key transcriptional role in the adaptation of cell physiology in relation with the oxygen content within a cell. In this current study, we provide a comprehensive review on the molecular mechanisms of oxygen sensing and homeostasis and the impacts of HIF-1 in hypoxic/anoxic conditions. Moreover, different molecular and biochemical responses of the cells to the surrounding environment are discussed in details. Finally, modern technological approaches for targeting the hypoxia related proteins are articulated. Copyright © 2017. Published by Elsevier B.V.

Expression of measles virus nucleoprotein induces apoptosis and modulates diverse functional proteins in cultured mammalian cells.

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Ashima Bhaskar

Full Text Available BACKGROUND: Measles virus nucleoprotein (N encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. PRINCIPAL FINDINGS: In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS was involved in the mechanism of cell death. Addition of ascorbic acid (AA or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. CONCLUSION: These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

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Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

2017-10-01

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Apoptosis in mammalian oocytes: a review.

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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.

Elabela-apelin receptor signaling pathway is functional in mammalian systems.

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Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

2015-02-02

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

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Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

2017-10-11

Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

12-oxo-phytodienoic acid, a plant-derived oxylipin, attenuates lipopolysaccharide-induced inflammation in microglia

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Taki-Nakano, Nozomi [Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Advanced Drug Research Laboratories, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda, Saitama 335-8505 (Japan); Kotera, Jun [Advanced Drug Research Laboratories, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda, Saitama 335-8505 (Japan); Ohta, Hiroyuki, E-mail: ohta.h.ab@m.titech.ac.jp [Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan)

2016-05-13

Jasmonates are plant lipid–derived oxylipins that act as key signaling compounds in plant immunity, germination, and development. Although some physiological activities of natural jasmonates in mammalian cells have been investigated, their anti-inflammatory actions in mammalian cells remain unclear. Here, we investigated whether jasmonates protect mouse microglial MG5 cells against lipopolysaccharide (LPS)–induced inflammation. Among the jasmonates tested, only 12-oxo-phytodienoic acid (OPDA) suppressed LPS-induced expression of the typical inflammatory cytokines interleukin-6 and tumor necrosis factor α. In addition, only OPDA reduced LPS-induced nitric oxide production through a decrease in the level of inducible nitric oxide synthase. Further mechanistic studies showed that OPDA suppressed neuroinflammation by inhibiting nuclear factor κB and p38 mitogen-activated protein kinase signaling in LPS-activated MG5 cells. In addition, OPDA induced expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of inflammation, in MG5 cells. Finally, we found that the nuclear factor erythroid 2-related factor 2 signaling cascade induced by OPDA is not involved in the anti-inflammatory effects of OPDA. These results demonstrate that OPDA inhibited LPS-induced cell inflammation in mouse microglial cells via multiple pathways, including suppression of nuclear factor κB, inhibition of p38, and activation of SOCS-1 signaling. -- Highlights: •OPDA attenuates LPS-induced inflammatory cytokines such as IL-6 and TNF-α. •OPDA reduces LPS-induced iNOS expression and NO production. •OPDA suppresses NF-κB and p38 pathways and activates SOCS-1 signaling.

12-oxo-phytodienoic acid, a plant-derived oxylipin, attenuates lipopolysaccharide-induced inflammation in microglia

International Nuclear Information System (INIS)

Taki-Nakano, Nozomi; Kotera, Jun; Ohta, Hiroyuki

2016-01-01

Jasmonates are plant lipid–derived oxylipins that act as key signaling compounds in plant immunity, germination, and development. Although some physiological activities of natural jasmonates in mammalian cells have been investigated, their anti-inflammatory actions in mammalian cells remain unclear. Here, we investigated whether jasmonates protect mouse microglial MG5 cells against lipopolysaccharide (LPS)–induced inflammation. Among the jasmonates tested, only 12-oxo-phytodienoic acid (OPDA) suppressed LPS-induced expression of the typical inflammatory cytokines interleukin-6 and tumor necrosis factor α. In addition, only OPDA reduced LPS-induced nitric oxide production through a decrease in the level of inducible nitric oxide synthase. Further mechanistic studies showed that OPDA suppressed neuroinflammation by inhibiting nuclear factor κB and p38 mitogen-activated protein kinase signaling in LPS-activated MG5 cells. In addition, OPDA induced expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of inflammation, in MG5 cells. Finally, we found that the nuclear factor erythroid 2-related factor 2 signaling cascade induced by OPDA is not involved in the anti-inflammatory effects of OPDA. These results demonstrate that OPDA inhibited LPS-induced cell inflammation in mouse microglial cells via multiple pathways, including suppression of nuclear factor κB, inhibition of p38, and activation of SOCS-1 signaling. -- Highlights: •OPDA attenuates LPS-induced inflammatory cytokines such as IL-6 and TNF-α. •OPDA reduces LPS-induced iNOS expression and NO production. •OPDA suppresses NF-κB and p38 pathways and activates SOCS-1 signaling.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

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WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Ghrelin: an emerging player in the regulation of reproduction in non-mammalian vertebrates.

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Unniappan, Suraj

2010-07-01

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of multiple endocrine factors mainly produced from the brain, pituitary, and gonads. In addition to these, several other tissues including the fat and gut produce factors that have reproductive effects. Ghrelin is one such gut/brain hormone with species-specific effects in the regulation of mammalian reproduction. Recent studies have shown that ghrelin and ghrelin receptor mRNAs, and protein are expressed in the ovary and testis of mammals, indicating a direct effect for ghrelin in the control of reproduction. Ghrelin regulates mammalian reproduction by modulating hormone secretion from the brain and pituitary, and by acting directly on the gonads to influence reproductive tissue development and steroid hormone release. Based on the studies reported so far, ghrelin seems to have a predominantly inhibitory role on mammalian reproduction. The presence of ghrelin and ghrelin receptor has been found in the brain, pituitary and gonads of several non-mammalian vertebrates. In contrast to mammals, ghrelin seems to have a stimulatory role in the regulation of non-mammalian reproduction. The main objective of this review is to do a perspective analysis of the comparative aspects of ghrelin regulation of reproduction. (c) 2009 Elsevier Inc. All rights reserved.

Radiation effects in mammalian cells in vitro

International Nuclear Information System (INIS)

Hill, C.K.; Han, A.; Elkind, M.M.; Wells, R.L.; Buess, E.M.; Lin, C.M.

1985-01-01

The purpose of this research effort is to elucidate the mechanisms for the radiation-induced changes in mammalian cells that lead to cell death, mutation, neoplastic transformation, DNA damage, and chromosomal alterations. Of particular interest are the effects of low-dose-rate and fractionated irradiation on these end points with respect to the mechanisms whereby these effects are influenced by cellular repair processes, inhibitors, and promoters that act at the genetic or biochemical level. 17 refs

Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels

International Nuclear Information System (INIS)

Saad, A.H.; Zhou, L.Y.; Lambe, E.K.; Hahn, G.M.

1994-01-01

In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K + ) currents, activated by ionizing radiation play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6±4.0 mutations per 100,000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5±1.5. This increase, however, is abolished if either K + channel blocker, CsCl or BaCl 2 , is present for 2h following irradiation of the cells. RbCl, chemically similar to CsCl but known not to block K + channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsCl or BaCl 2 had no effect on radiation-induced cell killing

Low concentration of exogenous carbon monoxide protects mammalian cells against proliferation induced by radiation-induced bystander effect

International Nuclear Information System (INIS)

Tong, Liping; Yu, K.N.; Bao, Lingzhi; Wu, Wenqing; Wang, Hongzhi; Han, Wei

2014-01-01

Highlights: • We show the possibility of modulate proliferation induced by radiation-induced bystander effect with low concentration carbon monoxide. • Carbon monoxide inhibited proliferation via modulating the transforming growth factor β1 (TGF-β1)/nitric oxide (NO) signaling pathway. • Exogenous carbon monoxide has potential application in clinical radiotherapy. - Abstract: Radiation-induced bystander effect (RIBE) has been proposed to have tight relationship with the irradiation-caused secondary cancers beyond the irradiation-treated area after radiotherapy. Our previous studies demonstrated a protective effect of low concentration carbon monoxide (CO) on the genotoxicity of RIBE after α-particle irradiation. In the present work, a significant inhibitory effect of low-dose exogenous CO, generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on both RIBE-induced proliferation and chromosome aberration was observed. Further studies on the mechanism revealed that the transforming growth factor β1/nitric oxide (NO) signaling pathway, which mediated RIBE signaling transduction, could be modulated by CO involved in the protective effects. Considering the potential of exogenous CO in clinical applications and its protective effect on RIBE, the present work aims to provide a foundation for potential application of CO in radiotherapy

Low concentration of exogenous carbon monoxide protects mammalian cells against proliferation induced by radiation-induced bystander effect

Energy Technology Data Exchange (ETDEWEB)

Tong, Liping [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Bao, Lingzhi; Wu, Wenqing; Wang, Hongzhi [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Han, Wei, E-mail: hanw@hfcas.cn [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China)

2014-01-15

Highlights: • We show the possibility of modulate proliferation induced by radiation-induced bystander effect with low concentration carbon monoxide. • Carbon monoxide inhibited proliferation via modulating the transforming growth factor β1 (TGF-β1)/nitric oxide (NO) signaling pathway. • Exogenous carbon monoxide has potential application in clinical radiotherapy. - Abstract: Radiation-induced bystander effect (RIBE) has been proposed to have tight relationship with the irradiation-caused secondary cancers beyond the irradiation-treated area after radiotherapy. Our previous studies demonstrated a protective effect of low concentration carbon monoxide (CO) on the genotoxicity of RIBE after α-particle irradiation. In the present work, a significant inhibitory effect of low-dose exogenous CO, generated by tricarbonyldichlororuthenium (II) dimer [CO-releasing molecule (CORM-2)], on both RIBE-induced proliferation and chromosome aberration was observed. Further studies on the mechanism revealed that the transforming growth factor β1/nitric oxide (NO) signaling pathway, which mediated RIBE signaling transduction, could be modulated by CO involved in the protective effects. Considering the potential of exogenous CO in clinical applications and its protective effect on RIBE, the present work aims to provide a foundation for potential application of CO in radiotherapy.

Radiosensitization of mammalian cells by misonidazole and oxygen: DNA damage exposed by Micrococcus luteus enzymes

International Nuclear Information System (INIS)

Skov, K.A.; Palcic, B.; Skarsgard, L.D.

1979-01-01

When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37 0 C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract

NRF2 Oxidative Stress Induced by Heavy Metals is Cell Type Dependent

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Exposure to metallic environmental toxicants has been demonstrated to induce a variety of oxidative stress responses in mammalian cells. The transcription factor Nrf2 is activated in response to oxidative stress and coordinates the expression of antioxidant gene products. In this...

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

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Kusumoto, Hiroki; Hirohashi, Yoshihiko; Nishizawa, Satoshi; Yamashita, Masamichi; Yasuda, Kazuyo; Murai, Aiko; Takaya, Akari; Mori, Takashi; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Kondo, Toru; Sato, Noriyuki; Hara, Isao; Torigoe, Toshihiko

2018-03-01

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Alpha radiation-induced alterations of the proliferation kinetics, chromatin structure and gene expression in mammalian cells

International Nuclear Information System (INIS)

Hieber, L.

1983-01-01

Exponentially growing mammalian cells were exposed to 3.4 MeV alpha particles. The chromatin of cells arrested in G2 by alpha irradiation was severely damaged, though all cells were still capable to condensate their chromatin after fusion with mitotic cells. In addition to the common types of aberrations (breaks, gaps, dicentrics and exchanges) cells were found possessing one or more chromosomes with long stretches of undercondensed chromatin. Repair of these lesions was indicated by site specific unscheduled DNA synthesis and by the observation that condensation of these regions improved during G2 arrest. Furthermore, during G2 arrest the synthesis of two cellular proteins was stimulated. This was studied by two-dimensional gel electrophoresis of 35 S-methionine labeled cellular proteins. All these findings provided evidence that radiation-induced G2 arrest is caused by chromatin damage, which prevents regular chromosome condensation for mitosis. (orig./MG) [de

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

International Nuclear Information System (INIS)

Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

1990-01-01

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

Science.gov (United States)

Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

2018-03-01

A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

Novel method to load multiple genes onto a mammalian artificial chromosome.

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Anna Tóth

Full Text Available Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

Novel method to load multiple genes onto a mammalian artificial chromosome.

Science.gov (United States)

Tóth, Anna; Fodor, Katalin; Praznovszky, Tünde; Tubak, Vilmos; Udvardy, Andor; Hadlaczky, Gyula; Katona, Robert L

2014-01-01

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

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Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y.

2016-01-01

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

[Mathematical modeling of synergistic interaction of sequential thermoradiation action on mammalian cells].

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Belkina, S V; Semkina, M A; Kritskiĭ, R O; Petin, V G

2010-01-01

Data obtained by other authors for mammalian cells treated by sequential action of ionizing radiation and hyperthermia were used to estimate the dependence of synergistic enhancement ratio on the ratio of damages induced by these agents. Experimental results were described and interpreted by means of the mathematical model of synergism in accordance with which the synergism is expected to result from the additional lethal damage arising from the interaction of sublesions induced by both agents.

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

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Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

2016-01-01

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

Antagonistic control of a dual-input mammalian gene switch by food additives.

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Xie, Mingqi; Ye, Haifeng; Hamri, Ghislaine Charpin-El; Fussenegger, Martin

2014-08-01

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sleep-inducing factors.

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García-García, Fabio; Acosta-Peña, Eva; Venebra-Muñoz, Arturo; Murillo-Rodríguez, Eric

2009-08-01

Kuniomi Ishimori and Henri Piéron were the first researchers to introduce the concept and experimental evidence for a chemical factor that would presumably accumulate in the brain during waking and eventually induce sleep. This substance was named hypnotoxin. Currently, the variety of substances which have been shown to alter sleep includes peptides, cytokines, neurotransmitters and some substances of lipidic nature, many of which are well known for their involvement in other biological activities. In this chapter, we describe the sleep-inducing properties of the vasoactive intestinal peptide, prolactin, adenosine and anandamide.

Possible involvement of SINEs in mammalian-specific brain formation.

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Sasaki, Takeshi; Nishihara, Hidenori; Hirakawa, Mika; Fujimura, Koji; Tanaka, Mikiko; Kokubo, Nobuhiro; Kimura-Yoshida, Chiharu; Matsuo, Isao; Sumiyama, Kenta; Saitou, Naruya; Shimogori, Tomomi; Okada, Norihiro

2008-03-18

Retroposons, such as short interspersed elements (SINEs) and long interspersed elements (LINEs), are the major constituents of higher vertebrate genomes. Although there are many examples of retroposons' acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of which constitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic functionality or was exapted after retropositioning in a mammalian ancestor. Here we identified 53 new AmnSINE1 loci and refined 124 total loci, two of which were further analyzed. Using a mouse enhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp from the gene FGF8 (fibroblast growth factor 8), is an enhancer that recapitulates FGF8 expression in two regions of the developing forebrain, namely the diencephalon and the hypothalamus. Our gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay center of the neocortex, suggesting a role for FGF8 in mammalian-specific forebrain patterning. Furthermore, we demonstrated that the locus, AS021, 392 kbp from the gene SATB2, controls gene expression in the lateral telencephalon, which is thought to be a signaling center during development. These results suggest important roles for SINEs in the development of the mammalian neuronal network, a part of which was initiated with the exaptation of AmnSINE1 in a common mammalian ancestor.

Tramadol Pretreatment Enhances Ketamine-Induced Antidepressant Effects and Increases Mammalian Target of Rapamycin in Rat Hippocampus and Prefrontal Cortex

Directory of Open Access Journals (Sweden)

Chun Yang

2012-01-01

Full Text Available Several lines of evidence have demonstrated that acute administration of ketamine elicits fast-acting antidepressant effects. Moreover, tramadol also has potential antidepressant effects. The aim of this study was to investigate the effects of pretreatment with tramadol on ketamine-induced antidepressant activity and was to determine the expression of mammalian target of rapamycin (mTOR in rat hippocampus and prefrontal cortex. Rats were intraperitoneally administrated with ketamine at the dose of 10 mg/kg or saline 1 h before the second episode of the forced swimming test (FST. Tramadol or saline was intraperitoneally pretreated 30 min before the former administration of ketamine or saline. The locomotor activity and the immobility time of FST were both measured. After that, rats were sacrificed to determine the expression of mTOR in hippocampus and prefrontal cortex. Tramadol at the dose of 5 mg/kg administrated alone did not elicit the antidepressant effects. More importantly, pretreatment with tramadol enhanced the ketamine-induced antidepressant effects and upregulated the expression of mTOR in rat hippocampus and prefrontal cortex. Pretreatment with tramadol enhances the ketamine-induced antidepressant effects, which is associated with the increased expression of mTOR in rat hippocampus and prefrontal cortex.

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

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Murata, Akihiko; Yoshino, Miya; Hikosaka, Mari; Okuyama, Kazuki; Zhou, Lan; Sakano, Seiji; Yagita, Hideo; Hayashi, Shin-Ichi

2014-01-01

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion. PMID:25255288

DNA damage-inducible transcripts in mammalian cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

1988-01-01

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.

Science.gov (United States)

Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

2001-04-01

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

Enhancer evolution across 20 mammalian species

DEFF Research Database (Denmark)

Villar, Diego; Berthelot, Camille; Aldridge, Sarah

2015-01-01

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders...... by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements....... These results provide important insight into the functional genetics underpinning mammalian regulatory evolution....

Two Ancient Gene Families Are Critical for Maintenance of the Mammalian Skin Barrier in Postnatal Life.

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Cangkrama, Michael; Darido, Charbel; Georgy, Smitha R; Partridge, Darren; Auden, Alana; Srivastava, Seema; Wilanowski, Tomasz; Jane, Stephen M

2016-07-01

The skin barrier is critical for mammalian survival in the terrestrial environment, affording protection against fluid loss, microbes, toxins, and UV exposure. Many genes indispensable for barrier formation in the embryo have been identified, but loss of these genes in adult mice does not induce barrier regression. We describe a complex regulatory network centered on two ancient gene families, the grainyhead-like (Grhl) transcription factors and the protein cross-linking enzymes (tissue transglutaminases [Tgms]), which are essential for skin permeability barrier maintenance in adult mice. Embryonic deletion of Grhl3 induces loss of Tgm1 expression, which disrupts the cornified envelope, thus preventing permeability barrier formation leading to neonatal death. However, gene deletion of Grhl3 in adult mice does not disrupt the preformed barrier, with cornified envelope integrity maintained by Grhl1 and Tgm5, which are up-regulated in response to postnatal loss of Grhl3. Concomitant deletion of both Grhl factors in adult mice induced loss of Tgm1 and Tgm5 expression, perturbation of the cornified envelope, and complete permeability barrier regression that was incompatible with life. These findings define the molecular safeguards for barrier function that accompany the transition from intrauterine to terrestrial life. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cloning of MASK, a novel member of mammalian germinal center kinase-III subfamily, with apoptosis-inducing properties

DEFF Research Database (Denmark)

Dan, Ippeita; Ong, Shao-En; Watanabe, Norinobu M

2002-01-01

We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK do...... apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells. Udgivelsesdato: 2002-Feb-22...

The food additive vanillic acid controls transgene expression in mammalian cells and mice.

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Gitzinger, Marc; Kemmer, Christian; Fluri, David A; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2012-03-01

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Functional consequences of inducible genetic elements from the p53 SOS response in a mammalian organ system.

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Guthrie, O'neil W

2017-10-01

In response to DNA damage from ultraviolet (UV) radiation, bacteria deploy the SOS response in order to limit cell death. This bacterial SOS response is characterized by an increase in the recA gene that transactivates expression of multiple DNA repair genes. The current series of experiments demonstrate that a mammalian organ system (the cochlea) that is not evolutionarily conditioned to UV radiation can elicit SOS responses that are reminiscent of that of bacteria. This mammalian SOS response is characterized by an increase in the p53 gene with activation of multiple DNA repair genes that harbor p53 response elements in their promoters. Furthermore, the experimental results provide support for the notion of a convergent trigger paradox, where independent SOS triggers facilitate disparate physiologic sequelae (loss vs. recovery of function). Therefore, it is proposed that the mammalian SOS response is multifunctional and manipulation of this endogenous response could be exploited in future biomedical interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

Energy Technology Data Exchange (ETDEWEB)

Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.; Kong, Betty Y.; Gleber, Sophie C.; Vogt, Stefan; Chen, Si; Garwin, Seth A.; Bayer, Amanda R.; Dravid, Vinayak P.; Woodruff, Teresa K.; O' Halloran, Thomas V.

2014-12-15

Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes

An analysis of the factors that influence the level and scaling of mammalian BMR.

Science.gov (United States)

McNab, Brian Keith

2008-09-01

The factors influencing the basal rate of metabolism (BMR) in 639 species of mammals include body mass, food habits, climate, habitat, substrate, a restriction to islands or highlands, use of torpor, and type of reproduction. They collectively account for 98.8% of the variation in mammalian BMR, but often interact in complex ways. The factor with the greatest impact on BMR, as always, is body mass (accounting for 96.8% of its variation), the extent of its impact reflecting the 10(6.17)-fold range of mass in measured species. The attempt to derive mathematically the power relationship of BMR in mammals is complicated by the necessity to include all of the factors that influence BMR that are themselves correlated with body mass. BMR also correlates with taxonomic affiliation because many taxa are distinguished by their ecological and behavioral characteristics. Phylogeny, reflecting previous commitments, may influence BMR either through a restriction on the realized range of behaviors or by opening new behavioral and ecological opportunities. A new opportunity resulted from the evolution by eutherians of a type of reproduction that permitted species feeding on high quality resources to have high BMRs. These rates facilitated high rates of gas, nutrient, and waste exchange between a pregnant eutherian and her placental offspring. This pattern led to high rates of reproduction in some eutherians, a response denied all monotremes and marsupials, thereby permitting eutherians to occupy cold-temperate and polar environments and to dominate other mammals in all environments to which ecologically equivalent eutherians had access.

Algal autolysate medium to label proteins for NMR in mammalian cells.

Science.gov (United States)

Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

2016-04-01

In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

Algal autolysate medium to label proteins for NMR in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia [University of Florence, Magnetic Resonance Center (CERM) (Italy); Neri, Sara [Giotto Biotech S.R.L. (Italy); Fragai, Marco, E-mail: fragai@cerm.unifi.it [University of Florence, Magnetic Resonance Center (CERM) (Italy)

2016-04-15

In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in {sup 15}N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells.

Science.gov (United States)

Angelici, Bartolomeo; Mailand, Erik; Haefliger, Benjamin; Benenson, Yaakov

2016-08-30

One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D

DEFF Research Database (Denmark)

Vinggaard, Anne Marie; Jensen, T.; Morgan, C.P.

1996-01-01

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially...... purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5'-[¿-thio]triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report...... that the use of didecanoyl phosphatidylcholine (C-PC) in mammalian PLD assays considerably increases the detection limit. C-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from...

DNA synthesis in irradiated mammalian cells

International Nuclear Information System (INIS)

Painter, R.B.; California Univ., San Francisco; Young, B.R.

1987-01-01

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v. -induced inhibition of replicon initiation. (author

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors | Office of Cancer Genomics

Science.gov (United States)

Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.

Risk scaling factors from inactivation to chromosome aberrations, mutations and oncogenic transformations in mammalian cells

International Nuclear Information System (INIS)

Alkaharam, A.S.; Watt, D.E.

1997-01-01

Analyses of bio-effect mechanisms of damage to mammalian cells in terms of the quality parameter 'mean free path for primary ionisation', for heavy charged particles, strongly suggests that there is a common mechanism for the biological endpoints of chromosome aberrations, mutations and oncogenic transformation. The lethal lesions are identified as unrepaired double-strand breaks in the intracellular DNA. As data for the various endpoints studied can be represented in a unified scheme, for any radiation type, it follows that radiation risk factors can be determined on the basis of simple ratios to the inactivation cross sections. There are intrinsic physical reasons why neutrons can never reach the saturation level of heavier particles for equal fluences. The probabilities of risk with respect to inactivation, for chromosome dicentrics, mutation of the HPRT gene and of oncogenic transformation are respectively 0.24, 5.8 x 10 -5 , and 4.1 x 10 -3 . (author)

Molecular nature of X-ray-induced mutations compared with that of spontaneous ones in human c-hprt gene integrated into mammalian chromosomal DNA

International Nuclear Information System (INIS)

Kimura, Hiroshi; Kato, Takesi.

1992-01-01

X-ray-induced mutations were analysed at molecular levels in comparison with spontaneous mutations. Altered sequences were determined tentatively of 30 independent X-ray-induced mutations in a cDNA of the human hprt gene which was integrated into mammalian chromosome as a part of a shuttle vector. Mutations consisted of base substitutions (37 %), frameshifts (27 %), deletions (27 %) and others (10 %). All these mutational events were distributed randomly over the gene without there being hot spots. The spectrum and distribution of X-ray-induced mutations resembled those of spontaneous mutations. Among base substitutions, transversions were predominant and base substitution mutations occurred more at A:T sites than at G:C sites, which is also the case in spontaneous mutations. Most of the frameshift and deletion mutations induced by X-rays, as well as those spontaneously arising, were characterized by the existence of short direct repeats of several identical bases in a row at the sites of the mutations. A slippage misalignment mechanism in replication well accounts for the generation of these classes of mutations. Judging from the data accumulated so far, it can be concluded that X-ray-induced mutations at molecular levels are similar to those spontaneously occurring. (author)

Risk factors for amiodarone-induced thyroid dysfunction in Japan

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Sayoko Kinoshita

2016-12-01

Conclusion: DCM and cardiac sarcoidosis were identified as risk factors for amiodarone-induced hyperthyroidism. Risk factors for amiodarone-induced hypothyroidism included higher baseline TSH level and lower baseline free T4 level, suggesting that subclinical hypothyroidism may be a potential risk factor for the development of amiodarone-induced hypothyroidism.

Mammalian synthetic biology: emerging medical applications.

Science.gov (United States)

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. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Mammalian development in space

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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

Mammalian cell biology

International Nuclear Information System (INIS)

Elkind, M.M.

1975-01-01

Progress is reported on the following research projects: the effects of N-ethyl-maleimide and hydroxyurea on hamster cells in culture; sensitization of synchronized human cells to x rays by N-ethylmaleimide; sensitization of hypoxic mammalian cells with a sulfhydryl inhibitor; damage interaction due to ionizing and nonionizing radiation in mammalian cells; DNA damage relative to radioinduced cell killing; spurious photolability of DNA labeled with methyl- 14 C-thymidine; radioinduced malignant transformation of cultured mouse cells; a comparison of properties of uv and near uv light relative to cell function and DNA damage; Monte Carlo simulation of DNA damage and repair mechanisms; and radiobiology of fast neutrons

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

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Poppi, Lauren A; Tabatabaee, Hessam; Drury, Hannah R; Jobling, Phillip; Callister, Robert J; Migliaccio, Americo A; Jordan, Paivi M; Holt, Joseph C; Rabbitt, Richard D; Lim, Rebecca; Brichta, Alan M

2018-01-01

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted

Effect of temperature on spontaneous release of transmitter at the mammalian neuromuscular junction

Energy Technology Data Exchange (ETDEWEB)

Duncan, C J; Statham, H E

1977-01-01

Temperature has a multifactorial effect on miniature endplate potential (MEPP) frequency at the mammalian neuromuscular junction, with a negative Q/sub 10/ at 14--28/sup 0/C. The results are explained in terms of the effect of temperature on the various factors that control (Ca/sup 2/+sub i/ at the presynaptic terminals. The temperature-sensitivity of the Ca/sup 2 +/-transport enzyme is believed to be of particular significance and accounts for the observed differences between the amphibian and mammalian neuromuscular junctions.

Hypoxia regulates the expression of the neuromedin B receptor through a mechanism dependent on hypoxia-inducible factor-1α.

Directory of Open Access Journals (Sweden)

Hyun-Joo Park

Full Text Available The neuromedin B receptor (NMB-R, a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α. We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

A biotin-triggered genetic switch in mammalian cells and mice.

Science.gov (United States)

Weber, Wilfried; Lienhart, Cédric; Baba, Marie Daoud-El; Fussenegger, Martin

2009-03-01

Adjustable and reversible transgene expression systems enabling precise control of metabolic pathways and tunable production of specific target proteins have been essential for conditional reprogramming of mammalian cells to achieve progress in basic and applied bioengineering disciplines. Most of the currently available transgene control modalities have been designed to be responsive to clinically licensed pharmacologically active drugs which were expected to prevail in future clinical trials yet raised concerns about side effects when administered long term at subclinical doses. We have chosen vitamin H, also known as biotin, to control target gene transcription in mammalian cells in a potentially side effect-free manner. BirA, the Escherichia coli repressor of the biotin biosynthesis operon, was fused to the Herpes simplex transactivation domain to generate a biotin-dependent transactivator(BIT), which, in the presence of biotin, binds and activates chimeric target promoters (P(BIT)) harboring BirA-specific operator sites 5' of a minimal promoter. Biotin-inducible transgene expression was functional in a variety of rodent, monkey and human cell lines, showed excellent adjustability and reversibility in transgenic Chinese hamster ovary cell lines, provided precise product gene control in standard bioreactor cultures and enabled dose-dependent vitamin H control of a human glycoprotein in mice. The combination of a side effect-free inducer, precise and reversible transcription tunability and broad functionality in different cell types as well as in entire animals represents a unique asset for the use of biotin-inducible transgene control in future gene therapy, tissue engineering and biopharmaceutical manufacturing scenarios.

Regulation of Autophagy by Glucose in Mammalian Cells

OpenAIRE

Moruno, Félix; Pérez-Jiménez, Eva; Knecht, Erwin

2012-01-01

Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focu...

Effect of Marine Omega 3 Fatty Acids on Methylmercury-Induced Toxicity in Fish and Mammalian Cells In Vitro

Directory of Open Access Journals (Sweden)

O. J. Nøstbakken

2012-01-01

Full Text Available Methylmercury (MeHg is a ubiquitous environmental contaminant which bioaccumulates in marine biota. Fish constitute an important part of a balanced human diet contributing with health beneficial nutrients but may also contain contaminants such as MeHg. Interactions between the marine n-3 fatty acids eicosapentaenoic acid (20:5n-3, EPA and docosahexaenoic acid (22:6n-3, DHA with MeHg-induced toxicity were investigated. Different toxic and metabolic responses were studied in Atlantic salmon kidney (ASK cell line and the mammalian kidney-derived HEK293 cell line. Both cell lines were preincubated with DHA or EPA prior to MeHg-exposure, and cell toxicity was assessed differently in the cell lines by MeHg-uptake in cells (ASK and HEK293, proliferation (HEK293 and ASK, apoptosis (ASK, oxidation of the red-ox probe roGFP (HEK293, and regulation of selected toxicological and metabolic transcriptional markers (ASK. DHA was observed to decrease the uptake of MeHg in HEK293, but not in ASK cells. DHA also increased, while EPA decreased, MeHg-induced apoptosis in ASK. MeHg exposure induced changes in selected metabolic and known MeHg biomarkers in ASK cells. Both DHA and MeHg, but not EPA, oxidized roGFP in HEK293 cells. In conclusion, marine n-3 fatty acids may ameliorate MeHg toxicity, either by decreasing apoptosis (EPA or by reducing MeHg uptake (DHA. However, DHA can also augment MeHg toxicity by increasing oxidative stress and apoptosis when combined with MeHg.

A platform for rapid prototyping of synthetic gene networks in mammalian cells

Science.gov (United States)

Duportet, Xavier; Wroblewska, Liliana; Guye, Patrick; Li, Yinqing; Eyquem, Justin; Rieders, Julianne; Rimchala, Tharathorn; Batt, Gregory; Weiss, Ron

2014-01-01

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. PMID:25378321

A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

Directory of Open Access Journals (Sweden)

Zhang Tingting

2012-12-01

Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

Science.gov (United States)

Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

2017-09-15

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

ASVCP quality assurance guidelines: control of preanalytical and analytical factors for hematology for mammalian and nonmammalian species, hemostasis, and crossmatching in veterinary laboratories.

Science.gov (United States)

Vap, Linda M; Harr, Kendal E; Arnold, Jill E; Freeman, Kathleen P; Getzy, Karen; Lester, Sally; Friedrichs, Kristen R

2012-03-01

In December 2009, the American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance and Laboratory Standards committee published the updated and peer-reviewed ASVCP Quality Assurance Guidelines on the Society's website. These guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports: (1) general analytical factors for veterinary laboratory performance and comparisons; (2) hematology, hemostasis, and crossmatching; and (3) clinical chemistry, cytology, and urinalysis. This particular report is one of 3 reports and provides recommendations for control of preanalytical and analytical factors related to hematology for mammalian and nonmammalian species, hemostasis testing, and crossmatching and is adapted from sections 1.1 and 2.3 (mammalian hematology), 1.2 and 2.4 (nonmammalian hematology), 1.5 and 2.7 (hemostasis testing), and 1.6 and 2.8 (crossmatching) of the complete guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimal guidelines for quality assurance and quality control for veterinary laboratory testing and a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts. © 2012 American Society for Veterinary Clinical Pathology.

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

International Nuclear Information System (INIS)

Grether-Beck, S.; Olaizola-Horn, S.; Schmitt, H.; Grewe, M.

1996-01-01

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing OCAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response. 38 refs., 3 figs., 3 tabs

YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung, E-mail: keejung@skku.edu

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.

YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

International Nuclear Information System (INIS)

Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

2015-01-01

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

Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages.

Science.gov (United States)

Jinadasa, Rasika N; Bloom, Stephen E; Weiss, Robert S; Duhamel, Gerald E

2011-07-01

Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.

Enhancer Evolution across 20 Mammalian Species

Science.gov (United States)

Villar, Diego; Berthelot, Camille; Aldridge, Sarah; Rayner, Tim F.; Lukk, Margus; Pignatelli, Miguel; Park, Thomas J.; Deaville, Robert; Erichsen, Jonathan T.; Jasinska, Anna J.; Turner, James M.A.; Bertelsen, Mads F.; Murchison, Elizabeth P.; Flicek, Paul; Odom, Duncan T.

2015-01-01

Summary The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution. PMID:25635462

Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Ventura, A M; Ortega, J M; Schumacher, R I; Meneghini, R

1987-01-01

In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. Using metabolic inhibitors, it has been shown that (1) even the low repair rate exhibited by V79 cells is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of small replicons.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

Science.gov (United States)

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

Science.gov (United States)

Levine, Zebulon G; Walker, Suzanne

2016-06-02

O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.

The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis.

Science.gov (United States)

Liou, M L; Liou, H C

1999-04-09

The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins. By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1. Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells. In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line. Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS. Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD. Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector.

Mammalian gastrointestinal parasites in rainforest remnants

Indian Academy of Sciences (India)

Here, we studied the gastrointestinal parasites of nonhuman mammalian hosts living in 10 rainforest patches of the Anamalai Tiger Reserve, India. We examined 349 faecal samples of 17 mammalian species and successfully identified 24 gastroin-testinal parasite taxa including 1 protozoan, 2 trematode, 3 cestode and 18 ...

Dedifferentiation and proliferation of mammalian cardiomyocytes.

Directory of Open Access Journals (Sweden)

Yiqiang Zhang

2010-09-01

Full Text Available It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1 cardiomyocyte purification from rat hearts, and 2 genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs, while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+.Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness

Retinoic Acid-Induced Epidermal Transdifferentiation in Skin

Directory of Open Access Journals (Sweden)

Yoshihiro Akimoto

2014-06-01

Full Text Available Retinoids function as important regulatory signaling molecules during development, acting in cellular growth and differentiation both during embryogenesis and in the adult animal. In 1953, Fell and Mellanby first found that excess vitamin A can induce transdifferentiation of chick embryonic epidermis to a mucous epithelium (Fell, H.B.; Mellanby, E. Metaplasia produced in cultures of chick ectoderm by high vitamin A. J. Physiol. 1953, 119, 470–488. However, the molecular mechanism of this transdifferentiation process was unknown for a long time. Recent studies demonstrated that Gbx1, a divergent homeobox gene, is one of the target genes of all-trans retinoic acid (ATRA for this transdifferentiation. Furthermore, it was found that ATRA can induce the epidermal transdifferentiation into a mucosal epithelium in mammalian embryonic skin, as well as in chick embryonic skin. In the mammalian embryonic skin, the co-expression of Tgm2 and Gbx1 in the epidermis and an increase in TGF-β2 expression elicited by ATRA in the dermis are required for the mucosal transdifferentiation, which occurs through epithelial-mesenchymal interaction. Not only does retinoic acid (RA play an important role in mucosal transdifferentiation, periderm desquamation, and barrier formation in the developing mammalian skin, but it is also involved in hair follicle downgrowth and bending by its effect on the Wnt/β-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families.

Localization of two mammalian cyclin dependent kinases during mammalian meiosis

NARCIS (Netherlands)

Ashley, T.; Walpita, D.; de rooij, D. G.

2001-01-01

Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids)

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

DEFF Research Database (Denmark)

Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

2008-01-01

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

Science.gov (United States)

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Identification of mammalian proteins cross-linked to DNA by ionizing radiation.

Science.gov (United States)

Barker, Sharon; Weinfeld, Michael; Zheng, Jing; Li, Liang; Murray, David

2005-10-07

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1α-dependent pathway.

Science.gov (United States)

Kang, Min-Jung; Song, Eun-Jung; Kim, Bo-Yeon; Kim, Dong-Jae; Park, Jong-Hwan

2014-12-01

Although Helicobacter pylori have been known to induce vascular endothelial growth factor (VEGF) production in gastric epithelial cells, the precise mechanism for cellular signaling is incompletely understood. In this study, we investigated the role of bacterial virulence factor and host cellular signaling in VEGF production of H. pylori-infected gastric epithelial cells. We evaluated production of VEGF, activation of nuclear factor nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) and hypoxia-inducible factor-1α (HIF-1α) stabilization in gastric epithelial cells infected with H. pylori WT or isogenic mutants deficient in type IV secretion system (T4SS). H. pylori induced VEGF production in gastric epithelial cells via both T4SS-dependent and T4SS-independent pathways, although T4SS-independent pathway seems to be the dominant signaling. The inhibitor assay implicated that activation of NF-κB and MAPKs is dispensable for H. pylori-induced VEGF production in gastric epithelial cells. H. pylori led to HIF-1α stabilization in gastric epithelial cells independently of T4SS, NF-κB, and MAPKs, which was essential for VEGF production in these cells. N-acetyl-cysteine (NAC), a reactive oxygen species (ROS) inhibitor, treatment impaired H. pylori-induced HIF-1α stabilization and VEGF production in gastric epithelial cells. We defined the important role of ROS-HIF-1α axis in VEGF production of H. pylori-infected gastric epithelial cells, and bacterial T4SS has a minor role in H. pylori-induced VEGF production of gastric epithelial cells. © 2014 John Wiley & Sons Ltd.

Focusing on RISC assembly in mammalian cells.

Science.gov (United States)

Hong, Junmei; Wei, Na; Chalk, Alistair; Wang, Jue; Song, Yutong; Yi, Fan; Qiao, Ren-Ping; 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.

Focusing on RISC assembly in mammalian cells

International Nuclear Information System (INIS)

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

2008-01-01

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

Regulation of mutagenesis by exogenous biological factors in the eukaryotic cell systems

Directory of Open Access Journals (Sweden)

Lukash L. L.

2013-07-01

Full Text Available The representations of the mutations and the nature of spontaneous mutation process and mutagenesis induced by exogenous oncoviruses, DNAs and proteins-mitogens are analysed. Exogenous biological factors induce DNA damages in regulatory-informational way, acting on the cellular systems for maintenance of genetical stability. Molecular mechanisms are the same as at spontaneous mutagenesis but they are realized with the participation of alien genetical material. Among biological mutagens, the oncoviruses and mobile genetic elements (MGEs are distinguished as the strongest destabilizing factors which direct tumor transformation of somatic mammalian cells. Genetical reprogramming or changing the programs of gene expression at the differentiation of stem and progenitor cells under growth factors and citokines is probably followed by mutations and recombinations as well.

Specificity of chicken and mammalian transferrins in myogenesis

International Nuclear Information System (INIS)

Beach, R.L.; Popiela, Heinz; Festoff, B.W.

1985-01-01

Chicken transferrins isolated from eggs, embryo extract, serum or ischiatic-peroneal nerves are able to stimulate incorporation of ( 3 H)thymidine, and promote myogenesis by primary chicken muscles cells in vitro. Mammalian transferrins (bovine, rat, mouse, horse, rabbit, and human) do not promote ( 3 H)thymidine incorporation or myotube development. Comparison of the peptide fragments obtained after chemical or limited proteolytic cleavage demonstrates that the four chicken transferrins are all indistinguishable, but they differ considerably from the mammalian transferrins. The structural differences between chicken and mammalian transferrins probably account for the inability of mammalian transferrins to act as mitogens for, and to support myogenesis of, primary chicken muscle cells. (author)

Mammalian cell biology

International Nuclear Information System (INIS)

Elkind, M.M.

1979-01-01

This section contains summaries of research on mechanisms of lethality and radioinduced changes in mammalian cell properties, new cell systems for the study of the biology of mutation and neoplastic transformation, and comparative properties of ionizing radiations

Applications of stable isotope analysis in mammalian ecology.

Science.gov (United States)

Walter, W David; Kurle, Carolyn M; Hopkins, John B

2014-01-01

In this editorial, we provide a brief introduction and summarize the 10 research articles included in this Special Issue on Applications of stable isotope analysis in mammalian ecology. The first three articles report correction and discrimination factors that can be used to more accurately estimate the diets of extinct and extant mammals using stable isotope analysis. The remaining seven applied research articles use stable isotope analysis to address a variety of wildlife conservation and management questions from the oceans to the mountains.

Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis.

Science.gov (United States)

Vizzini, Aiti; Di Falco, Felicia; Parrinello, Daniela; Sanfratello, Maria Antonietta; Cammarata, Matteo

2016-02-01

Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that CiTGF-β was transcriptionally upregulated in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Radiation equivalence of genotoxic chemicals - Validation in cultered mammalian cell lines

International Nuclear Information System (INIS)

Murthy, M.S.S.

1982-01-01

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated. REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human. (orig.)

Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

International Nuclear Information System (INIS)

Huberman, E.; Langenbach, R.

1977-01-01

We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

Mosaic evolution of the mammalian auditory periphery.

Science.gov (United States)

Manley, Geoffrey A

2013-01-01

The classical mammalian auditory periphery, i.e., the type of middle ear and coiled cochlea seen in modern therian mammals, did not arise as one unit and did not arise in all mammals. It is also not the only kind of auditory periphery seen in modern mammals. This short review discusses the fact that the constituents of modern mammalian auditory peripheries arose at different times over an extremely long period of evolution (230 million years; Ma). It also attempts to answer questions as to the selective pressures that led to three-ossicle middle ears and the coiled cochlea. Mammalian middle ears arose de novo, without an intermediate, single-ossicle stage. This event was the result of changes in eating habits of ancestral animals, habits that were unrelated to hearing. The coiled cochlea arose only after 60 Ma of mammalian evolution, driven at least partly by a change in cochlear bone structure that improved impedance matching with the middle ear of that time. This change only occurred in the ancestors of therian mammals and not in other mammalian lineages. There is no single constellation of structural features of the auditory periphery that characterizes all mammals and not even all modern mammals.

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells.

Science.gov (United States)

Kennedy, Allyson E; Kandalam, Suraj; Olivares-Navarrete, Rene; Dickinson, Amanda J G

2017-01-01

Since electronic cigarette (ECIG) introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM) in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product.

Inhibition of Mammalian Target of Rapamycin Complex 1 Attenuates Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

Science.gov (United States)

Kumar, Vikash; Wollner, Clayton; Kurth, Theresa; Bukowy, John D; Cowley, Allen W

2017-10-01

The goal of the present study was to explore the protective effects of mTORC1 (mammalian target of rapamycin complex 1) inhibition by rapamycin on salt-induced hypertension and kidney injury in Dahl salt-sensitive (SS) rats. We have previously demonstrated that H 2 O 2 is elevated in the kidneys of SS rats. The present study showed a significant upregulation of renal mTORC1 activity in the SS rats fed a 4.0% NaCl for 3 days. In addition, renal interstitial infusion of H 2 O 2 into salt-resistant Sprague Dawley rats for 3 days was also found to stimulate mTORC1 activity independent of a rise of arterial blood pressure. Together, these data indicate that the salt-induced increases of renal H 2 O 2 in SS rats activated the mTORC1 pathway. Daily administration of rapamycin (IP, 1.5 mg/kg per day) for 21 days reduced salt-induced hypertension from 176.0±9.0 to 153.0±12.0 mm Hg in SS rats but had no effect on blood pressure salt sensitivity in Sprague Dawley treated rats. Compared with vehicle, rapamycin reduced albumin excretion rate in SS rats from 190.0±35.0 to 37.0±5.0 mg/d and reduced the renal infiltration of T lymphocytes (CD3 + ) and macrophages (ED1 + ) in the cortex and medulla. Renal hypertrophy and cell proliferation were also reduced in rapamycin-treated SS rats. We conclude that enhancement of intrarenal H 2 O 2 with a 4.0% NaCl diet stimulates the mTORC1 pathway that is necessary for the full development of the salt-induced hypertension and kidney injury in the SS rat. © 2017 American Heart Association, Inc.

Novel Drosophila receptor that binds multiple growth factors

International Nuclear Information System (INIS)

Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

1986-01-01

The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

Clotting of mammalian fibrinogens by papain: A re-examination

OpenAIRE

Doolittle, RF

2014-01-01

© 2014 American Chemical Society. Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be du...

Investigation of X-ray-induced chromosome aberrations in 'preleukaemic' mammalian cells

International Nuclear Information System (INIS)

Szollar, J.

1977-01-01

A study was done on the frequency of numerical and structural aberrations induced by different doses of X-ray irradiation in spontaneously leukaemic AKR mice, compared with the values of healthy control CBA/H-T 6 T 6 mice. Both were irradiated under the same conditions, but their chromosomes were affected in a different way. The number of cells containing aneuploid sets, rings, fragments, or metacentric chromosomes was significantly higher in the 2-month-old AKR mice than in the control CBA group. The increased chromosomal fragility found in AKR bone marrow cells 5-7 months before the manifestation of lymphoid leukaemia might be an important factor in the development of malignant condition. This genetic imbalance could provide a possible reason for an increase of spontaneous malfunction of the cellular system, as well as for an increased sensitivity to external factors. Thus it might be connected directly with the predisposition to malignant growth, or it has an indirect role helping virus activation, or acting together with the immune deficiency, by creating a weaker system that is more sensitive to carcinogenic agents

Nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective.

Science.gov (United States)

Wang, Pei-Hui; Weng, Shao-Ping; He, Jian-Guo

2015-02-01

Nucleic acids derived from viral pathogens are typical pathogen associated molecular patterns (PAMPs). In mammals, the recognition of viral nucleic acids by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs) and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), induces the release of inflammatory cytokines and type I interferons (IFNs) through the activation of nuclear factor κB (NF-κB) and interferon regulatory factor (IRF) 3/7 pathways, triggering the host antiviral state. However, whether nucleic acids can induce similar antiviral immunity in invertebrates remains ambiguous. Several studies have reported that nucleic acid mimics, especially dsRNA mimic poly(I:C), can strongly induce non-specific antiviral immune responses in insects, shrimp, and oyster. This behavior shows multiple similarities to the hallmarks of mammalian IFN responses. In this review, we highlight the current understanding of nucleic acid-induced antiviral immunity in invertebrates. We also discuss the potential recognition and regulatory mechanisms that confer non-specific antiviral immunity on invertebrate hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

DEFF Research Database (Denmark)

Tornøe, Jens; Kusk, P.; Johansen, T.E.

2002-01-01

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C...

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

Science.gov (United States)

Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

2014-10-16

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Homogenization of Mammalian Cells.

Science.gov (United States)

de Araújo, Mariana E G; Lamberti, Giorgia; Huber, Lukas A

2015-11-02

Homogenization is the name given to the methodological steps necessary for releasing organelles and other cellular constituents as a free suspension of intact individual components. Most homogenization procedures used for mammalian cells (e.g., cavitation pump and Dounce homogenizer) rely on mechanical force to break the plasma membrane and may be supplemented with osmotic or temperature alterations to facilitate membrane disruption. In this protocol, we describe a syringe-based homogenization method that does not require specialized equipment, is easy to handle, and gives reproducible results. The method may be adapted for cells that require hypotonic shock before homogenization. We routinely use it as part of our workflow to isolate endocytic organelles from mammalian cells. © 2015 Cold Spring Harbor Laboratory Press.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

Science.gov (United States)

Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells.

Directory of Open Access Journals (Sweden)

Alden C Klarer

Full Text Available Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta, is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE, the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

Science.gov (United States)

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Evolutionary patterns of RNA-based duplication in non-mammalian chordates.

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Ming Chen

Full Text Available The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

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Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

2014-11-10

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hypoxia-inducible factor signalling mechanisms in the central nervous system.

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Corcoran, A; O'Connor, J J

2013-08-01

In the CNS, neurones are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased, neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma and neurodegenerative diseases. Cellular responses to oxygen deprivation are complex and result in activation of short- and long-term mechanisms to conserve energy and protect cells. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute effects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, pre-synaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurones to hypoxia is the activation of transcription factors such as hypoxia-inducible factor. The activation of hypoxia-inducible factor is governed by a family of dioxygenases called hypoxia-inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia-inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia-inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia-inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

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Vidya P Nair

2016-04-01

Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

Mammalian diversity: gametes, embryos and reproduction.

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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.

Mesozoic mammals from Arizona: new evidence on Mammalian evolution.

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Jenkins, F A; Crompton, A W; Downs, W R

1983-12-16

Knowledge of early mammalian evolution has been based on Old World Late Triassic-Early Jurassic faunas. The discovery of mammalian fossils of approximately equivalent age in the Kayenta Formation of northeastern Arizona gives evidence of greater diversity than known previously. A new taxon documents the development of an angular region of the jaw as a neomorphic process, and represents an intermediate stage in the origin of mammalian jaw musculature.

Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.

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Erickson, Andrea K; Jesudhasan, Palmy R; Mayer, Melinda J; Narbad, Arjan; Winter, Sebastian E; Pfeiffer, Julie K

2018-01-10

RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections. Copyright © 2017 Elsevier Inc. All rights reserved.

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells.

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Allyson E Kennedy

Full Text Available Since electronic cigarette (ECIG introduction to American markets in 2007, vaping has surged in popularity. Many, including women of reproductive age, also believe that ECIG use is safer than traditional tobacco cigarettes and is not hazardous when pregnant. However, there are few studies investigating the effects of ECIG exposure on the developing embryo and nothing is known about potential effects on craniofacial development. Therefore, we have tested the effects of several aerosolized e-cigarette liquids (e-cigAM in an in vivo craniofacial model, Xenopus laevis, as well as a mammalian neural crest cell line. Results demonstrate that e-cigAM exposure during embryonic development induces a variety of defects, including median facial clefts and midface hypoplasia in two of e-cigAMs tested e-cigAMs. Detailed quantitative analyses of the facial morphology revealed that nicotine is not the main factor in inducing craniofacial defects, but can exacerbate the effects of the other e-liquid components. Additionally, while two different e-cigAMs can have very similar consequences on facial appearances, there are subtle differences that could be due to the differences in e-cigAM components. Further assessment of embryos exposed to these particular e-cigAMs revealed cranial cartilage and muscle defects and a reduction in the blood supply to the face. Finally, the expression of markers for vascular and cartilage differentiation was reduced in a mammalian neural crest cell line corroborating the in vivo effects. Our work is the first to show that ECIG use could pose a potential hazard to the developing embryo and cause craniofacial birth defects. This emphasizes the need for more testing and regulation of this new popular product.

ATF1 Modulates the Heat Shock Response by Regulating the Stress-Inducible Heat Shock Factor 1 Transcription Complex

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Takii, Ryosuke; Fujimoto, Mitsuaki; Tan, Ke; Takaki, Eiichi; Hayashida, Naoki; Nakato, Ryuichiro; Shirahige, Katsuhiko

2014-01-01

The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation. PMID:25312646

Granulocyte colony-stimulating factor induces in vitro lymphangiogenesis

International Nuclear Information System (INIS)

Lee, Ae Sin; Kim, Dal; Wagle, Susbin Raj; Lee, Jung Eun; Jung, Yu Jin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

2013-01-01

Highlights: •G-CSF induces tube formation, migration and proliferation of lymphatic cells. •G-CSF increases phosphorylation of MAPK and Akt in lymphatic endothelial cells. •MAPK and Akt pathways are linked to G-CSF-induced in vitro lymphangiogenesis. •G-CSF increases sprouting of a lymphatic ring. •G-CSF produces peritoneal lymphangiogenesis. -- Abstract: Granulocyte-colony stimulating factor (G-CSF) is reported to induce differentiation in cells of the monocyte lineage and angiogenesis in vascular endothelial cells, but its effects on lymphangiogenesis is uncertain. Here we examined the effects and the mechanisms of G-CSF-induced lymphangiogenesis using human lymphatic endothelial cells (hLECs). Our results showed that G-CSF induced capillary-like tube formation, migration and proliferation of hLECs in a dose- and time-dependent manner and enhanced sprouting of thoracic duct. G-CSF increased phosphorylation of Akt and ERK1/2 in hLECs. Supporting the observations, specific inhibitors of phosphatidylinositol 3′-kinase and MAPK suppressed the G-CSF-induced in vitro lymphangiogenesis and sprouting. Intraperitoneal administration of G-CSF to mice also stimulated peritoneal lymphangiogenesis. These findings suggest that G-CSF is a lymphangiogenic factor

Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

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Takeshi Azuma

2011-02-01

Full Text Available Previously, we reported that vitamin K3 (VK3, but not VK1 or VK2 (=MK-4, inhibits the activity of human DNA polymerase γ (pol γ. In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF-α induced by lipopolysaccharide (LPS. Moreover, MK-2 was found to inhibit the action of nuclear factor (NF-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.

TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

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Tien-Ling Liao

2014-09-01

Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

Overexpression of a transcription factor LYL1 induces T- and B-cell lymphoma in mice.

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Zhong, Y; Jiang, L; Hiai, H; Toyokuni, S; Yamada, Y

2007-10-18

LYL1, a member of the class II basic helix-loop-helix transcription factors, is aberrantly expressed in a fraction of human T-cell acute lymphoblastic leukemia. Here, we generated transgenic mice ubiquitously overexpressing LYL1 using a construct expressing full-length cDNA driven by a human elongation factor 1alpha promoter. Four independent lines exhibiting high LYL1 expression were established. Of these transgenic mice, 96% displayed loss of hair with a short kinked tail. Furthermore, 30% of them developed malignant lymphoma, with an average latent period of 352 days. In these mice, histological examination revealed tumor cell infiltration in multiple organs and immunohistochemical analysis showed that the infiltrated tumor cells were either CD3 or CD45R/B220-positive; fluorescence-activated cell sorter analysis indicated that each tumor consisted either of mainly CD4, CD8 double-positive T cells or mature B cells; the clonality of LYL1-induced lymphoma was confirmed by T-cell receptor rearrangement and immunoglobulin heavy-chain gene rearrangement analyses. Mammalian two-hybrid analysis and luciferase assay suggested that excess LYL1 blocked the dimerization of E2A and thus inhibited the regulatory activity of E2A on the CD4 promoter. Reverse transcription-polymerase chain reaction results showed that the expression of certain E2A/HEB target genes was downregulated. Taken together, our results provide direct evidence that aberrant expression of LYL1 plays a role in lymphomagenesis.

Radiation-induced cell mutations as a function of dose rate

International Nuclear Information System (INIS)

Kiefer, J.

1987-01-01

A brief review of the data in the literature is presented and forms the background of the experimental data given by the author obtained with exponential long-term cultures of V79 hamster cells exposed over a period of up to 35 days to different dose rates of gamma radiation. The experimental results show that at a dose rate of 40 mGy/hour the number of induced mutations is reduced, - which is in agreement with literature data - , but a dose rate of less than 30 mGy/hour makes the induced mutations leap to a value clearly higher than those induced by acute irradiation. As in addition to the mutations recombination is a significant factor of the radiation risk, experiments with a heterozygotic yeast strain have been made, as there is to date no reliable mammalian cell system available for this kind of research. Long-term radiation exposure of the yeast cells over a period of six weeks drastically increased the rate of recombinations, to a value higher by a factor of about 4 than that induced by acute irradiation. (orig.) [de

Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

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Lebar, Tina; Jerala, Roman

2018-01-01

Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

[Research progress of mammalian synthetic biology in biomedical field].

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Yang, Linfeng; Yin, Jianli; Wang, Meiyan; Ye, Haifeng

2017-03-25

Although still in its infant stage, synthetic biology has achieved remarkable development and progress during the past decade. Synthetic biology applies engineering principles to design and construct gene circuits uploaded into living cells or organisms to perform novel or improved functions, and it has been widely used in many fields. In this review, we describe the recent advances of mammalian synthetic biology for the treatment of diseases. We introduce common tools and design principles of synthetic gene circuits, and then we demonstrate open-loop gene circuits induced by different trigger molecules used in disease diagnosis and close-loop gene circuits used for biomedical applications. Finally, we discuss the perspectives and potential challenges of synthetic biology for clinical applications.

A hybrid mammalian cell cycle model

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Vincent Noël

2013-08-01

Full Text Available Hybrid modeling provides an effective solution to cope with multiple time scales dynamics in systems biology. Among the applications of this method, one of the most important is the cell cycle regulation. The machinery of the cell cycle, leading to cell division and proliferation, combines slow growth, spatio-temporal re-organisation of the cell, and rapid changes of regulatory proteins concentrations induced by post-translational modifications. The advancement through the cell cycle comprises a well defined sequence of stages, separated by checkpoint transitions. The combination of continuous and discrete changes justifies hybrid modelling approaches to cell cycle dynamics. We present a piecewise-smooth version of a mammalian cell cycle model, obtained by hybridization from a smooth biochemical model. The approximate hybridization scheme, leading to simplified reaction rates and binary event location functions, is based on learning from a training set of trajectories of the smooth model. We discuss several learning strategies for the parameters of the hybrid model.

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

International Nuclear Information System (INIS)

Veelen, Lieneke R. van; Essers, Jeroen; Rakt, Mandy W.M.M. van de; Odijk, Hanny; Pastink, Albert; Zdzienicka, MaIgorzata Z.; Paulusma, Coen C.; Kanaar, Roland

2005-01-01

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response

Localization of RNA and translation in the mammalian oocyte and embryo

Czech Academy of Sciences Publication Activity Database

Jansová, Denisa; Tětková, Anna; Končická, Markéta; Kubelka, Michal; Šušor, Andrej

2018-01-01

Roč. 13, č. 3 (2018), č. článku e0192544. E-ISSN 1932-6203 R&D Projects: GA ČR GA13-12291S; GA ČR GA15-22765S; GA MŠk EF15_003/0000460 Institutional support: RVO:67985904 Keywords : RNA localization * mammalian oocyte Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 2.806, year: 2016

Surgical manipulation of mammalian embryos in vitro.

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Naruse, I; Keino, H; Taniguchi, M

1997-04-01

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor-κB in osteoblasts.

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Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

2017-01-01

Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity.

Dexamethasone impairs hypoxia-inducible factor-1 function

International Nuclear Information System (INIS)

Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

2008-01-01

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of α- and β-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1α levels in the cytosol of HepG2 cells, while nuclear HIF-1α levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients

E2F8 is essential for polyploidization in mammalian cells.

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Pandit, Shusil K; Westendorp, Bart; Nantasanti, Sathidpak; van Liere, Elsbeth; Tooten, Peter C J; Cornelissen, Peter W A; Toussaint, Mathilda J M; Lamers, Wouter H; de Bruin, Alain

2012-11-01

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

Mini Screening of Kinase Inhibitors Affecting Period-length of Mammalian Cellular Circadian Clock

International Nuclear Information System (INIS)

Yagita, Kazuhiro; Yamanaka, Iori; Koinuma, Satoshi; Shigeyoshi, Yasufumi; Uchiyama, Yasuo

2009-01-01

In mammalian circadian rhythms, the transcriptional-translational feedback loop (TTFL) consisting of a set of clock genes is believed to elicit the circadian clock oscillation. The TTFL model explains that the accumulation and degradation of mPER and mCRY proteins control the period-length (tau) of the circadian clock. Although recent studies revealed that the Casein Kinase Iεδ (CKIεδ) regurates the phosphorylation of mPER proteins and the circadian period-length, other kinases are also likely to contribute the phosphorylation of mPER. Here, we performed small scale screening using 84 chemical compounds known as kinase inhibitors to identify candidates possibly affecting the circadian period-length in mammalian cells. Screening by this high-throughput real-time bioluminescence monitoring system revealed that the several chemical compounds apparently lengthened the cellular circadian clock oscillation. These compounds are known as inhibitors against kinases such as Casein Kinase II (CKII), PI3-kinase (PI3K) and c-Jun N-terminal Kinase (JNK) in addition to CKIεδ. Although these kinase inhibitors may have some non-specific effects on other factors, our mini screening identified new candidates contributing to period-length control in mammalian cells

Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells

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Fujita Masatoshi

2006-10-01

Full Text Available Abstract In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1 functional and SCFSkp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2 replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.

Growth factor involvement in tension-induced skeletal muscle growth

Science.gov (United States)

Vandenburgh, Herman H.

1993-01-01

Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

Science.gov (United States)

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Building the mammalian testis

DEFF Research Database (Denmark)

Svingen, Terje; Koopman, Peter

2013-01-01

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial...

Synthetic RNA Controllers for Programming Mammalian Cell Fate and Function

Science.gov (United States)

2015-11-04

Final report for “Synthetic RNA controllers for programming mammalian cell fate and function” Principal Investigator: Christina D. Smolke...SUBTITLE Synthetic RNA controllers for programming mammalian cell fate and function 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18   2 Synthetic RNA controllers for programming mammalian cell fate and function Task 1

Radiation induced reproductive death as a function of mammalian cell ploidy

International Nuclear Information System (INIS)

Philbrick, D.A.

1976-09-01

Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37 0 C, 4 0 C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated 3 HTdR and 125 IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37 0 C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death

DNA repair in non-mammalian animals

International Nuclear Information System (INIS)

Mitani, Hiroshi

1984-01-01

Studies on DNA repair have been performed using microorganisms such as Escherichia coli and cultured human and mammalian cells. However, it is well known that cultured organic cells differ from each other in many respects, although DNA repair is an extremely fundamental function of organisms to protect genetic information from environmental mutagens such as radiation and 0 radicals developing in the living body. To answer the question of how DNA repair is different between the animal species, current studies on DNA repair of cultured vertebrate cells using the methods similar to those in mammalian experiments are reviewed. (Namekawa, K.)

Incorporation of mammalian actin into microfilaments in plant cell nucleus

Directory of Open Access Journals (Sweden)

Paves Heiti

2004-04-01

Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

Amino acids in the cultivation of mammalian cells.

Science.gov (United States)

Salazar, Andrew; Keusgen, Michael; von Hagen, Jörg

2016-05-01

Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

Plasma treatment of mammalian vascular cells : A quantitative description

NARCIS (Netherlands)

Kieft, IE; Darios, D; Roks, AJM; Stoffels, E

For the first time, quantitative data was obtained on plasma treatment of living mammalian cells. The nonthermal atmospheric discharge produced by the plasma needle was used for treatment of mammalian endothelial and smooth muscle cells. The influence of several experimental parameters on cell

Plasma treatment of mammalian vascular cells: a quantitative description

NARCIS (Netherlands)

Kieft, I.E.; Darios, D.; Roks, A.J.M.; Stoffels - Adamowicz, E.

2005-01-01

For the first time, quantitative data was obtained on plasma treatment of living mammalian cells. The nonthermal atmospheric discharge produced by the plasma needle was used for treatment of mammalian endothelial and smooth muscle cells. The influence of several experimental parameters on cell

Mammalian Herbivores in the Boreal Forests: Their Numerical Fluctuations and Use by Man

Directory of Open Access Journals (Sweden)

Kjell Danell

1998-12-01

Full Text Available Within the boreal zone, there are about 50 native mammalian herbivore species that belong to the orders Artiodactyla, Rodentia, and Lagomorpha. Of these species, 31 occur in the Nearctic and 24 in the Palaearctic. Only six species occur in both regions. Species of the family Cervidae have probably been, and still are, the most important group for man, as they provide both meat and hides. Pelts from squirrels, muskrats, and hares were commercially harvested at the beginning of the century, but have less value today. The semi-domestic reindeer in the Palaearctic produces meat and hides on a commercial basis. It is also used for milking, to a limited extent, as is the semi-domestic moose in Russia. The Siberian musk deer is used for its musk and is raised in captivity in China. All species heavier than 1 kg are utilized by man, those with a body mass in the range 1 kg - 1 hg are sometimes used, and species lighter than 1 hg are rarely used. Here, we review the numerical fluctuations in terms of periodicity and amplitude, based on an extensive data set found in the literature, especially from the former Soviet Union. Current understanding of the underlying factors behind the population fluctuations is briefly reviewed. Management and conservation aspects of the mammalian herbivores in the boreal zone are also discussed. We conclude that there is a challenge to manage the forests for the mammalian herbivores, but there is also a challenge to manage the populations of mammalian herbivores for the forests.

Mammalian collection on Noah's Ark: the effects of beauty, brain and body size.

Directory of Open Access Journals (Sweden)

Daniel Frynta

Full Text Available The importance of today's zoological gardens as the so-called "Noah's Ark" grows as the natural habitat of many species quickly diminishes. Their potential to shelter a large amount of individuals from many species gives us the opportunity to reintroduce a species that disappeared in nature. However, the selection of animals to be kept in zoos worldwide is highly selective and depends on human decisions driven by both ecological criteria such as population size or vulnerability and audience-driven criteria such as aesthetic preferences. Thus we focused our study on the most commonly kept and bred animal class, the mammals, and we asked which factors affect various aspects of the mammalian collection of zoos. We analyzed the presence/absence, population size, and frequency per species of each of the 123 mammalian families kept in the worldwide zoo collection. Our aim was to explain these data using the human-perceived attractiveness of mammalian families, their body weight, relative brain size and species richness of the family. In agreement with various previous studies, we found that the body size and the attractiveness of mammals significantly affect all studied components of the mammalian collection of zoos. There is a higher probability of the large and attractive families to be kept. Once kept, these animals are presented in larger numbers in more zoos. On the contrary, the relative mean brain size only affects the primary selection whether to keep the family or not. It does not affect the zoo population size or the number of zoos that keep the family.

Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein

OpenAIRE

Han, Seong-Jeong; Lee, Byung Cheon; Yim, Sun Hee; Gladyshev, Vadim N.; Lee, Seung-Rock

2014-01-01

Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells ...

Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

DEFF Research Database (Denmark)

Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

2010-01-01

Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer  Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc.1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... Hospital, Vejle, Denmark4Institute of Regional Health Services Research, University of Southern Denmark, Odense DenmarkBackgroundPrognostic and predictive markers are needed for individualizing the treatment of colorectal cancer. Hypoxia-inducible factor 1α (HIF-1α) is a transcription-inducing factor which...... the predictive and prognostic value of HIF-1α in colorectal cancer.Materials and MethodsThe project is divided into 3 substudies:1. Biological and methodological aspects. The expression of HIF-1α measured by immunohistochemistry in paraffin embedded tissue is related to single nucleotide polymorphism (SNP...

The effects of selenium on glutathione peroxidase activity and radioprotection in mammalian cells

International Nuclear Information System (INIS)

Diamond, A.M.; Murray, J.L.; Dale, P.; Tritz, R.; Grdina, D.J.

1995-01-01

The media of representative mammalian cell lines were supplemented with low levels of selenium in the form of sodium selenite in order to investigate the effects of selenium on mammalian cells. Following incubation in 30 nM sodium selenite, these cells were assayed for changes in glutathione peroxidase (GPx) activity. The cells examined included NIH 3T3 mouse fibroblasts, PC12 rat sympathetic precursor cells, SupT-1 human lymphocytes, MCF-7 adr human breast carcinoma cells and AA8 Chinese hamster ovary cells. Selenium supplementation resulted in a marginal increase in GPx activity for the NIH 3T3, MCF-7 adr and Supt-1 cells but stimulated GPx activity approximately 5-fold in PC12 and AA8 cells. AA8 cells were selected to evaluate whether selenium supplementation was radioprotective against 60 cobalt gamma irradiation. Protection against radiation-induced mutation was measured by evaluating mutation frequency at the hprt locus. In this assay, preincubation of AA8 CHO cells significantly protected these cells from exposure to 8 Gy

Next-generation mammalian genetics toward organism-level systems biology.

Science.gov (United States)

Susaki, Etsuo A; Ukai, Hideki; Ueda, Hiroki R

2017-01-01

Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

Science.gov (United States)

Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

1997-01-01

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

Mammalian DNA Repair. Final Report

Energy Technology Data Exchange (ETDEWEB)

Wood, Richard D.

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.

Mammalian Synthetic Biology: Engineering Biological Systems.

Science.gov (United States)

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.

Structure and function of stem cell pools in mammalian cell renewal systems

International Nuclear Information System (INIS)

Fliedner, T.M.; Nothdurft, W.

1979-01-01

Stem cells play a key-role in the maintenance of the equilibrium between cell loss and cell production in cell renewal systems as well as in the understanding of the radiation pathophysiology of mammalian organisms. The integrity of mammalian organisms with the need to maintain a constant ''millieu interior'' is depending on the normal functioning of cell renewal systems, especially those of epithelial surfaces and blood cell forming organs. All cell renewal systems of bodies have a very similar functional structure consisting of functional, proliferative - amplifying and stem cell compartments. They differ in transit and cell cycle times and in the number of amplification division - aside from the difference in their functional and biochemical make-up. The stem cell pools are providing the cells capable of differentiation without depleting their own kind. This can be achieved by symmetrical or assymmetrical stem cell division. In normal steady state, 50% of the stem cell division remain in the stem cell pool, while the other 50% leave it to differentiate, proliferate and mature, hemopoietic system is distributed throughout bodies. This is an important factor in the radiation biology of mammalian organisms since the loss of function in one area can be compensated for by more production in other areas, and locally depleted sites can be reseeded with the stem cells migrating in from blood. (Yamashita, S.)

Chemical determination of free radical-induced damage to DNA.

Science.gov (United States)

Dizdaroglu, M

1991-01-01

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

International Nuclear Information System (INIS)

Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

2014-01-01

Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

Positive Selection Linked with Generation of Novel Mammalian Dentition Patterns.

Science.gov (United States)

Machado, João Paulo; Philip, Siby; Maldonado, Emanuel; O'Brien, Stephen J; Johnson, Warren E; Antunes, Agostinho

2016-09-11

A diverse group of genes are involved in the tooth development of mammals. Several studies, focused mainly on mice and rats, have provided a detailed depiction of the processes coordinating tooth formation and shape. Here we surveyed 236 tooth-associated genes in 39 mammalian genomes and tested for signatures of selection to assess patterns of molecular adaptation in genes regulating mammalian dentition. Of the 236 genes, 31 (∼13.1%) showed strong signatures of positive selection that may be responsible for the phenotypic diversity observed in mammalian dentition. Mammalian-specific tooth-associated genes had accelerated mutation rates compared with older genes found across all vertebrates. More recently evolved genes had fewer interactions (either genetic or physical), were associated with fewer Gene Ontology terms and had faster evolutionary rates compared with older genes. The introns of these positively selected genes also exhibited accelerated evolutionary rates, which may reflect additional adaptive pressure in the intronic regions that are associated with regulatory processes that influence tooth-gene networks. The positively selected genes were mainly involved in processes like mineralization and structural organization of tooth specific tissues such as enamel and dentin. Of the 236 analyzed genes, 12 mammalian-specific genes (younger genes) provided insights on diversification of mammalian teeth as they have higher evolutionary rates and exhibit different expression profiles compared with older genes. Our results suggest that the evolution and development of mammalian dentition occurred in part through positive selection acting on genes that previously had other functions. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Development of a model system to study leukotriene-induced modification of radiation sensitivity in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Walden, Jr, T L; Holahan, Jr, E V; Catravas, G N

1986-01-01

Leukotrienes (LT) are an important class of biological mediators for which no information exists concerning their synthesis following a radiation insult or on their ability to modify cellular response to a subsequent radiation exposure. Results are presented which illustrate that the Chinese hamster lung fibroblast cell line, V79A03, is useful as a model system to study the metabolic fate of leukotrienes and the effect of LT on radiation sensitivity of mammalian cells in vitro. (U.K.).

Acoustophoretic Synchronization of Mammalian Cells in Microchannels

DEFF Research Database (Denmark)

Thévoz, P.; Adams, J.D.; Shea, H.

2010-01-01

We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel to selec......We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel...

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

DEFF Research Database (Denmark)

Tornøe, Jens; Kusk, P.; Johansen, T.E.

2002-01-01

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C......, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture....

Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

Science.gov (United States)

Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

2017-05-01

Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

Radiation induced reproductive death as a function of mammalian cell ploidy

Energy Technology Data Exchange (ETDEWEB)

Philbrick, D.A.

1976-09-01

Mammalian cells containing different multiples of the diploid chromosome set were created through drug induction and cell fusion. In all cell strains used the chromosome number was determined from metaphase spreads, as well as from DNA content and cell size. The survival of cells as a function of radiation dose was determined for cell lines with differing chromosome complements at 37/sup 0/C, 4/sup 0/C, in hypertonic media, while frozen, and with increasing levels of incorporated IUdR. Survival of frozen diploid and hypotetraploid Chinese hamster cells was determined following varying numbers of decays of incorporated /sup 3/HTdR and /sup 125/IUdR. The percent of reproductively viable cells following irradiation is a function of the cell ploidy, i.e., the number of haploid sets of chromosomes contained in the cell genome. At 37/sup 0/C and in hypertonic media, the Chinese hamster cells of progressively higher ploidies are increasingly sensitive to irradiation. As the number of chromosomes per unit cell volume increases the radiosensitivity increases. Both trends suggest interaction between chromosomes as an important cause of cell death.

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

International Nuclear Information System (INIS)

Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

1984-01-01

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

Four simple rules that are sufficient to generate the mammalian blastocyst

DEFF Research Database (Denmark)

Nissen, Silas Boye; Perera Pérez, Marta; Martin Gonzalez, Javier

2017-01-01

requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells’ competence of fibroblast growth factor (FGF)/extracellular signal—regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively......Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs...

Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

Science.gov (United States)

Rebollo, Rita; Mager, Dixie L

2016-01-01

Endogenous retroviruses are repetitive sequences found abundantly in mammalian genomes which are capable of modulating host gene expression. Nevertheless, most endogenous retrovirus copies are under tight epigenetic control via histone-repressive modifications and DNA methylation. Here we describe a common method used in our laboratory to detect, quantify, and compare mammalian endogenous retrovirus DNA methylation. More specifically we describe methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR.

SEPT12–NDC1 Complexes Are Required for Mammalian Spermiogenesis

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Tsung-Hsuan Lai

2016-11-01

Full Text Available Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12 is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identified as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1. NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12–NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the filament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck

Inducing and Aggravating Factors of Gastroesophageal Reflux Symptoms

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Radhiyatam Mardhiyah

2016-12-01

Full Text Available Gastroesophageal reflux disease (subsequently abbreviated as GERD is a disease commonly found in the community. Several factors have been recognized as inducing and aggravating factors of GERD symptoms such as older age, female gender, obesity, smoking habit, alcohol consumption, certain diet and poor eating habit like eating fatty, spicy, and acid food.

Hereditary Factors Involved in Radiation-Induced Leukaemogenesis

International Nuclear Information System (INIS)

Duplan, J.F.

1969-01-01

The hereditary factors involved in radiation-induced leukaemogenesis were studied in pure AKR and C57BL strains, their first-generation hybrids and their back-crosses. It is known that the heredity of spontaneous lymphoid leukaemias is attributable to hereditary factors, of which only some are chromosomal, and the same situation can be considered to exist as regards the heredity of radiation-induced leukoses. In order to identify the various chromosomal and non-chromosomal factors concerned, three types of experiment were conducted with the pure strains and with each of the crosses, intended to evaluate (a) the incidence of spontaneous lymphoid leukoses, (b) the incidence of radiation-induced leukoses and (c) the inhibition of radioleukaemo- genesis by the injection of isogenic haematopoietic cells. The results show that the main non-chromosomal factor is the leukaemogenic Gross virus (VG) in the case of the AKR strain and the radioleukaemia virus (VRL) in that of the C57BL strain; these two agents are transmitted by the mother to her progeny. The VG may be responsible for radioleukaemias as well as for spontaneous leukoses, but the VRL does not produce spontaneous leukaemias even in back-crosses possessing a substantial fraction of the AKR genome, which is particularly conducive to leukaemogenesis. Restoration using C57BL bone marrow brings about a distinct inhibition of leukaemogenesis in all animals deriving from crossings for which this material is histocompatible; AKR marrow, however, never exhibits any restorative activity. Three hypotheses may be put forward to explain these results. The first is that C57BL bone marrow contains many more precursor elements than AKR marrow, these cells being necessary for inhibition of the leukaemogenic process. The second hypothesis is that the AKR strain lacks a factor which is essential for the utilization of these precursors. Finally the third hypothesis, which seems the least probable, is that AKR cells are much more

A mammalianized synthetic nitroreductase gene for high-level expression

International Nuclear Information System (INIS)

Grohmann, Maik; Paulmann, Nils; Fleischhauer, Sebastian; Vowinckel, Jakob; Priller, Josef; Walther, Diego J

2009-01-01

The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans

Distributional congruence of mammalian herbivores in the Trans-Himalayan Mountains

NARCIS (Netherlands)

Namgail, T.; Wieren, van S.E.; Prins, H.H.T.

2013-01-01

Large-scale distribution and diversity patterns of mammalian herbivores, especially less charismatic species in alpine environments remain little understood. We studied distributional congruence of mammalian herbivores in the Trans-Himalayan region of Ladakh to see if the distributions of less

Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and Cas9 Nucleases

NARCIS (Netherlands)

Vriend, Lianne E. M.; Jasin, Maria; Krawczyk, Przemek M.

2014-01-01

Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the

Base Composition Characteristics of Mammalian miRNAs

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Bin Wang

2013-01-01

Full Text Available MicroRNAs (miRNAs are short RNA sequences that repress protein synthesis by either inhibiting the translation of messenger RNA (mRNA or increasing mRNA degradation. Endogenous miRNAs have been found in various organisms, including animals, plants, and viruses. Mammalian miRNAs are evolutionarily conserved, are scattered throughout chromosomes, and play an important role in the immune response and the onset of cancer. For this study, the author explored the base composition characteristics of miRNA genes from the six mammalian species that contain the largest number of known miRNAs. It was found that mammalian miRNAs are evolutionarily conserved and GU-rich. Interestingly, in the miRNA sequences investigated, A residues are clearly the most frequent occupants of positions 2 and 3 of the 5′ end of miRNAs. Unlike G and U residues that may pair with C/U and A/G, respectively, A residues can only pair with U residues of target mRNAs, which may augment the recognition specificity of the 5′ seed region.

The mammalian ovary from genesis to revelation.

Science.gov (United States)

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

2009-10-01

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.

Angiogenesis is induced by airway smooth muscle strain.

Science.gov (United States)

Hasaneen, Nadia A; Zucker, Stanley; Lin, Richard Z; Vaday, Gayle G; Panettieri, Reynold A; Foda, Hussein D

2007-10-01

Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

Science.gov (United States)

Qi, Li; Pujanauski, Lindsey M; Davis, A Sally; Schwartzman, Louis M; Chertow, Daniel S; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L; Slemons, Richard D; Walters, Kathie-Anne; Kash, John C; Taubenberger, Jeffery K

2014-11-18

Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections

Factors Associated with Induced Abortion among Women in Hohoe ...

African Journals Online (AJOL)

In Hohoe, Ghana, induced abortion is the second highest cause of hospital admissions. We aimed to describe factors influencing induced abortion among 408 randomly selected women aged 15-49 years. 21% of the women had had an abortion; of those, 36% said they did not want to disrupt their education or employment; ...

Using energy budgets to combine ecology and toxicology in a mammalian sentinel species

Science.gov (United States)

Desforges, Jean-Pierre W.; Sonne, Christian; Dietz, Rune

2017-04-01

Process-driven modelling approaches can resolve many of the shortcomings of traditional descriptive and non-mechanistic toxicology. We developed a simple dynamic energy budget (DEB) model for the mink (Mustela vison), a sentinel species in mammalian toxicology, which coupled animal physiology, ecology and toxicology, in order to mechanistically investigate the accumulation and adverse effects of lifelong dietary exposure to persistent environmental toxicants, most notably polychlorinated biphenyls (PCBs). Our novel mammalian DEB model accurately predicted, based on energy allocations to the interconnected metabolic processes of growth, development, maintenance and reproduction, lifelong patterns in mink growth, reproductive performance and dietary accumulation of PCBs as reported in the literature. Our model results were consistent with empirical data from captive and free-ranging studies in mink and other wildlife and suggest that PCB exposure can have significant population-level impacts resulting from targeted effects on fetal toxicity, kit mortality and growth and development. Our approach provides a simple and cross-species framework to explore the mechanistic interactions of physiological processes and ecotoxicology, thus allowing for a deeper understanding and interpretation of stressor-induced adverse effects at all levels of biological organization.

Role of hypoxia-inducible factor in diabetic myocardial hypertrophy ...

African Journals Online (AJOL)

Purpose: This study was carried out to investigate the role of hypoxia-inducible factor (HIF) in diabetic cardiomyopathy in vitro. Methods: Hypoxia was induced chemically in H9C2 cells (cardiac hypertrophy model), and the cells were treated with phenylephrine (PE), deferoxamine (DFO), PE + DFO, and HIF-1α siRNA under ...

Mitochondria are the target organelle of differentiation-inducing factor-3, an anti-tumor agent isolated from Dictyostelium discoideum [corrected].

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Yuzuru Kubohara

Full Text Available Differentiation-inducing factor-3 (DIF-3, found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3 are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY, and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5-20 µ M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 µM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1-3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.

Identification of human genes involved in cellular responses to ionizing radiation: molecular and cellular studies of gene encoding the p68 helicase in mammalian cells

International Nuclear Information System (INIS)

Menaa, F.

2003-12-01

Cells submitted to genotoxic factors -like IR- activate several and important mechanisms such as repair, cell cycle arrest or 'apoptosis' to maintain genetic integrity. So, the damaged cells will induce many and different genes. The human transcriptome analysis by 'SSH' method in a human breast carcinoma cell line MCF7 γ-irradiated versus not irradiated, allowed to identify about one hundred genes. Among of these genes, we have focused our study on a radio-induced gene encoding the p68 helicase. In the conditions of irradiation used, our results show that the kinetic and the regulation of this gene expression differs between the nature of radiations used. Indeed, in γ-irradiated mammalian cells, ATM, a protein kinase activated by DSB and IR, is required to induce quickly P68 gene via the important transcription factor p53 stabilized by IR. In the case of UVC-irradiated cells, the P68 gene induction is late and the intracellular signalling pathway that lead to this induction is independent from the p53 protein. Finally, we show that the p68 protein under-expression is responsible for an increased radiosensitivity of MCF7 cells. Consequently, we can postulate that the p68 protein is involved in cellular responses to radiations to reduce the increased radiosensitivity of cells exposed to γ-rays. (author)

Factors that modify risks of radiation-induced cancer

International Nuclear Information System (INIS)

Fabrikant, J.I.

1988-11-01

The collective influence of biologic and physical factors that modify risks of radiation-induced cancer introduces uncertainties sufficient to deny precision of estimates of human cancer risk that can be calculated for low-dose radiation in exposed populations. The important biologic characteristics include the tissue sites and cell types, baseline cancer incidence, minimum latent period, time-to-tumor recognition, and the influence of individual host (age and sex) and competing etiologic influences. Physical factors include radiation dose, dose rate, and radiation quality. Statistical factors include time-response projection models, risk coefficients, and dose-response relationships. Other modifying factors include other carcinogens, and other biological sources (hormonal status, immune status, hereditary factors)

The shape of mammalian phylogeny

DEFF Research Database (Denmark)

Purvis, Andy; Fritz, Susanne A; Rodríguez, Jesús

2011-01-01

an assemblage, ecoregion or larger area always tends to be more unbalanced than expected from the phylogeny of species at the next more inclusive spatial scale. We conclude with a verbal model of mammalian macroevolution, which emphasizes the importance to diversification of accessing new regions...

Technology of mammalian cell encapsulation

NARCIS (Netherlands)

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

2000-01-01

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

The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity

International Nuclear Information System (INIS)

Cole, Russell D.; Anderson, Gary L.; Williams, Phillip L.

2004-01-01

Fifteen organic phosphate pesticides were tested by computer tracking for their acute behavioral toxicity with the nematode Caenorhabditis elegans. Thirteen of these 15 chemicals are used as insecticides and are anticholesterase agents. The other two chemicals are used as herbicides. EC50 values for each chemical were compared to the corresponding LD50 acute lethality value in rats and mice. Order of toxicity was found to be significantly correlated in comparisons of C. elegans to both rats and mice. Mechanistic investigations were conducted by assaying 8 of the 15 chemicals for anticholinesterase activity in C. elegans. Significant cholinesterase inhibition was confirmed for five chemicals that had displayed high behavioral toxicity, while three chemicals of low behavioral toxicity showed no significant decrease in cholinesterase activity. Toxicity for two chemicals that do not inhibit cholinesterase in mammals was linked to pH effects. Detailed comparison of individual chemicals and metabolic issues are discussed. These results have positive implications for the use of C. elegans as a mammalian neurological model and support the use of C. elegans in early rounds of chemical toxicity screening

Mammalian RNA polymerase II core promoters: insights from genome-wide studies

DEFF Research Database (Denmark)

Sandelin, Albin; Carninci, Piero; Lenhard, Boris

2007-01-01

The identification and characterization of mammalian core promoters and transcription start sites is a prerequisite to understanding how RNA polymerase II transcription is controlled. New experimental technologies have enabled genome-wide discovery and characterization of core promoters, revealing...... in the mammalian transcriptome and proteome. Promoters can be described by their start site usage distribution, which is coupled to the occurrence of cis-regulatory elements, gene function and evolutionary constraints. A comprehensive survey of mammalian promoters is a major step towards describing...

Paraquat-induced radiosensitization of mammalian cells

International Nuclear Information System (INIS)

Miller, R.C.; Fujikura, Toshio; Hiraoka, Toshio; Tenou, Hiromi.

1983-06-01

The herbicide, paraquat (methyl viologen, 1-1' dimethy1-4, 4'-bipyridinium dichloride), stimulates the production of superoxide anion (O 2 sup(-.)) in aerobic cells and therefore mimics some effects of ionizing radiation. In addition, concentrations of cellular glutathione are reduced by reaction with O 2 sup(-.). It is reported here that paraquat, toxic in its own right to aerobic cells, acts as a radiosensitizer when cells are exposed to nontoxic concentrations of the drug prior to and during irradiation. The radiomimetic effect of paraquat, alone and in combination with X-rays, was examined. Paraquat affects aerated cells (hamster lung V79 cells) in a dose-dependent manner. Doses in excess of 1 mM for two hours cause significant cell killing. In combination with radiation, sublethal doses of paraquat, given for two hours prior to irradiation, enhance the lethal effects of radiation. However, if cells are exposed to the same concentration of paraquat following irradiation, no additional lethal effect is observed. Paraquat is a useful tool to study the effects of O 2 sup(-.) and may lead to better understanding of the mechanisms of radiation-induced energy deposition in cells. (author)

Adult Neurogenesis in the Mammalian Hippocampus: Why the Dentate Gyrus?

Science.gov (United States)

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…

Hypoxia-Inducible Factor-1α in carcinogenesis and progression of breast cancer

NARCIS (Netherlands)

Bos, R.

2004-01-01

This thesis is primarily focused on the previously hardly explored role of HIF-1 in breast cancer. HIF-1 is a transcription factor induced by hypoxia, but also by some oncogenes, tumor suppressor genes and growth factors. Activated HIF-1 can induce angiogenesis, glycolysis, erythropoiesis, and other

Perflurooctanoic Acid Induces Developmental Cardiotoxicity in Chicken Embryos and Hatchlings

Science.gov (United States)

Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxi...

Viral Cre-LoxP tools aid genome engineering in mammalian cells.

Science.gov (United States)

Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

2017-01-01

Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

An initial biochemical and cell biological characterization of the mammalian homologue of a central plant developmental switch, COP1

Directory of Open Access Journals (Sweden)

Wang Haiyang

2002-12-01

Full Text Available Abstract Background Constitutive photomorphogenic 1 (COP1 has been defined as a central regulator of photomorphogenic development in plants, which targets key transcription factors for proteasome-dependent degradation. Although COP1 mammalian homologue has been previously reported, its function and distribution in animal kingdom are not known. Results Here we report the characterization of full-length human and mouse COP1 cDNAs and the genomic structures of the COP1 genes from several different species. Mammalian COP1 protein binds to ubiquitinated proteins in vivo and is itself ubiquitinated. Furthermore, mammalian COP1 is predominately nuclear localized and exists primarily as a complex of over 700 kDa. Through mutagenesis studies, we have defined a leucine-rich nuclear export signal (NES within the coiled-coil domain of mammalian COP1 and a nuclear localization signal (NLS, which is composed of two clusters of positive-charged amino acids, bridged by the RING finger. Disruption of the RING finger structure abolishes the nuclear import, while deletion of the entire RING finger restores the nuclear import. Conclusions Our data suggest that mammalian COP1, similar to its plant homologue, may play a role in ubiquitination. Mammalian COP1 contains a classic leucine-rich NES and a novel bipartite NLS bridged by a RING finger domain. We propose a working model in which the COP1 RING finger functions as a structural scaffold to bring two clusters of positive-charged residues within spatial proximity to mimic a bipartite NLS. Therefore, in addition to its well-characterized role in ubiquitination, the RING finger domain may also play a structural role in nuclear import.

Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice: involvement of tissue factor, inducible NO synthase and nuclear factor (NF)-κB.

Science.gov (United States)

Sun, Qi; Chen, Ling; Gao, Mengyu; Jiang, Wenwen; Shao, Fangxian; Li, Jingjing; Wang, Jun; Kou, Junping; Yu, Boyang

2012-01-01

Acute lung injury is still a significant clinical problem with a high mortality rate and there are few effective therapies in clinic. Here, we studied the inhibitory effect of ruscogenin, an anti-inflammatory and anti-thrombotic natural product, on lipopolysaccharide (LPS)-induced acute lung injury in mice basing on our previous studies. The results showed that a single oral administration of ruscogenin significantly decreased lung wet to dry weight (W/D) ratio at doses of 0.3, 1.0 and 3.0 mg/kg 1 h prior to LPS challenge (30 mg/kg, intravenous injection). Histopathological changes such as pulmonary edema, coagulation and infiltration of inflammatory cells were also attenuated by ruscogenin. In addition, ruscogenin markedly decreased LPS-induced myeloperoxidase (MPO) activity and nitrate/nitrite content, and also downregulated expression of tissue factor (TF), inducible NO synthase (iNOS) and nuclear factor (NF)-κB p-p65 (Ser 536) in the lung tissue at three doses. Furthermore, ruscogenin reduced plasma TF procoagulant activity and nitrate/nitrite content in LPS-induced ALI mice. These findings confirmed that ruscogenin significantly attenuate LPS-induced acute lung injury via inhibiting expressions of TF and iNOS and NF-κB p65 activation, indicating it as a potential therapeutic agent for ALI or sepsis. Copyright © 2011 Elsevier B.V. All rights reserved.

Bioenergetics of mammalian sperm capacitation.

Science.gov (United States)

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.

Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

Science.gov (United States)

Mitani, Akihisa; Ito, Kazuhiro; Vuppusetty, Chaitanya; Barnes, Peter J; Mercado, Nicolas

2016-01-15

Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae.

Science.gov (United States)

Muzaffar, Suhail; Chattoo, Bharat B

2017-03-01

Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.

Mammalian-specific genomic functions: Newly acquired traits generated by genomic imprinting and LTR retrotransposon-derived genes in mammals.

Science.gov (United States)

Kaneko-Ishino, Tomoko; Ishino, Fumitoshi

2015-01-01

Mammals, including human beings, have evolved a unique viviparous reproductive system and a highly developed central nervous system. How did these unique characteristics emerge in mammalian evolution, and what kinds of changes did occur in the mammalian genomes as evolution proceeded? A key conceptual term in approaching these issues is "mammalian-specific genomic functions", a concept covering both mammalian-specific epigenetics and genetics. Genomic imprinting and LTR retrotransposon-derived genes are reviewed as the representative, mammalian-specific genomic functions that are essential not only for the current mammalian developmental system, but also mammalian evolution itself. First, the essential roles of genomic imprinting in mammalian development, especially related to viviparous reproduction via placental function, as well as the emergence of genomic imprinting in mammalian evolution, are discussed. Second, we introduce the novel concept of "mammalian-specific traits generated by mammalian-specific genes from LTR retrotransposons", based on the finding that LTR retrotransposons served as a critical driving force in the mammalian evolution via generating mammalian-specific genes.

Fish Lymphocytes: An Evolutionary Equivalent of Mammalian Innate-Like Lymphocytes?

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Giuseppe Scapigliati

2018-05-01

Full Text Available Lymphocytes are the responsible of adaptive responses, as they are classically described, but evidence shows that subpopulations of mammalian lymphocytes may behave as innate-like cells, engaging non-self rapidly and without antigen presentation. The innate-like lymphocytes of mammals have been mainly identified as γδT cells and B1-B cells, exert their activities principally in mucosal tissues, may be involved in human pathologies and their functions and tissue(s of origin are not fully understood. Due to similarities in the morphology and immunobiology of immune system between fish and mammals, and to the uniqueness of having free-living larval stages where the development can be precisely monitored and engineered, teleost fish are proposed as an experimental model to investigate human immunity. However, the homology between fish lymphocytes and mammalian innate-like lymphocytes is an issue poorly considered in comparative immunology. Increasing experimental evidence suggests that fish lymphocytes could have developmental, morphological, and functional features in common with innate-like lymphocytes of mammals. Despite such similarities, information on possible links between conventional fish lymphocytes and mammalian innate-like lymphocytes is missing. The aim of this review is to summarize and describe available findings about the similarities between fish lymphocytes and mammalian innate-like lymphocytes, supporting the hypothesis that mammalian γδT cells and B1-B cells could be evolutionarily related to fish lymphocytes.

Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

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Masayuki Nakano

Full Text Available Branched-chain amino acids (BCAAs have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

Dynamics of radioecological and genetic processes in populations of mammalian model species at contamination of ecosystems

International Nuclear Information System (INIS)

Ryabokon', N.I.; Goncharova, R.I.

2008-01-01

A short review of data on the time course of radiobiological and genetic processes in natural populations of mammalian model species inhabiting radiocontaminated ecosystems over many generations is presented here. The described time-courses of biological end-points in these populations do not reflect the time course of the whole-body dose rates, but do the outcome of multiple processes, including the direct response to individual irradiation, the transgeneration transmission and accumulation of induced damages and the development of adaptation. (authors)

La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts.

Science.gov (United States)

Blakqori, Gjon; Delhaye, Sophie; Habjan, Matthias; Blair, Carol D; Sánchez-Vargas, Irma; Olson, Ken E; Attarzadeh-Yazdi, Ghassem; Fragkoudis, Rennos; Kohl, Alain; Kalinke, Ulrich; Weiss, Siegfried; Michiels, Thomas; Staeheli, Peter; Weber, Friedemann

2007-05-01

La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

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Zaagsma Johan

2005-07-01

Full Text Available Abstract Background In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction. Methods Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK and cyclooxygenase (COX to these reponses was established, using the inhibitors Y-27632 (1 μM, U-0126 (3 μM and indomethacin (3 μM, respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α and prostaglandin E2 (PGE2 was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM and the selective EP1-antagonist AH-6809 (10 μM on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay. Results Epidermal growth factor (EGF-and platelet-derived growth factor (PDGF-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM significantly

Formation and repair of gamma-ray induced nucleic acid base damage in bacteria and mammalian cells. Final report, September 1, 1973--August 31, 1976

International Nuclear Information System (INIS)

Cerutti, P.A.

1976-01-01

Results are summarized from a three-year study of the formation and repair of γ-ray induced thymine damage in bacteria and mammalian cells. A systematic study was made of the formation of a specific type of ionizing radiation induced base damage under in vivo conditions. Assay for the determination of γ-ray products of the 5,6-dihydroxy-dihydrothymine type (alkaline-acid degradation assay) and a method for the determination of the formation of 5-methylene-uracil radicals (formation of ( 3 H)H 2 O from thymine-methyl ( 3 H)) are discussed. The radiation-chemical reactivity of thymine decreased according to the following pattern in different biological systems: phi X174-DNA greater than E. coli DNA = phi X174 phage much greater than HeLa chromatin greater than E. coli cells greater than human fibroblasts WI-38. In WI-38 the efficiency of formation of 5-methylene-uracil radicals was 1.6 x 10 -3 per Krad and 10 6 daltons DNA and of products of the 5,6-dihydroxy-dihydrothymine type 0.54 x 10 -3 per Krad per 10 6 daltons DNA (uncorrected). It was concluded that γ-rays produce DNA single strand breaks and (total) base damage with comparable efficiencies under in vivo conditions in cultured cells. A list is included of 18 published papers that report the findings in detail

The architecture of mammalian ribosomal protein promoters

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Perry Robert P

2005-02-01

Full Text Available Abstract Background Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. Results A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y2C+1TY(T2(Y3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. Conclusions This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.

Functional role of CCCTC binding factor (CTCF) in stress-induced apoptosis

International Nuclear Information System (INIS)

Li Tie; Lu Luo

2007-01-01

CTCF, a nuclear transcriptional factor, is a multifunctional protein and involves regulation of growth factor- and cytokine-induced cell proliferation/differentiation. In the present study, we investigated the role of CTCF in protecting stress-induced apoptosis in various human cell types. We found that UV irradiation and hyper-osmotic stress induced human corneal epithelial (HCE) and hematopoietic myeloid cell apoptosis detected by significantly increased caspase 3 activity and decreased cell viability. The stress-induced apoptotic response in these cells requires down-regulation of CTCF at both mRNA and protein levels, suggesting that CTCF may play an important role in downstream events of stress-induced signaling pathways. Inhibition of NFκB activity prevented stress-induced down-regulation of CTCF and increased cell viability against stress-induced apoptosis. The anti-apoptotic effect of CTCF was further studied by manipulating CTCF activities in HCE and hematopoietic cells. Transient transfection of cDNAs encoding full-length human CTCF markedly suppressed stress-induced apoptosis in these cells. In contrast, knocking down of CTCF mRNA using siRNA specific to CTCF significantly promoted stress-induced apoptosis. Thus, our results reveal that CTCF is a down stream target of stress-induced signaling cascades and it plays a significant anti-apoptotic role in regulation of stress-induced cellular responses in HCE and hematopoietic myeloid cells

Systemic anti-tumor necrosis factor antibody treatment exacerbates endotoxin-induced uveitis in the rat

NARCIS (Netherlands)

de Vos, A. F.; van Haren, M. A.; Verhagen, C.; Hoekzema, R.; Kijlstra, A.

1995-01-01

Tumor necrosis factor is released in the circulation and aqueous humor during endotoxin-induced uveitis, and induces acute uveitis when injected intraocularly in rats. To elucidate the role of tumor necrosis factor in the development of endotoxin-induced uveitis we analysed the effect of

Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

Science.gov (United States)

Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-08-01

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.

Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

OpenAIRE

Qi, Li; Pujanauski, Lindsey M.; Davis, A. Sally; Schwartzman, Louis M.; Chertow, Daniel S.; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L.; Slemons, Richard D.; Walters, Kathie-Anne; Kash, John C.; Taubenberger, Jeffery K.

2014-01-01

ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backb...

Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

Science.gov (United States)

Zhang, Qiuxia; Na, Quan; Song, Weiwei

2017-10-01

Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

Role of cyclins in controlling progression of mammalian spermatogenesis

OpenAIRE

WOLGEMUTH, DEBRA J.; MANTEROLA, MARCIA; VASILEVA, ANA

2013-01-01

Cyclins are key regulators of the mammalian cell cycle, functioning primarily in concert with their catalytic partners, the cyclin-dependent kinases (Cdks). While their function during mitosis in somatic cells has been extensively documented, their function during both mitosis and meiosis in the germ line is poorly understood. From the perspective of cell cycle regulation there are several aspects of mammalian spermatogenesis that suggest unique modes of regulation and hence, possible unique ...

Antioxidation activities of pteridines in mammalian cell lines

Energy Technology Data Exchange (ETDEWEB)

Zhang, Y.; Shen, R. (Univ. of Texas, Galveston (United States))

1991-03-11

L-erythro-5,6,7,8-Tetrahydrobiopterin (BH{sub 4}), the cofactor for aromatic amino acid hydroxylases (AAA-H), is a predominant form of pteridines which occur ubiquitously in nature. When BH{sub 4} is oxidized to quinonoid dihydrobiopterin by AAA-H, it is regenerated by dihydropteridine reductase (DHPR) at the expense of NADH. The role of BH{sub 4} other than serving as the hydroxylase cofactor is not clear. The existence of BH{sub 4} and DHPR in tissues which are devoid of AAA-H suggests that BH{sub 4} may play an as yet undiscovered physiological function. This study demonstrates a BH{sub 4}-mediated antioxidation system, which consists of BH{sub 4}, DHPR, peroxidase and NADH in rat pheochromocytoma PC 12 cells and mouse macrophages J774A.1. This system was as effective as catalase and ascorbic acid in protecting cells against H{sub 2}O{sub 2} and xanthine/xanthine oxidase-induced toxicity and was more effective than catalase in defense against nitrofurantoin-induced toxicity. The antioxidation effect of this system was not due to peroxidase and was improved when synthetic pteridines were substituted for BH{sub 4}. Since BH{sub 4}, DHPR, peroxidases and NADH are widely distributed in major organs and blood cells, they may constitute an as yet little known antioxidation system in mammalian cells.

Comparison of amphibian and mammalian thyroperoxidase ...

Science.gov (United States)

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

Familial factors responsible for persistent crying-induced asthma: a case report.

Science.gov (United States)

Weinstein, A G

1987-10-01

Crying behavior of the asthmatic child may induce wheezing symptoms. This may be a clinical problem for families with asthmatic children who exhibit frequent and persistent crying behavior. This case report identifies behaviors by the child and parents that may be responsible for continual crying. Child factors include (1) "spoiled" personality, (2) poor self-image, (3) biologic sensitivity to foods, medication, and environmental allergens producing irritability. Parental factors include poor disciplinary practices secondary to (1) disrupted home life, (2) guilt, and (3) overprotective behavior. Identification of these factors may be helpful in establishing clinical management strategies to reduce crying-induced asthma.

mammalian brain system

Directory of Open Access Journals (Sweden)

Alan Kania

2014-06-01

Full Text Available Relaxin-3, a member of the relaxin peptide family, was discovered in 2001 as a homologue of relaxin – a well-known reproductive hormone. However, it is the brain which turned out to be a major expression site of this newly discovered peptide. Both its molecular structure and expression pattern were shown to be very conserved among vertebrates. Extensive research carried out since the discovery of relaxin-3 contributed to the significant progress in our knowledge regarding this neuropeptide. The endogenous relaxin-3 receptor (RXFP3 was identified and the anatomy of the yet uncharacterized mammalian brain system was described, with nucleus incertus as the main center of relaxin-3 expression. Not only its diffusive projections throughout the whole brain, which reach various brain structures such as the hippocampus, septum, intergeniculate leaflet or amygdala, but also functional studies of the relaxin-3/RXFP3 signaling system, allowed this brain network to be classified as one of the ascending nonspecific brain systems. Thus far, research depicts the connection of relaxin-3 with phenomena such as feeding behavior, spatial memory, sleep/wake cycle or modulation of pituitary gland hormone secretion. Responsiveness of relaxin-3 neurons to stress factors and the strong orexigenic effect exerted by this peptide suggest its participation in modulation of feeding by stress, in particular of the chronic type. The discovery of relaxin-3 opened a new research field which will contribute to our better understanding of the neurobiological basis of feeding disorders.

[Importance of age and other risk factors in NSAID-induced gastropathy].

Science.gov (United States)

Lee, Hang Lak; Han, Dong Soo; Kim, Jin Bae; Kim, Jong Pyo; Jeon, Yong Chul; Sohn, Joo Hyun; Hahm, Joon Soo

2004-11-01

It is clinically important to analyze the risk factors of NSAID-induced gastropathy because there could be no symptoms. Age is the most important risk factor according to previous reports. The aim of this study was to find risk factors of NSAID-induced gastropathy and to confirm the association between NSAID-induced gastropathy and age. We retrospectively assessed 300 patients who conducted an upper gastroscopy during the course of chronic NSAID treatment. Median age of patients group is 51.4 +/- 12.2 years. In multivariate analysis, age and ulcer history are two significant risk factors. Median age is 46.7 +/- 10.7 years for the patients with nonspecific gastroscopic finding, 53.0 +/- 12.5 for those with erosion, 57.6 +/- 10.0 for those with ulcer, and 63.2 +/- 8.9 for those with hemorrhage. The proportion of ulcer patients is as follows: 6% in the patients of under 40 years old, 14.9% in patients of the 40s, 20% in patients of the 50s, 30.9% in patients of the 60s, 33.3% in patients over 70 years. The proportion of nonspecific findings is 62.2% in patients of the 40s, 37.8% in patients of the 50s, and 29% in patients over 60 years. Age is the most important risk factor of the NSAID-induced gastrointestinal mucosal injury. A larger randomized prospective control study will be required in the future for more conclusive results.

Do prey densities determine preferences of mammalian predators for habitat edges in an agricultural landscape?

Czech Academy of Sciences Publication Activity Database

Šálek, Martin; Kreisinger, Jakub; Sedláček, František; Albrecht, Tomáš

2010-01-01

Roč. 98, č. 2 (2010), s. 86-91 ISSN 0169-2046 R&D Projects: GA MŠk LC06073; GA MŠk 1P05OC078 Institutional research plan: CEZ:AV0Z60870520; CEZ:AV0Z60930519 Keywords : mammalian predators * fragmentation * small mammals * edge effect * predator–prey * distribution * Mustelids Subject RIV: EH - Ecology, Behaviour Impact factor: 2.004, year: 2010

Mammalian Sperm Motility: Observation and Theory

KAUST Repository

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

2011-01-01

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

1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

International Nuclear Information System (INIS)

NONE

1999-01-01

This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair

1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

Energy Technology Data Exchange (ETDEWEB)

NONE

1999-02-12

This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.

Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

Directory of Open Access Journals (Sweden)

Lv Y

2015-07-01

Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

Evolutionary dynamics of mammalian karyotypes

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Carlo Alberto Redi

2012-12-01

Full Text Available This special volume of Cytogenetic and Genome Research (edited by Roscoe Stanyon, University of Florence and Alexander Graphodatsky, Siberian division of the Russian Academy of Sciences is dedicated to the fascinating long search of the forces behind the evolutionary dynamics of mammalian karyotypes, revealed after the hypotonic miracle of the 1950s....

A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications.

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Shu-Bai Liu

2008-03-01

Full Text Available In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens betagamma-crystallin (alpha-subunit and trefoil factor (beta-subunit, named betagamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains, with a non-covalently linked form of alphabeta(2, show significant sequence homology to ep37 proteins, a group of non-lens betagamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. betagamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. betagamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+ efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, betagamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of betagamma-CAT (25-50 pM were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50 10 nM and apoptosis. betagamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase and nocodazole (an agent of microtuble depolymerizing, while inhibited betagamma-CAT induced vacuole formation, significantly inhibited betagamma-CAT induced cell detachment, suggesting

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.

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Katona, Robert L

2015-02-01

Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.

Masticatory loading, function, and plasticity: a microanatomical analysis of mammalian circumorbital soft-tissue structures.

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Jasarević, Eldin; Ning, Jie; Daniel, Ashley N; Menegaz, Rachel A; Johnson, Jeffrey J; Stack, M Sharon; Ravosa, Matthew J

2010-04-01

In contrast to experimental evidence regarding the postorbital bar, postorbital septum, and browridge, there is exceedingly little evidence regarding the load-bearing nature of soft-tissue structures of the mammalian circumorbital region. This hinders our understanding of pronounced transformations during primate origins, in which euprimates evolved a postorbital bar from an ancestor with the primitive mammalian condition where only soft tissues spanned the lateral orbital margin between frontal bone and zygomatic arch. To address this significant gap, we investigated the postorbital microanatomy of rabbits subjected to long-term variation in diet-induced masticatory stresses. Rabbits exhibit a masticatory complex and feeding behaviors similar to primates, yet retain a more primitive mammalian circumorbital region. Three cohorts were obtained as weanlings and raised on different diets until adult. Following euthanasia, postorbital soft tissues were dissected away, fixed, and decalcified. These soft tissues were divided into inferior, intermediate, and superior units and then dehydrated, embedded, and sectioned. H&E staining was used to characterize overall architecture. Collagen orientation and complexity were evaluated via picrosirius-red staining. Safranin-O identified proteoglycan content with additional immunostaining performed to assess Type-II collagen expression. Surprisingly, the ligament along the lateral orbital wall was composed of elastic fibrocartilage. A more degraded organization of collagen fibers in this postorbital fibrocartilage is correlated with increased masticatory forces due to a more fracture-resistant diet. Furthermore, the lack of marked changes in the extracellular composition of the lateral orbital wall related to tissue viscoelasticity suggests it is unlikely that long-term exposure to elevated masticatory stresses underlies the development of a bony postorbital bar. (c) 2010 Wiley-Liss, Inc.

The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

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Mehtap Kilic Eren

Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

Proinflammatory Factors Mediate Paclitaxel-Induced Impairment of Learning and Memory

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Zhao Li

2018-01-01

Full Text Available The chemotherapeutic agent paclitaxel is widely used for cancer treatment. Paclitaxel treatment impairs learning and memory function, a side effect that reduces the quality of life of cancer survivors. However, the neural mechanisms underlying paclitaxel-induced impairment of learning and memory remain unclear. Paclitaxel treatment leads to proinflammatory factor release and neuronal apoptosis. Thus, we hypothesized that paclitaxel impairs learning and memory function through proinflammatory factor-induced neuronal apoptosis. Neuronal apoptosis was assessed by TUNEL assay in the hippocampus. Protein expression levels of tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β in the hippocampus tissue were analyzed by Western blot assay. Spatial learning and memory function were determined by using the Morris water maze (MWM test. Paclitaxel treatment significantly increased the escape latencies and decreased the number of crossing in the MWM test. Furthermore, paclitaxel significantly increased the number of TUNEL-positive neurons in the hippocampus. Also, paclitaxel treatment increased the expression levels of TNF-α and IL-1β in the hippocampus tissue. In addition, the TNF-α synthesis inhibitor thalidomide significantly attenuated the number of paclitaxel-induced TUNEL-positive neurons in the hippocampus and restored the impaired spatial learning and memory function in paclitaxel-treated rats. These data suggest that TNF-α is critically involved in the paclitaxel-induced impairment of learning and memory function.

Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

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Mahendran Botlagunta

2011-03-01

Full Text Available DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

Vertebrate brains and evolutionary connectomics: on the origins of the mammalian ‘neocortex’

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Karten, Harvey J.

2015-01-01

The organization of the non-mammalian forebrain had long puzzled neurobiologists. Unlike typical mammalian brains, the telencephalon is not organized in a laminated ‘cortical’ manner, with distinct cortical areas dedicated to individual sensory modalities or motor functions. The two major regions of the telencephalon, the basal ventricular ridge (BVR) and the dorsal ventricular ridge (DVR), were loosely referred to as being akin to the mammalian basal ganglia. The telencephalon of non-mammalian vertebrates appears to consist of multiple ‘subcortical’ groups of cells. Analysis of the nuclear organization of the avian brain, its connections, molecular properties and physiology, and organization of its pattern of circuitry and function relative to that of mammals, collectively referred to as ‘evolutionary connectomics’, revealed that only a restricted portion of the BVR is homologous to the basal ganglia of mammals. The remaining dorsal regions of the DVR, wulst and arcopallium of the avian brain contain telencephalic inputs and outputs remarkably similar to those of the individual layers of the mammalian ‘neocortex’, hippocampus and amygdala, with instances of internuclear connections strikingly similar to those found between cortical layers and within radial ‘columns’ in the mammalian sensory and motor cortices. The molecular properties of these ‘nuclei’ in birds and reptiles are similar to those of the corresponding layers of the mammalian neocortex. The fundamental pathways and cell groups of the auditory, visual and somatosensory systems of the thalamus and telencephalon are homologous at the cellular, circuit, network and gene levels, and are of great antiquity. A proposed altered migration of these homologous neurons and circuits during development is offered as a mechanism that may account for the altered configuration of mammalian telencephalae. PMID:26554047

Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer

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Kristine von Maltzan

2016-02-01

Full Text Available The Slug transcription factor plays an important role in ultraviolet radiation (UVR-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2 pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.

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Harlow, Philippa H; Perry, Simon J; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A; Flemming, Anthony J

2016-03-18

To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives Activate Hypoxia-Inducible Factor via Inhibition of Factor Inhibiting Hypoxia-Inducible Factor-1

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Shin-ichi Kawaguchi

2018-04-01

Full Text Available Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions, many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1 has also been shown to have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were evaluated in SK-N-BE(2c cells by measuring HIF response element (HRE promoter activity. Several furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological HIF-α/HRE transcriptional activity.

Macrophage migration inhibitory factor induces vascular leakage via autophagy

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Hong-Ru Chen

2015-01-01

Full Text Available Vascular leakage is an important feature of acute inflammatory shock, which currently has no effective treatment. Macrophage migration inhibitory factor (MIF is a pro-inflammatory cytokine that can induce vascular leakage and plays an important role in the pathogenesis of shock. However, the mechanism of MIF-induced vascular leakage is still unclear. In this study, using recombinant MIF (rMIF, we demonstrated that MIF induced disorganization and degradation of junction proteins and increased the permeability of human endothelial cells in vitro. Western blotting analysis showed that rMIF treatment induced LC3 conversion and p62 degradation. Inhibition of autophagy with a PI3K inhibitor (3-MA, a ROS scavenger (NAC or autophagosomal-lysosomal fusion inhibitors (bafilomycin A1 and chloroquine rescued rMIF-induced vascular leakage, suggesting that autophagy mediates MIF-induced vascular leakage. The potential involvement of other signaling pathways was also studied using different inhibitors, and the results suggested that MIF-induced vascular leakage may occur through the ERK pathway. In conclusion, we showed that MIF triggered autophagic degradation of endothelial cells, resulting in vascular leakage. Inhibition of MIF-induced autophagy may provide therapeutic targets against vascular leakage in inflammatory shock.

The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety

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Juozaityte, Vaida; Pladevall-Morera, David; Podolska, Agnieszka

2017-01-01

Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in Caenorhabditis elegans Here we report that the C. elegans transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety......-induced quiescence. Nutritional status has a major influence on C. elegans behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food, C. elegans become sated and enter quiescence. We show......-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in C. elegans, and establish a paradigm for the elucidation of obesity-relevant mechanisms....

The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

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Appukuttan, Binoy; McFarland, Trevor J.; Stempel, Andrew; Kassem, Jean B.; Hartzell, Matthew; Zhang, Yi; Bond, Derek; West, Kelsey; Wilson, Reid; Stout, Andrew; Pan, Yuzhen; Ilias, Hoda; Robertson, Kathryn; Klein, Michael L.; Wilson, David; Smith, Justine R.; Stout, J. Timothy

2012-01-01

Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases. PMID:22761647

Quantification of mammalian tumor cell state plasticity with digital holographic cytometry

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Hejna, Miroslav; Jorapur, Aparna; Zhang, Yuntian; Song, Jun S.; Judson, Robert L.

2018-02-01

Individual cells within isogenic tumor populations can exhibit distinct cellular morphologies, behaviors, and molecular profiles. Cell state plasticity refers to the propensity of a cell to transition between these different morphologies and behaviors. Elevation of cell state plasticity is thought to contribute to critical stages in tumor evolution, including metastatic dissemination and acquisition of therapeutic resistance. However, methods for quantifying general plasticity in mammalian cells remain limited. Working with a HoloMonitor M4 digital holographic cytometry platform, we have established a machine learning-based pipeline for high accuracy and label-free classification of adherent cells. We use twenty-six morphological and optical density-derived features for label-free identification of cell state in heterogeneous cultures. The system is housed completely within a mammalian cell incubator, permitting the monitoring of changes in cell state over time. Here we present an application of our approach for studying cell state plasticity. Human melanoma cell lines of known metastatic potential were monitored in standard growth conditions. The rate of feature change was quantified for each individual cell in the populations. We observed that cells of higher metastatic potential exhibited more rapid fluctuation of cell state in homeostatic conditions. The approach we demonstrate will be advantageous for further investigations into the factors that influence cell state plasticity.

Evaluation of Mammalian Interspersed Repeats to investigate the goat genome

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P. Mariani

2010-01-01

Full Text Available Among the repeated sequences present in most eukaryotic genomes, SINEs (Short Interspersed Nuclear Elements are widely used to investigate evolution in the mammalian order (Buchanan et al., 1999. One family of these repetitive sequences, the MIR (Mammalian Interspersed Repeats; Jurka et al., 1995, is ubiquitous in all mammals.MIR elements are tRNA-derived SINEs and are identifiable by a conserved core region of about 70 nucleotides.

Structure and function of mammalian cilia

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Satir, Peter; Christensen, Søren T

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...

Troglitazone induces differentiation in Trypanosoma brucei

International Nuclear Information System (INIS)

Denninger, Viola; Figarella, Katherine; Schoenfeld, Caroline; Brems, Stefanie; Busold, Christian; Lang, Florian; Hoheisel, Joerg; Duszenko, Michael

2007-01-01

Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor γ. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator

Mel-18, a mammalian Polycomb gene, regulates angiogenic gene expression of endothelial cells.

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Jung, Ji-Hye; Choi, Hyun-Jung; Maeng, Yong-Sun; Choi, Jung-Yeon; Kim, Minhyung; Kwon, Ja-Young; Park, Yong-Won; Kim, Young-Myeong; Hwang, Daehee; Kwon, Young-Guen

2010-10-01

Mel-18 is a mammalian homolog of Polycomb group (PcG) genes. Microarray analysis revealed that Mel-18 expression was induced during endothelial progenitor cell (EPC) differentiation and correlates with the expression of EC-specific protein markers. Overexpression of Mel-18 promoted EPC differentiation and angiogenic activity of ECs. Accordingly, silencing Mel-18 inhibited EC migration and tube formation in vitro. Gene expression profiling showed that Mel-18 regulates angiogenic genes including kinase insert domain receptor (KDR), claudin 5, and angiopoietin-like 2. Our findings demonstrate, for the first time, that Mel-18 plays a significant role in the angiogenic function of ECs by regulating endothelial gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

Mammalian Sperm Motility: Observation and Theory

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Gaffney, E.A.

2011-01-21

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. © 2011 by Annual Reviews. All rights reserved.

Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione.

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Wiraswati, Hesti Lina; Hangen, Emilie; Sanz, Ana Belén; Lam, Ngoc-Vy; Reinhardt, Camille; Sauvat, Allan; Mogha, Ariane; Ortiz, Alberto; Kroemer, Guido; Modjtahedi, Nazanine

2016-11-22

Mitochondrial apoptosis inducing factor (AIF) is a redox-active enzyme that participates to the biogenesis/maintenance of complex I of the respiratory chain, yet also contributes to catabolic reactions in the context of regulated cell death when AIF translocates to the cytosol and to the nucleus. Here we explore the contribution of AIF to cell death induced by menadione (2-methyl-1,4-naphtoquinone; also called vitamin K3) in conditions in which this pro-oxidant does not cause the mitochondrial release of AIF, yet causes caspase-independent cell killing. Depletion of AIF from human cancer cells reduced the cytotoxicity of menadione. This cytoprotective effect was accompanied by the maintenance of high levels of reduced glutathione (GSH), which are normally depleted by menadione. In addition, AIF depletion reduced the arylation of cellular proteins induced by menadione. This menadione-triggered arylation, which can be measured by a fluorescence assay, is completely suppressed by addition of exogenous glutathione or N-acetyl cysteine. Complex I inhibition by Rotenone did not mimic the cytoprotective action of AIF depletion. Altogether, these results are compatible with the hypothesis that mitochondrion-sessile AIF facilitates lethal redox cycling of menadione, thereby precipitating protein arylation and glutathione depletion.

Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

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Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

2017-04-01

To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

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Velia Siciliano

2011-06-01

Full Text Available Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL by generating a clonal population of mammalian cells (CHO carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA, whose expression is regulated by a tTA responsive promoter (CMV-TET, thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP, thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL, by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off, and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the "switch off" times, as compared to the non-autoregulated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour.

Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

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Siciliano, Velia; Menolascina, Filippo; Marucci, Lucia; Fracassi, Chiara; Garzilli, Immacolata; Moretti, Maria Nicoletta; di Bernardo, Diego

2011-06-01

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the "switch off" times, as compared to the non-autoregulated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour.

Archetype, adaptation and the mammalian heart

NARCIS (Netherlands)

Meijler, F.L.; Meijler, T.D.

2011-01-01

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

The fine-scale utilization of forest edges by mammalian mesopredators related to patch size and conservation issues in Central European farmland

Czech Academy of Sciences Publication Activity Database

Červinka, J.; Šálek, Martin; Pavluvčík, P.; Kreisinger, J.

2011-01-01

Roč. 20, č. 14 (2011), s. 3459-3475 ISSN 0960-3115 R&D Projects: GA MŠk LC06073 Keywords : Edge effect * Fragment size * Mammalian mesopredators * Fragmentation * Scent stations * Czech Republic Subject RIV: EH - Ecology, Behaviour Impact factor: 2.238, year: 2011

Factors associated with repeat induced abortion in Kenya.

Science.gov (United States)

Maina, Beatrice W; Mutua, Michael M; Sidze, Estelle M

2015-10-12

Over six million induced abortions were reported in Africa in 2008 with over two million induced abortions occurring in Eastern Africa. Although a significant proportion of women in the region procure more than one abortion during their reproductive period, there is a dearth of research on factors associated with repeat abortion. Data for this study come from the Magnitude and Incidence of Unsafe Abortion Study conducted by the African Population and Health Research Center in Kenya in 2012. The study used a nationally-representative sample of 350 facilities (level II to level VI) that offer post-abortion services for complications following induced and spontaneous abortions. A prospective morbidity survey tool was used by health providers in 328 facilities to collect information on socio-demographic charateristics, reproductive health history and contraceptive use at conception for all patients presenting for post-abortion services. Our analysis is based on data recorded on 769 women who were classified as having had an induced abortion. About 16 % of women seeking post abortion services for an induced abortion reported to have had a previous induced abortion. Being separated or divorced or widowed, having no education, having unwanted pregnancy, having 1-2 prior births and using traditional methods of contraception were associated with a higher likelihood of a repeat induced abortion. The findings point to the need to address the reasons why women with first time induced abortion do not have the necessary information to prevent unintended pregnancies and further induced abortions. Possible explanations linked to the quality of post-abortion family planning and coverage of long-acting methods should be explored.

Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

Science.gov (United States)

2015-09-01

Summary of Results. Task 1. To identify mammalian PC- PLC . Based on results published by other groups, we proposed to identify candidate PC- PLC mRNAs by...establishing the role of the elusive mammalian protein, phosphatidycholine- specific phospholipase C (PC- PLC ) in the inflammatory processes involved in...progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC- PLC gene and protein; and 2. to test PC- PLC

Hypoxia Inducible Factors and Hypertension: Lessons from Sleep Apnea Syndrome

Science.gov (United States)

Nanduri, Jayasri; Peng, Ying-Jie; Yuan, Guoxiang; Kumar, Ganesh K.; Prabhakar, Nanduri R.

2015-01-01

Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity is a major contributor to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2- mediated transcription. Dysregulation of HIF-1- and HIF-2- mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive species (ROS) generation in the chemosensory reflex which is central for developing hypertension. PMID:25772710

Quantitative determination of the contribution of indirect and direct radiation action to the production of lethal lesions in mammalian cells

International Nuclear Information System (INIS)

Pohlit, W.; Drenkard, S.

1985-01-01

For quantitative models of radiation action in living cells it is necessary to know what fraction of the absorbed dose affects the target molecule by direct radiation action and what fraction by indirect radiation action. Mammalian cells were irradiated in suspension, saturated with N 2 O or CO 2 . With these gases the production of OH-radicals is changed by a factor of two in aqueous solutions and a corresponding change in cell survival would be expected, if only indirect radiation action is involved in the production of lethal lesions in the living cell. No difference could be detected, however, and it is concluded that indirect radiation action does not contribute to radiation lethality in mammalian cells. (author)

Factors affecting drug-induced liver injury: antithyroid drugs as instances

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Reza Heidari

2014-09-01

Full Text Available Methimazole and propylthiouracil have been used in the management of hyperthyroidism for more than half a century. However, hepatotoxicity is one of the most deleterious side effects associated with these medications. The mechanism(s of hepatic injury induced by antithyroid agents is not fully recognized yet. Furthermore, there are no specific tools for predicting the occurrence of hepatotoxicity induced by these drugs. The purpose of this article is to give an overview on possible susceptibility factors in liver injury induced by antithyroid agents. Age, gender, metabolism characteristics, alcohol consumption, underlying diseases, immunologic mechanisms, and drug interactions are involved in enhancing antithyroid drugs-induced hepatic damage. An outline on the clinically used treatments for antithyroid drugs-induced hepatotoxicity and the potential therapeutic strategies found to be effective against this complication are also discussed.

Rapid and preferential activation of the c-jun gene during the mammalian UV response

International Nuclear Information System (INIS)

Devary, Y.; Gottlieb, R.A.; Lau, L.F.; Karin, M.

1991-01-01

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase

Hypoxic induction of the regulator of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor pathway.

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Sam W Z Olechnowicz

Full Text Available The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2 in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4 protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered.

Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway

Science.gov (United States)

Olechnowicz, Sam W. Z.; Fedele, Anthony O.; Peet, Daniel J.

2012-01-01

The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered. PMID:22970249

Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification.

Science.gov (United States)

Mokas, Sophie; Larivière, Richard; Lamalice, Laurent; Gobeil, Stéphane; Cornfield, David N; Agharazii, Mohsen; Richard, Darren E

2016-09-01

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Dimethyl sulfoxide-inducible cytoplasmic factor involved in erythroid differentiation in mouse erythroleukemia (Friend) cells

International Nuclear Information System (INIS)

Watanabe, T.; Oishi, M.

1987-01-01

A previous report described an intracellular factor (differentiation-inducing factor I, or DIF-I) that seem to play a role in erythroid differentiation in mouse erythroleukemia (MEL) cells. The authors have detected another erythroid-inducing factor in cell-free extracts from dimethyl sulfoxide- or hexamethylenebis(acetamide)-treated MEL cells, which acts synergistically with DIF-I. The partially purified factor (termed DIF-II) triggered erythroid differentiation when introduced into undifferentiated MEL cells that had been potentiated by the induction of DIF-I. The activity in the extracts appeared in an inducible manner after addition of dimethyl sulfoxide or hexamethylenebis(acetamide), reached a maximum at 6 hr, and then rapidly decreased. The induction was inhibited by phorbol 12-myristate 13-acetate and also by cycloheximide. No induction was observed in a mutant MEL cell line defective in erythroid differentiation. These characteristics are consistent with the supposition that DIF-II is one of the putative dimethyl sulfoxide-inducible factors detected in previously reported cell-fusion and cytoplast-fusion experiments. The role of DIF-II in MEL-cell differentiation and in vitro differentiation in general is discussed

InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

Science.gov (United States)

Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

2007-10-15

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

Cell functional enviromics: Unravelling the function of environmental factors

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Alves Paula M

2011-06-01

Full Text Available Abstract Background While functional genomics, focused on gene functions and gene-gene interactions, has become a very active field of research in molecular biology, equivalent methodologies embracing the environment and gene-environment interactions are relatively less developed. Understanding the function of environmental factors is, however, of paramount importance given the complex, interactive nature of environmental and genetic factors across multiple time scales. Results Here, we propose a systems biology framework, where the function of environmental factors is set at its core. We set forth a "reverse" functional analysis approach, whereby cellular functions are reconstructed from the analysis of dynamic envirome data. Our results show these data sets can be mapped to less than 20 core cellular functions in a typical mammalian cell culture, while explaining over 90% of flux data variance. A functional enviromics map can be created, which provides a template for manipulating the environmental factors to induce a desired phenotypic trait. Conclusion Our results support the feasibility of cellular function reconstruction guided by the analysis and manipulation of dynamic envirome data.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Science.gov (United States)

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

A Novel Endometriosis Inducing Factor In Women with Endometriosis

OpenAIRE

Ramzy A,; Bibars M; El-Sawaf A; Selim M; Sabry D; Azmy O; Taha TF; Atta H; Rasheed K; El-Garf W; Anwar M

2010-01-01

Aim: To confirm the hypothesis of the presence of a possible endometriosis inducing factor(s) (EIF) in the blood of women with endometriosis. Patients and Methods: Forty infertile women were studied. The study group compromised of fifteen women of each three different degrees of endometriosis and fifteen women without endometriosis as a control group. Stem cells are characterized by being spindle shaped and proliferate in appropriate culture indefinitely. The women sera were co-cultured with ...

Current perspectives of CASA applications in diverse mammalian spermatozoa.

Science.gov (United States)

van der Horst, Gerhard; Maree, Liana; du Plessis, Stefan S

2018-03-26

Since the advent of computer-aided sperm analysis (CASA) some four decades ago, advances in computer technology and software algorithms have helped establish it as a research and diagnostic instrument for the analysis of spermatozoa. Despite mammalian spermatozoa being the most diverse cell type known, CASA is a great tool that has the capacity to provide rapid, reliable and objective quantitative assessment of sperm quality. This paper provides contemporary research findings illustrating the scientific and commercial applications of CASA and its ability to evaluate diverse mammalian spermatozoa (human, primates, rodents, domestic mammals, wildlife species) at both structural and functional levels. The potential of CASA to quantitatively measure essential aspects related to sperm subpopulations, hyperactivation, morphology and morphometry is also demonstrated. Furthermore, applications of CASA are provided for improved mammalian sperm quality assessment, evaluation of sperm functionality and the effect of different chemical substances or pathologies on sperm fertilising ability. It is clear that CASA has evolved significantly and is currently superior to many manual techniques in the research and clinical setting.

Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

Science.gov (United States)

Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

2014-10-01

Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm. © FASEB.

Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing

Energy Technology Data Exchange (ETDEWEB)

Dumortier, Jerome; Streblow, Daniel N.; Moses, Ashlee V.; Jacobs, Jon M.; Kreklywich, Craig N.; Camp, David G.; Smith, Richard D.; Orloff, Susan L.; Nelson, Jay

2008-07-01

Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.

‘The octet’: eight protein kinases that control mammalian DNA replication

Directory of Open Access Journals (Sweden)

Melvin L. Depamphilis

2012-09-01

Full Text Available Development of a fertilized human egg into an average sized adult requires about 29 trillion cell divisions, thereby producing enough DNA to stretch to the Sun and back 200 times (DePamphilis and Bell, 2011! Even more amazing is the fact that throughout these mitotic cell cycles, the human genome is duplicated once and only once each time a cell divides. If a cell accidentally begins to re-replicate its nuclear DNA prior to cell division, checkpoint pathways trigger apoptosis. And yet, some cells are developmentally programmed to respond to environmental cues by switching from mitotic cell cycles to endocycles, a process in which multiple S phases occur in the absence of either mitosis or cytokinesis. Endocycles allow production of viable, differentiated, polyploid cells that no longer proliferate. What is surprising is that among the 516 (Manning et al., 2002 to 557 (BioMart web site protein kinases encoded by the human genome, only eight regulate nuclear DNA replication directly. These are Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, Cdc7, Chk1 and Chk2. Even more remarkable is the fact that only four of these enzymes (Cdk1, Cdk7, Cdc7 and Chk1 are essential for mammalian development. Here we describe how these protein kinases determine when DNA replication occurs during mitotic cell cycles, how mammalian cells switch from mitotic cell cycles to endocycles, and how cancer cells can be selectively targeted for destruction by inducing them to begin a second S phase before mitosis is complete.

Analysis of the factors in determining radiosensitivity in mammalian cells by using radio-sensitive and -resistant clones isolated from HeLa S3 cells in vitro

International Nuclear Information System (INIS)

Nikaido, Osamu; Horikawa, Masakatsu

1976-01-01

The factors in determining radiosensitivity of cultured mammalian cells were analysed by using two clones each having different radiosensitivities. The radiosensitive clones were isolated from HeLa S3 cells by the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treatment, X-irradiation (200 R) and 5-bromodeoxyuridine (BUdR)-visible light method. On the other hand, the radioresistant clone was isolated by single X-irradiation (2000 R) from MNNG-treated HeLa S3 cell population. The radiosensitivities expressed in D sub(o) and D sub(q) values were 110 and 140 R in radiosensitive SM-1a clone and 180 and 230 R in radioresistant RM-1b clone respectively. The biological and biochemical characteristics of both clones such as the distribution of chromosome numbers, formation and rejoining of single strand breaks in DNA caused by X-irradiation, non-protein sulfhydryl (NPSH) and apparent total sulfhydryl (APSH) contents were measured. Among the characteristics analysed, different contents of NPSH in the cell were well correlated to their daiosensitivities among the original HeLa S3 cells, SM-1a and RM-1b clone. Additionally, it was found that the radioresistant L.P3 Co-3 cells isolated by Tsuboi et al. from the original mouse L.P3 cells by means of serial irradiation with 60 Co γ-rays have more abundant NPSH than the original L.P3 cells. From these results, it can be concluded that the amount of NPSH play the main role in determining radiosensitivity in cultured mammalian cells. (auth.)

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish

Science.gov (United States)

Mokalled, Mayssa H.; Patra, Chinmoy; Dickson, Amy L.; Endo, Toyokazu; Stainier, Didier Y. R.; Poss, Kenneth D.

2016-01-01

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration, a relatively unexplored process. Here, we performed a genome-wide profiling screen for secreted factors that are upregulated during zebrafish spinal cord regeneration. We find that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupt spinal cord repair, while transgenic ctgfa overexpression and local human CTGF recombinant protein delivery accelerate bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration. PMID:27811277

Non-linear leak currents affect mammalian neuron physiology

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Shiwei eHuang

2015-11-01

Full Text Available In their seminal works on squid giant axons, Hodgkin and Huxley approximated the membrane leak current as Ohmic, i.e. linear, since in their preparation, sub-threshold current rectification due to the influence of ionic concentration is negligible. Most studies on mammalian neurons have made the same, largely untested, assumption. Here we show that the membrane time constant and input resistance of mammalian neurons (when other major voltage-sensitive and ligand-gated ionic currents are discounted varies non-linearly with membrane voltage, following the prediction of a Goldman-Hodgkin-Katz-based passive membrane model. The model predicts that under such conditions, the time constant/input resistance-voltage relationship will linearize if the concentration differences across the cell membrane are reduced. These properties were observed in patch-clamp recordings of cerebellar Purkinje neurons (in the presence of pharmacological blockers of other background ionic currents and were more prominent in the sub-threshold region of the membrane potential. Model simulations showed that the non-linear leak affects voltage-clamp recordings and reduces temporal summation of excitatory synaptic input. Together, our results demonstrate the importance of trans-membrane ionic concentration in defining the functional properties of the passive membrane in mammalian neurons as well as other excitable cells.

Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus.

Science.gov (United States)

Cassone, V M; Speh, J C; Card, J P; Moore, R Y

1988-01-01

A detailed analysis of the cytoarchitecture, retinohypothalamic tract (RHT) projections, and immunohistochemical localization of major cell and fiber types within the hypothalamic suprachiasmatic nuclei (SCN) was conducted in five mammalian species: two species of opossum, the domestic cat, the guinea pig, and the house mouse. Cytoarchitectural and immunohistochemical studies were conducted in three additional species of marsupial mammals and in the domestic pig. The SCN in this diverse transect of mammalian taxonomy bear striking similarities. First, the SCN are similar in location, lying close to the third ventricle (3V) dorsal to the optic chiasm (OC), with a cytoarchitecture characterized by small, tightly packed neurons. Second, in all groups studied, the SCN receive bilateral retinal input. Third, the SCN contain immunohistochemically similar elements. These similarities suggest that the SCN developed characteristic features early in mammalian phylogeny. Some details of SCN organization vary among the species studied. In marsupials, vasopressin-like immunoreactive (VP-LI) and vasoactive intestinal polypeptide-like immunoreactive (VIP-LI) cells codistribute primarily in the dorsomedial aspects of the SCN, while in eutherians, VP-LI and VIP-LI cells are separated into SCN subnuclei. Furthermore, the marsupial RHT projects to the periventricular dorsomedial region, whereas the eutherian RHT projects more ventrally in the SCN into the zone that typically contains VIP-LI perikarya.

Musculoskeletal networks reveal topological disparity in mammalian neck evolution.

Science.gov (United States)

Arnold, Patrick; Esteve-Altava, Borja; Fischer, Martin S

2017-12-13

The increase in locomotor and metabolic performance during mammalian evolution was accompanied by the limitation of the number of cervical vertebrae to only seven. In turn, nuchal muscles underwent a reorganization while forelimb muscles expanded into the neck region. As variation in the cervical spine is low, the variation in the arrangement of the neck muscles and their attachment sites (i.e., the variability of the neck's musculoskeletal organization) is thus proposed to be an important source of neck disparity across mammals. Anatomical network analysis provides a novel framework to study the organization of the anatomical arrangement, or connectivity pattern, of the bones and muscles that constitute the mammalian neck in an evolutionary context. Neck organization in mammals is characterized by a combination of conserved and highly variable network properties. We uncovered a conserved regionalization of the musculoskeletal organization of the neck into upper, mid and lower cervical modules. In contrast, there is a varying degree of complexity or specialization and of the integration of the pectoral elements. The musculoskeletal organization of the monotreme neck is distinctively different from that of therian mammals. Our findings reveal that the limited number of vertebrae in the mammalian neck does not result in a low musculoskeletal disparity when examined in an evolutionary context. However, this disparity evolved late in mammalian history in parallel with the radiation of certain lineages (e.g., cetartiodactyls, xenarthrans). Disparity is further facilitated by the enhanced incorporation of forelimb muscles into the neck and their variability in attachment sites.

Distinct radioprotective activities of major heat shock proteins in irradiated mammalian cells

International Nuclear Information System (INIS)

Kabakov, Alexander; Malyutina, Yana; Kudryavtsev, Vladimir

2008-01-01

Full text: Several years ago we have suggested that heat shock proteins (Hsps) can be involved in cellular and tissue mechanisms of protection from ionizing radiation. At present, the accumulated experimental data do allow us to characterize three major mammalian Hsps, Hsp70, Hsp27 and Hsp90, as specific endogenous radioprotectors which are able to prevent or minimize cell death resulting from the radiation exposure. It follows from the many findings that the radioprotective effect of these Hsps is particularly manifested in their ability to attenuate apoptosis in various normal and tumor cells irradiated in vivo or in vitro. The obtained data already enable to suggest three main mechanisms of the radioprotection conferred by the excess Hsps: 1) Modulation of the intracellular signaling so that the apoptotic signal transduction is blocked, whereas the 'cell survival' signal transduction is stimulated; 2) Suppression of the radiation-associated free radical generation and apoptosis induced by reactive oxygen species (ROS); 3) Attenuation of the genotoxic impact of ionizing radiation. The latter suggested mechanism seems particularly intriguing and implies that the excess Hsps can somehow contribute to protection/repair of genomic DNA from radiation-induced damage. According to our recent results, Hsp90 is indeed involved in the post-irradiation repair of nuclear DNA, while excess Hsp70 can beneficially affect the p53-mediated DNA damage response in irradiated cells to ensure their long-term survival and recovery. As for Hsp27, we found that its accumulation in target cells increases their radioresistance by enhancing the irradiation-responsive activation of anti apoptotic pathways. While the Hsp70 and Hsp27 seem to perform different functions in irradiated cells, the synergistic enhancement of radioprotection was clearly observed in the cells enriched by the both the Hsps. In vivo, such radioprotective activities of the major mammalian Hsps may play a role in

The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release

International Nuclear Information System (INIS)

Kim, Bong Yoon; Sahara, Yoshinori; Yamamoto, Akitsugu; Kominami, Eiki; Kohsaka, Shinichi; Akazawa, Chihiro

2006-01-01

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca 2+ -dependent exocytosis, which was abolished by the Ca 2+ -channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A

Understanding mammalian genetic systems: the challenge of phenotyping in the mouse.

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Steve D M Brown

2006-08-01

Full Text Available Understanding mammalian genetic systems is predicated on the determination of the relationship between genetic variation and phenotype. Several international programmes are under way to deliver mutations in every gene in the mouse genome. The challenge for mouse geneticists is to develop approaches that will provide comprehensive phenotype datasets for these mouse mutant libraries. Several factors are critical to success in this endeavour. It will be important to catalogue assay and environment and where possible to adopt standardised procedures for phenotyping tests along with common environmental conditions to ensure comparable datasets of phenotypes. Moreover, the scale of the task underlines the need to invest in technological development improving both the speed and cost of phenotyping platforms. In addition, it will be necessary to develop new informatics standards that capture the phenotype assay as well as other factors, genetic and environmental, that impinge upon phenotype outcome.

Social factors modulate restraint stress induced hyperthermia in mice.

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Watanabe, Shigeru

2015-10-22

Stress-induced hyperthermia (SIH) was examined in three different social conditions in mice by thermographic measurement of the body surface temperature. Placing animals in cylindrical holders induced restraint stress. I examined the effect of the social factors in SIH using the thermograph (body surface temperature). Mice restrained in the holders alone showed SIH. Mice restrained in the holders at the same time as other similarly restrained cage mates (social equality condition) showed less hyperthermia. Interestingly, restrained mice with free moving cage mates (social inequality condition) showed the highest hyperthermia. These results are consistent with a previous experiment measuring the memory-enhancing effects of stress and the stress-induced elevation of corticosterone, and suggest that social inequality enhances stress. Copyright © 2015 Elsevier B.V. All rights reserved.

The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction.

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Wale, Petra L; Gardner, David K

2016-01-01

Although laboratory procedures, along with culture media formulations, have improved over the past two decades, the issue remains that human IVF is performed in vitro (literally 'in glass'). Using PubMed, electronic searches were performed using keywords from a list of chemical and physical factors with no limits placed on time. Examples of keywords include oxygen, ammonium, volatile organics, temperature, pH, oil overlays and incubation volume/embryo density. Available clinical and scientific evidence surrounding physical and chemical factors have been assessed and presented here. Development of the embryo outside the body means that it is constantly exposed to stresses that it would not experience in vivo. Sources of stress on the human embryo include identified factors such as pH and temperature shifts, exposure to atmospheric (20%) oxygen and the build-up of toxins in the media due to the static nature of culture. However, there are other sources of stress not typically considered, such as the act of pipetting itself, or the release of organic compounds from the very tissue culture ware upon which the embryo develops. Further, when more than one stress is present in the laboratory, there is evidence that negative synergies can result, culminating in significant trauma to the developing embryo. It is evident that embryos are sensitive to both chemical and physical signals within their microenvironment, and that these factors play a significant role in influencing development and events post transfer. From the viewpoint of assisted human reproduction, a major concern with chemical and physical factors lies in their adverse effects on the viability of embryos, and their long-term effects on the fetus, even as a result of a relatively brief exposure. This review presents data on the adverse effects of chemical and physical factors on mammalian embryos and the importance of identifying, and thereby minimizing, them in the practice of human IVF. Hence, optimizing the

Chern-Simons induced spin factors in noncovariant gauges

International Nuclear Information System (INIS)

Tanaka, I.

1993-01-01

We study Chern-Simons induced spin factors in noncovariant metric-independent gauges, such as the axial gauge and the Coulomb gauge. These spin factors are defined without loop splitting. We find that they are equal to integers and have particular geometrical meanings. In the axial gauge, this integer is the writhe number of a link diagram defined by the projection of a loop to the time direction. In the Coulomb gauge, it is suggested that this integer is also the writhe number of a link diagram, defined by the projection of a loop to a spatial plane

Mammalian niche conservation through deep time.

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Larisa R G DeSantis

Full Text Available 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

Comparative transcriptome analyses indicate molecular homology of zebrafish swimbladder and mammalian lung.

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Weiling Zheng

Full Text Available The fish swimbladder is a unique organ in vertebrate evolution and it functions for regulating buoyancy in most teleost species. It has long been postulated as a homolog of the tetrapod lung, but the molecular evidence is scarce. In order to understand the molecular function of swimbladder as well as its relationship with lungs in tetrapods, transcriptomic analyses of zebrafish swimbladder were carried out by RNA-seq. Gene ontology classification showed that genes in cytoskeleton and endoplasmic reticulum were enriched in the swimbladder. Further analyses depicted gene sets and pathways closely related to cytoskeleton constitution and regulation, cell adhesion, and extracellular matrix. Several prominent transcription factor genes in the swimbladder including hoxc4a, hoxc6a, hoxc8a and foxf1 were identified and their expressions in developing swimbladder during embryogenesis were confirmed. By comparison of enriched transcripts in the swimbladder with those in human and mouse lungs, we established the resemblance of transcriptome of the zebrafish swimbladder and mammalian lungs. Based on the transcriptomic data of zebrafish swimbladder, the predominant functions of swimbladder are in its epithelial and muscular tissues. Our comparative analyses also provide molecular evidence of the relatedness of the fish swimbladder and mammalian lung.

Presence of abscisic acid, a phytohormone, in the mammalian brain

International Nuclear Information System (INIS)

Le Page-Degivry, M.T.; Bidard, J.N.; Rouvier, E.; Bulard, C.; Lazdunski, M.

1986-01-01

This paper reports the presence of abscisic acid, one of the most important phytohormones, in the central nervous system of pigs and rats. The identification of this hormone in brain was made after extensive purification by using a radioimmunoassay that is very specific for (+)-cis-abscisic acid. The final product of purification from mammalian brain has the same properties as authentic abscisic acid: it crossreacts in the radioimmunoassay for the phytohormone and it has the same retention properties and the same gas chromatography/mass spectrometry characteristics. Moreover, like (+)-cis-abscisic acid itself, the brain factor inhibits stomatal apertures of abaxial epidermis strips of Setcreasea purpurea Boom (Commelinaceae). The presence of abscisic acid conjugates that are present in plants has also been identified in brain

Mammalian cell biology

International Nuclear Information System (INIS)

Elkind, M.M.

1975-01-01

Studies of the action of N-ethylmaleimide (NEM), as an inhibitor of repair of x radioinduced injuries were extended from synchronous Chinese hamster cells to synchronous human HeLa cells. These studies showed a similar mode of action in both cell types lending support to the notion that conclusions may be extracted from such observations that are of fairly general applicability to mammalian cells. Radiation studies with NEM are being extended to hypoxic cells to inquire if NEM is effective relative to oxygen-independent damage. Observations relative to survival, DNA synthesis, and DNA strand elongation resulting from the addition products to DNA when cells were exposed to near uv in the presence of psoralen were extended. (U.S.)

Inducible nucleosome depletion at OREBP-binding-sites by hypertonic stress.

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Edith H Y Tong

Full Text Available BACKGROUND: Osmotic Response Element-Binding Protein (OREBP, also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown. METHODOLOGY: Using hypertonic induction of the aldose reductase (AR gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s around the OREs. The loss of nucleosome(s was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBP-dependent hyperacetylation of histones that spanned the 5' upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone. SIGNIFICANCE: Our findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled.

Ecology and evolution of mammalian biodiversity.

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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.

Ecology and evolution of mammalian biodiversity

Science.gov (United States)

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

Detection of DNA strand breaks in mammalian cells using the radioresistant bacterium PprA protein

International Nuclear Information System (INIS)

Satoh, Katsuya; Wada, Seiichi; Narumi, Issay; Kikuchi, Masahiro; Funayama, Tomoo; Kobayashi, Yasuhiko

2003-01-01

We have previously found that the PprA protein from Deinococcus radiodurans possesses ability to recognize DNA carrying strand breaks. In the present study, we attempted to visualize radiation-induced DNA strand breaks with PprA protein using immunofluorescence technique to elucidate the DNA damage response mechanism in mammalian cultured cells. As a result, colocalization of Cy2 and DAPI fluorescent signals was observed. This observation suggests that DNA strand breaks in the nucleus of CHO-K1 cells were effectively detected using the PprA protein. The amount of DNA strand breaks (integrated density of Cy2 fluorescent signals) was increased with the increase in the radiation dose. (author)

Molecular characterization and expression profile of nanos in Schistosoma japonicum and its influence on the expression several mammalian stem cell factors.

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Giri, Bikash Ranjan; Du, Xiaoli; Xia, Tianqi; Chen, Yongjun; Li, Hao; Cheng, Guofeng

2017-07-01

Pluripotent stem cells, called neoblasts, are well known for the regenerative capability and developmental plasticity in flatworms. Impressive advancement has been made in free-living flatworms, while in case of its parasitic counterpart, neoblast-like stem cells have attracted recent attention for its self-renewal and differentiation capacity. Nanos is a key conserved post-transcriptional regulator critical for the formation, development, and/or maintenance of the pluripotent germ line stem cell systems in many metazoans including schistosomes. In the present study, we report the molecular cloning and expression of nanos orthologous genes nanos in Schistosoma japonicum (Sjnanos). The cDNA of Sjnanos is 826 bp long, containing an open reading frame (ORF) for 223 amino acid long protein. qRT-PCR analysis shown that Sjnanos was differently expressed in several stages of schistosomes with relatively high level in schistosomula. Additionally, Sjnanos was expressed highly in adult females compared to adult males. Transfection of recombinant plasmid for expressing Sjnanos resulted in significant proliferation and increased expression of several stem cell factors in mammalian cells. Overall, our preliminary study provides the molecular basis to further functionally characterize Sjnanos in S. japonicum.

On activation of cholesterologenesis under the effect of ionizing radiation on mammalian body

International Nuclear Information System (INIS)

Kolomijtseva, I.K.

1986-01-01

The assumption is made that ionizing radiation induces cholesterologenesis activation in different cells of mammalian organism as an early reaction to the harmful effect necessary for restoration of biomembranes. Liver cells activate adaptively the cholesterol synthesis in the animal body irradiated with lethal doses in response to the injury to radiosensitive cells in order to make them recover and compensate for their functions (with the gastrointestinal syndrome, for instance, to compensate for the cholesterol-producing function of the intestine and to make it recover). With lethal radiation doses, a change in the lipid content and metabolism of microsomal membrane lipids of the liver is associated with activation of synthetic functions of the liver due to compensation of the injury to radiosensitive tissues

Non-lethal effects of low- and high-LET radiation on cultured mammalian cells

International Nuclear Information System (INIS)

Walker, J.T.

1982-01-01

In analyzing post-irradiation growth kinetics of cultured mammalian cells, specifically T1-E human cells, this investigation shows that the shift in post-irradiation clone-size distributions toward small colonies is due to both radiation-induced division delay and increased generation times of the irradiated population. Evidence also indicates that the final shape of the final clone-size distribution is influenced by the age density distribution of the parent cells at the time of plating. From computer-generated delay time distributions it was determined that a large percentage of the parent population was found to be in the plateau phase at early growth times and evidence indicates that these cells may contribute heavily to the total population response to radiation

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

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Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

2012-12-01

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)*

Science.gov (United States)

Elguindy, Mahmoud M.; Nakamaru-Ogiso, Eiko

2015-01-01

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. PMID:26063804

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

Science.gov (United States)

Elguindy, Mahmoud M; Nakamaru-Ogiso, Eiko

2015-08-21

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Granulocyte Colony-Stimulating Factor in the Treatment of Acute Radiation Syndrome: A Concise Review

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Michal Hofer

2014-04-01

Full Text Available This article concisely summarizes data on the action of one of the principal and best known growth factors, the granulocyte colony-stimulating factor (G-CSF, in a mammalian organism exposed to radiation doses inducing acute radiation syndrome. Highlighted are the topics of its real or anticipated use in radiation accident victims, the timing of its administration, the possibilities of combining G-CSF with other drugs, the ability of other agents to stimulate endogenous G-CSF production, as well as of the capability of this growth factor to ameliorate not only the bone marrow radiation syndrome but also the gastrointestinal radiation syndrome. G-CSF is one of the pivotal drugs in the treatment of radiation accident victims and its employment in this indication can be expected to remain or even grow in the future.

Prdm9 controls activation of mammalian recombination hotspots.

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Parvanov, Emil D; Petkov, Petko M; Paigen, Kenneth

2010-02-12

Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, ensures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse chromosome 17, which controls activation of recombination at multiple distant hotspots, has been mapped within a 181-kilobase interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is expressed in early meiosis and whose deficiency results in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and multiple minor alleles. The identification of Prdm9 as a protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system.

Locomotor circuits in the mammalian spinal cord

DEFF Research Database (Denmark)

Kiehn, Ole

2006-01-01

Intrinsic spinal networks, known as central pattern generators (CPGs), control the timing and pattern of the muscle activity underlying locomotion in mammals. This review discusses new advances in understanding the mammalian CPGs with a focus on experiments that address the overall network struct...

Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.

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Fredriksson, Lisa; Herpers, Bram; Benedetti, Giulia; Matadin, Quraisha; Puigvert, Jordi C; de Bont, Hans; Dragovic, Sanja; Vermeulen, Nico P E; Commandeur, Jan N M; Danen, Erik; de Graauw, Marjo; van de Water, Bob

2011-06-01

Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity. Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis. Copyright © 2011 American Association for the Study of Liver Diseases.

Assays for mammalian tyrosinase: a comparative study

International Nuclear Information System (INIS)

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

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α.

Science.gov (United States)

Nagaya, Yoshiaki; Aoyama, Mineyoshi; Tamura, Tetsuya; Kakita, Hiroki; Kato, Shin; Hida, Hideki; Saitoh, Shinji; Asai, Kiyofumi

2014-12-01

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

Science.gov (United States)

Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

2005-01-01

Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

Induced DNA repair pathway in mammalian cells

International Nuclear Information System (INIS)

Overberg, R.

1985-01-01

The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

Clotting of mammalian fibrinogens by papain: a re-examination.

Science.gov (United States)

Doolittle, Russell F

2014-10-28

Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

The involvement of mRNA processing factors TIA-1, TIAR, and PABP-1 during mammalian hibernation.

Science.gov (United States)

Tessier, Shannon N; Audas, Timothy E; Wu, Cheng-Wei; Lee, Stephen; Storey, Kenneth B

2014-11-01

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

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Danielle K Offerdahl

Full Text Available Tick-borne flaviviruses (TBFV are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero and Ixodes scapularis tick cells (ISE6 to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM, and electron tomography (ET, we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60-100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol

International Nuclear Information System (INIS)

Mitchell, J.B.; DeGraff, W.; Kaufman, D.; Krishna, M.C.; Samuni, A.; Finkelstein, E.; Ahn, M.S.; Hahn, S.M.; Gamson, J.; Russo, A.

1991-01-01

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress

Endogenous retrovirus sequences expressed in male mammalian ...

African Journals Online (AJOL)

Objectives: To review the research findings on the expression of endogenous retroviruses and retroviral-related particles in male mammalian reproductive tissues, and to discuss their possible role in normal cellular events and association with disease conditions in male reproductive tissues. Data sources: Published ...

[Cellular adhesion signal transduction network of tumor necrosis factor-alpha induced hepatocellular carcinoma cells].

Science.gov (United States)

Zheng, Yongchang; Du, Shunda; Xu, Haifeng; Xu, Yiyao; Zhao, Haitao; Chi, Tianyi; Lu, Xin; Sang, Xinting; Mao, Yilei

2014-11-18

To systemically explore the cellular adhesion signal transduction network of tumor necrosis factor-alpha (TNF-α)-induced hepatocellular carcinoma cells with bioinformatics tools. Published microarray dataset of TNF-α-induced HepG2, human transcription factor database HTRI and human protein-protein interaction database HPRD were used to construct and analyze the signal transduction network. In the signal transduction network, MYC and SP1 were the key nodes of signaling transduction. Several genes from the network were closely related with cellular adhesion.Epidermal growth factor receptor (EGFR) is a possible key gene of effectively regulating cellular adhesion during the induction of TNF-α. EGFR is a possible key gene for TNF-α-induced metastasis of hepatocellular carcinoma.

Retention of the Native Epigenome in Purified Mammalian Chromatin.

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Andreas H Ehrensberger

Full Text Available A protocol is presented for the isolation of native mammalian chromatin as fibers of 25-250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.

A role for carbohydrate recognition in mammalian sperm-egg binding

International Nuclear Information System (INIS)

Clark, Gary F.

2014-01-01

Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented

Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth

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Javier Enrione

2017-12-01

Full Text Available In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter and biocompatibility, permitting myoblast cell adhesion (~40% and growth (~24 h duplication time. The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

A role for carbohydrate recognition in mammalian sperm-egg binding

Energy Technology Data Exchange (ETDEWEB)

Clark, Gary F., E-mail: clarkgf@health.missouri.edu

2014-08-01

Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

Natural radiation dose of small mammalians in beech forest of Rokkasho, Japan

International Nuclear Information System (INIS)

Ohtsuka, Yoshihito; Iyogi, Takashi; Takaku, Yuichi; Hisamatsu, Shun'ichi

2008-01-01

Full text: Protection of the environment itself from radiation hazards is recognized as important as that of humans. Actual data on the background natural radiation dose, which is necessary to evaluate the effect of radiation, are very few, especially in the terrestrial environment. Forests around the Japan's first large scale nuclear fuel reprocessing plant in Rokkasho were selected as the research field for evaluating the background natural radiation to the environment. Data observed will be used in future for the comparison with the radiation dose from radionuclides released from the plant. Small mammalians, mouse (Apodeums argentus) and mole (Urotrichus talpoides), were selected as representative animals of the forests in this study, and it was planned to measure their radiation doses from natural sources; environmental γ-rays, Rn and internal radionuclides. The forests around the plant were classified into three types: beech, oak and coniferous. We have been measuring the natural radiation to the mammalians in each of those forests one by one and report the data for the beech forest here. The mammalians caught in traps in the beech forest during June-October, 2006 were analyzed for their natural radionuclides burdens ( 210 Pb, 210 Po, 40 K, 87 Rb, 226 Ra, 238 U and 232 Th) in 11 organs and carcass. Radiation dose rates from environmental γ-rays and atmospheric concentration of Rn in the forest were also measured during August-December, 2006. Mean internal dose rates of mouse and mole caught in June, 2006 were estimated to be 0.036 μGy h -1 and 0.28 μGy h -1 , respectively, from mean concentrations of the nuclides in a total body and internal dose conversion coefficients by FASSET. The difference of the dose between the two mammalian species was attributed to higher contribution of 210 Po in mole, in which dose reached 0.25 μGy h -1 in contrast to that in mouse of 0.016 μGy h -1 . The concentration of 210 Po in kidney of mole (0.37 Bq g -1 wet) was

Mitomycin C induced alterations in antioxidant enzyme levels in a model insect species, Spodoptera eridania.

Science.gov (United States)

Batcabe, J P; MacGill, R S; Zaman, K; Ahmad, S; Pardini, R S

1994-05-01

1. An insect species, the southern armyworm Spodoptera eridania, was used as an in vivo model to examine mitomycin C's (MMC) pro-oxidant effect reflected in alterations of antioxidant enzymes. 2. Following a 2-day exposure to 0.01 and 0.05% w/w dietary concentrations, MMC only induced superoxide dismutase activity. All other enzyme activities were not affected, indicating oxidative stress was mild. 3. Following a 5-day exposure to 0.05% w/w dietary MMC, the activities of superoxide dismutase, glutathione-S-transferase and its peroxidase activity and DT-diaphorase were induced. GR activity was not altered. The high constitutive catalase activity was also not affected. These responses of S. eridania's antioxidant enzymes are analogous to those of mammalian systems in alleviating MMC-induced oxidative stress. 4. S. eridania emerges as an appropriate non-mammalian model for initial and cost-effective screening of drug-induced oxidative stress.

Proposal of a model of mammalian neural induction

Science.gov (United States)

Levine, Ariel J.; Brivanlou, Ali H.

2009-01-01

How does the vertebrate embryo make a nervous system? This complex question has been at the center of developmental biology for many years. The earliest step in this process – the induction of neural tissue – is intimately linked to patterning of the entire early embryo, and the molecular and embryological basis these processes are beginning to emerge. Here, we analyze classic and cutting-edge findings on neural induction in the mouse. We find that data from genetics, tissue explants, tissue grafting, and molecular marker expression support a coherent framework for mammalian neural induction. In this model, the gastrula organizer of the mouse embryo inhibits BMP signaling to allow neural tissue to form as a default fate – in the absence of instructive signals. The first neural tissue induced is anterior and subsequent neural tissue is posteriorized to form the midbrain, hindbrain, and spinal cord. The anterior visceral endoderm protects the pre-specified anterior neural fate from similar posteriorization, allowing formation of forebrain. This model is very similar to the default model of neural induction in the frog, thus bridging the evolutionary gap between amphibians and mammals. PMID:17585896

A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

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Fat-Moon Suk

2013-01-01

Full Text Available Activating transcription factor-(ATF- 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002 is a Taiwanese propolin G (PPG derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose polymerase (PARP. GS-002 also induced endoplasmic reticular (ER stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78, growth arrest- and DNA damage-inducible gene 153 (GADD153, phosphorylated eukaryotic initiation factor 2α (eIF2α, phosphorylated protein endoplasmic-reticular-resident kinase (PERK, and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

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Sarah Inwood

2018-01-01

Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

The related transcriptional enhancer factor-1 isoform, TEAD4(216, can repress vascular endothelial growth factor expression in mammalian cells.

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Binoy Appukuttan

Full Text Available Increased cellular production of vascular endothelial growth factor (VEGF is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4 protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4(216, which represses VEGF promoter activity. The TEAD4(216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE, which is the sequence critical to hypoxia inducible factor (HIF-mediated effects. The TEAD4(216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4(216 isoform can competitively repress the stimulatory activity of the TEAD4(434 and TEAD4(148 enhancers. Synthesis of the native VEGF(165 protein and cellular proliferation is suppressed by the TEAD4(216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4(216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

Fundamental aspects of the freezing of cells, with emphasis on mammalian ova and embryos

Energy Technology Data Exchange (ETDEWEB)

Mazur, P.

1980-01-01

The problem in cryobiology is how to cool cells to -196/sup 0/C and return them to normal temperatures without killing them. One important factor is the presence of a protective additive like glycerol or dimethyl sulfoxide. Mammalian cells rarely survive freezing to below -40/sup 0/C in its absence. In the presence of an additive, survival is critically dependent on the cooling rate. Supraoptimal rates and suboptimal rates are both damaging. Death at supraoptimal rates is the result of the formation of intracellular ice and its recrystallization during warming. Death at suboptimal rates is a consequence of the major alterations in aqueous solutions produced by ice formation. The chief effects are a major reduction in the fraction of the solution remaining unfrozen at a given temperature and a major increase in the solute concentration of that fraction. The introduction of molar concentrations of additive greatly reduces both the fraction frozen and the concentration of electrolytes in the unfrozen channels and in the cell interior. Usually, freezing either kills cells outright or it results in survivors that retain full capacity to function. But there is the possibility that in some cases survivors may in fact be impaired genetically or physiologically. All evidence indicates that genetic damage does not occur. But there are clear examples in which freezing does induce nonlethal physiological damage. (ERB)

Modulating factors in the expression of radiation-induced oncogenic transformation

International Nuclear Information System (INIS)

Hall, E.J.; Hei, T.K.

1990-01-01

Many assays for oncogenic transformation have been developed ranging from those in established rodent cell lines where morphological alteration is scored, to those in human cells growing in nude mice where tumor invasiveness is scored. In general, systems that are most quantitaive are also the least relevant in terms of human carcinogenesis and human risk estimation. The development of cell culture systems has made it possible to assess at the cellular level the oncogenic potential of a variety of chemical, physical and viral agents. Cell culture systems afford the opportunity to identify factors and conditions that may prevent or enhance cellular transformation by radiation and chemicals. Permissive and protective factors in radiation-induced transformation include thyroid hormone and the tumor promoter TPA that increase the transformation incidence for a given dose of radiation, and retinoids, selenium, vitamin E, and 5-aminobenzamide that inhibit the expression of transformation. Densely ionizing α-particles, similar to those emitted by radon daughters, are highly effective in inducing transformations and appear to interact in a supra-additive fashion with asbestos fibers. The activation of a known dominant oncogene has not yet been demonstrated in radiation-induced oncogenic transformation. The most likely mechanism for radiation activation of an oncogene would be via the production of a chromosomal translocation. Radiation also efficiently induces deletions and may thus lead to the loss of a suppressor gene

The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels?

DEFF Research Database (Denmark)

Jespersen, Thomas; Grunnet, Morten; Rasmussen, Hanne B

2006-01-01

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump gamma-subunit. Originally it was suggested that CHIF could induce the expression of the I(Ks) current when expressed in Xenopu...

Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

Science.gov (United States)

Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

2016-10-04

Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

Physicochemical Factors: Impact on Spermagglutination Induced by Escherichia coli

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Kiranjeet Kaur

2014-02-01

Full Text Available Motility is a sensitive parameter of sperm function which is predictive of its fertilization potential in vitro. The decrease in sperm motility may be associated with sperm agglutination and immobilization due to mere presence of bacteria or excretion of bacterial toxic products. Supplementation with various agents like sucrose, mannitol, calcium, and EDTA is well known to improve the sperm motility in vitro. The present study was designed to check any protective role exerted by the addition of different agents on spermatozoal motility against E. coli induced sperm agglutination. 52 semen specimens were screened for the presence of sperm-agglutinating strain of E. coli. Further, influence of various factors, namely, sugars, salts, and chelating agents was studied. Also, the impact of exposure to high temperature and alcohol on sperm-agglutinating efficiency of E. coli was observed. None of the factors could inhibit the sperm agglutination induced by E. coli, except high temperature suggesting the involvement of protein moiety. In addition, it was observed that agglutinating efficiency of E. coli was limited to spermatozoa and RBCs. It may be concluded that sperm-agglutinating property of E. coli is quite stable as various physicochemical factors tested did not show any negative effect on the same except high temperature.

Irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy.

Science.gov (United States)

Reza, Musarrat Maisha; Subramaniyam, Nathiya; Sim, Chu Ming; Ge, Xiaojia; Sathiakumar, Durgalakshmi; McFarlane, Craig; Sharma, Mridula; Kambadur, Ravi

2017-10-24

Exercise induces expression of the myokine irisin, which is known to promote browning of white adipose tissue and has been shown to mediate beneficial effects following exercise. Here we show that irisin induces expression of a number of pro-myogenic and exercise response genes in myotubes. Irisin increases myogenic differentiation and myoblast fusion via activation of IL6 signaling. Injection of irisin in mice induces significant hypertrophy and enhances grip strength of uninjured muscle. Following skeletal muscle injury, irisin injection improves regeneration and induces hypertrophy. The effects of irisin on hypertrophy are due to activation of satellite cells and enhanced protein synthesis. In addition, irisin injection rescues loss of skeletal muscle mass following denervation by enhancing satellite cell activation and reducing protein degradation. These data suggest that irisin functions as a pro-myogenic factor in mice.

Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

Science.gov (United States)

Kim, Eun-Ae; Jang, Ji-Hoon; Lee, Yun-Han; Sung, Eon-Gi; Song, In-Hwan; Kim, Joo-Young; Kim, Suji; Sohn, Ho-Yong; Lee, Tae-Jin

2014-07-01

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

FGT-1 is a mammalian GLUT2-like facilitative glucose transporter in Caenorhabditis elegans whose malfunction induces fat accumulation in intestinal cells.

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Shun Kitaoka

Full Text Available Caenorhabditis elegans (C. elegans is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS, that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT, we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (Km of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01, indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2 (mammalian insulin receptor homologue mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism.

Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration

International Nuclear Information System (INIS)

Guelden, Michael; Moerchel, Sabine; Seibert, Hasso

2005-01-01

Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC 50 values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC 50 values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC 50 values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

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Ying-Hung Lin

2017-01-01

Full Text Available According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16. RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1, were identified using co-immunoprecipitation (co-IP and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS. We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Factors Associated with Incidence of Induced Abortion in Hamedan, Iran.

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Hosseini, Hatam; Erfani, Amir; Nojomi, Marzieh

2017-05-01

There is limited reliable information on abortion in Iran, where abortion is illegal and many women of reproductive age seek clandestine abortion to end their unintended pregnancy. This study aims to examine the determinants of induced abortion in the city of Hamedan, Iran. The study utilizes recent data from the 2015 Hamedan Survey of Fertility, conducted in a representative sample of 3,000 married women aged 15-49 years in the city of Hamedan, Iran. Binary logistic regression models are used to examine factors associated with the incidence of abortion. Overall, 3.8% of respondents reported having had an induced abortion in their life. Multivariate results showed that the incidence of abortion was strongly associated with women's education, type of contraceptive and family income level, after controlling for confounding factors. Women using long-acting contraceptive methods, those educated under high school diploma or postsecondary education, and those with high level of income were more likely to report having an induced abortion. The high incidence of abortion among less or more educated women and those with high income level signifies unmet family planning needs among these women, which must be addressed by focused reproductive health and family planning programs.

Ovulation-inducing factor: a protein component of llama seminal plasma

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Huanca Wilfredo

2010-05-01

Full Text Available Abstract Background Previously, we documented the presence of ovulation-inducing factor (OIF in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s in seminal plasma responsible for inducing ovulation. Methods In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group were treated i.m. with whole seminal plasma (positive control, phosphate-buffered saline (negative control, or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or Results In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each, but none ovulated in the other groups (P Conclusions We conclude that ovulation-inducing factor (OIF in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

Mammalian Synthetic Biology: Time for Big MACs.

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Martella, Andrea; Pollard, Steven M; Dai, Junbiao; Cai, Yizhi

2016-10-21

The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-potentially up to chromosome scale-and the effective delivery of these large DNA payloads to the host cell. Random integration of large transgenes, encoding therapeutic proteins or genetic circuits into host chromosomes, has several drawbacks such as risks of insertional mutagenesis, lack of control over transgene copy-number and position-specific effects; these can compromise the intended functioning of genetic circuits. The development of a system orthogonal to the endogenous genome is therefore beneficial. Mammalian artificial chromosomes (MACs) are functional, add-on chromosomal elements, which behave as normal chromosomes-being replicating and portioned to daughter cells at each cell division. They are deployed as useful gene expression vectors as they remain independent from the host genome. MACs are maintained as a single-copy and can accommodate multiple gene expression cassettes of, in theory, unlimited DNA size (MACs up to 10 megabases have been constructed). MACs therefore enabled control over ectopic gene expression and represent an excellent platform to rapidly prototype and characterize novel synthetic gene circuits without recourse to engineering the host genome. This review describes the obstacles synthetic biologists face when working with mammalian systems and how the development of improved MACs can overcome these-particularly given the spectacular advances in DNA synthesis and assembly that are fuelling this research area.

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

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

2007-01-01

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

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

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Jin, Wan; Hua, Zheng; Honglei, Xiao; Zhenjue, She [Department of Anatomy, Histology and Embryology, Shanghai Medical School, Fudan University, 200032 Shanghai (China); Zhou Guomin [Department of Anatomy, Histology and Embryology, Shanghai Medical School, Fudan University, 200032 Shanghai (China)], E-mail: gmzhou185@yahoo.com.cn

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.

Vitamin C deficiency aggravates tumor necrosis factor α-induced insulin resistance.

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Qing, Zhou; Xiao-Hui, Wu; Xi-Mei, Wu; Chao-Chun, Zou

2018-06-15

Chronic low-grade inflammation plays a major role in the development of insulin resistance. The potential role and underlying mechanism of vitamin C, an antioxidant and anti-inflammatory agent, was investigated in tumor necrosis factor-α (TNF-α)-induced insulin resistance. Gulonolactone oxidase knockout (Gulo -/- ) mice genetically unable to synthesize vitamin C were used to induce insulin resistance by continuously pumping small doses of TNF-α for seven days, and human liver hepatocellular carcinoma cells (HepG2 cells) were used to induce insulin resistance by treatment with TNF-α. Vitamin C deficiency aggravated TNF-α-induced insulin resistance in Gulo -/- mice, resulting in worse glucose tolerance test (GTT) results, higher fasting plasma insulin level, and the inactivation of the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β) pathway in the liver. Vitamin C deficiency also worsened liver lipid accumulation and inflammation in TNF-α-treated Gulo -/- mice. In HepG2 cells, vitamin C reversed the TNF-α-induced reduction of glucose uptake and glycogen synthesis, which were mediated by increasing GLUT2 levels and the activation of the insulin receptor substrate (IRS-1)/AKT/GSK3β pathway. Furthermore, vitamin C inhibited the TNF-α-induced activation of not only the mitogen-activated protein kinase (MAPKs), but also nuclear factor-kappa B (NF-κB) signaling. Taken together, vitamin C is essential for preventing and improving insulin resistance, and the supplementing with vitamin C may be an effective therapeutic intervention for metabolic disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

Secondary osteons scale allometrically in mammalian humerus and femur.

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Felder, A A; Phillips, C; Cornish, H; Cooke, M; Hutchinson, J R; Doube, M

2017-11-01

Intra-cortical bone remodelling is a cell-driven process that replaces existing bone tissue with new bone tissue in the bone cortex, leaving behind histological features called secondary osteons. While the scaling of bone dimensions on a macroscopic scale is well known, less is known about how the spatial dimensions of secondary osteons vary in relation to the adult body size of the species. We measured the cross-sectional area of individual intact secondary osteons and their central Haversian canals in transverse sections from 40 stylopodal bones of 39 mammalian species (body mass 0.3-21 000 kg). Scaling analysis of our data shows that mean osteonal resorption area (negative allometry, exponent 0.23, R 2  0.54, p <0.005) and Haversian canal area (negative allometry, exponent 0.31, R 2  0.45, p <0.005) are significantly related to body mass, independent of phylogeny. This study is the most comprehensive of its kind to date, and allows us to describe overall trends in the scaling behaviour of secondary osteon dimensions, supporting the inference that the osteonal resorption area may be limited by the need to avoid fracture in smaller mammalian species, but the need to maintain osteocyte viability in larger mammalian species.

Epigenetic control of hypoxia inducible factor-1α-dependent expression of placental growth factor in hypoxic conditions.

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Tudisco, Laura; Della Ragione, Floriana; Tarallo, Valeria; Apicella, Ivana; D'Esposito, Maurizio; Matarazzo, Maria Rosaria; De Falco, Sandro

2014-04-01

Hypoxia plays a crucial role in the angiogenic switch, modulating a large set of genes mainly through the activation of hypoxia-inducible factor (HIF) transcriptional complex. Endothelial cells play a central role in new vessels formation and express placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, mainly involved in pathological angiogenesis. Despite several observations suggest a hypoxia-mediated positive modulation of PlGF, the molecular mechanism governing this regulation has not been fully elucidated. We decided to investigate if epigenetic modifications are involved in hypoxia-induced PlGF expression. We report that PlGF expression was induced in cultured human and mouse endothelial cells exposed to hypoxia (1% O 2), although DNA methylation at the Plgf CpG-island remains unchanged. Remarkably, robust hyperacetylation of histones H3 and H4 was observed in the second intron of Plgf, where hypoxia responsive elements (HREs), never described before, are located. HIF-1α, but not HIF-2α, binds to identified HREs. Noteworthy, only HIF-1α silencing fully inhibited PlGF upregulation. These results formally demonstrate a direct involvement of HIF-1α in the upregulation of PlGF expression in hypoxia through chromatin remodeling of HREs sites. Therefore, PlGF may be considered one of the putative targets of anti-HIF therapeutic applications.

Enamel formation and growth in non-mammalian cynodonts

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Dirks, Wendy; Martinelli, Agustín G.

2018-01-01

The early evolution of mammals is associated with the linked evolutionary origin of diphyodont tooth replacement, rapid juvenile growth and determinate adult growth. However, specific relationships among these characters during non-mammalian cynodont evolution require further exploration. Here, polarized light microscopy revealed incremental lines, resembling daily laminations of extant mammals, in histological sections of enamel in eight non-mammalian cynodont species. In the more basal non-probainognathian group, enamel extends extremely rapidly from cusp to cervix. By contrast, the enamel of mammaliamorphs is gradually accreted, with slow rates of crown extension, more typical of the majority of non-hypsodont crown mammals. These results are consistent with the reduction in dental replacement rate across the non-mammalian cynodont lineage, with greater rates of crown extension required in most non-probainognathians, and slower crown extension rates permitted in mammaliamorphs, which have reduced patterns of dental replacement in comparison with many non-probainognathians. The evolution of mammal-like growth patterns, with faster juvenile growth and more abruptly terminating adult growth, is linked with this reduction in dental replacement rates and may provide an additional explanation for the observed pattern in enamel growth rates. It is possible that the reduction in enamel extension rates in mammaliamorphs reflects an underlying reduction in skeletal growth rates at the time of postcanine formation, due to a more abruptly terminating pattern of adult growth in these more mammal-like, crownward species. PMID:29892415

Epidermal growth factor receptor signaling mediates aldosterone-induced profibrotic responses in kidney

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Sheng, Lili; Yang, Min; Ding, Wei [Department of Nephrology, Shanghai Fifth People' s Hospital, Fudan University, Shanghai 200240 (China); Zhang, Minmin [Department of Nephrology, Shanghai Huashan Hospital, Fudan University, Shanghai 200240 (China); Niu, Jianying [Department of Nephrology, Shanghai Fifth People' s Hospital, Fudan University, Shanghai 200240 (China); Qiao, Zhongdong [School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240 (China); Gu, Yong, E-mail: yonggu@vip.163.com [Department of Nephrology, Shanghai Fifth People' s Hospital, Fudan University, Shanghai 200240 (China); Department of Nephrology, Shanghai Huashan Hospital, Fudan University, Shanghai 200240 (China)

2016-08-01

Aldosterone has been recognized as a risk factor for the development of chronic kidney disease (CKD). Studies have indicated that enhanced activation of epidermal growth factor receptor (EGFR) is associated with the development and progression of renal fibrosis. But if EGFR is involved in aldosterone-induced renal fibrosis is less investigated. In the present study, we examined the effect of erlotinib, an inhibitor of EGFR tyrosine kinase activity, on the progression of aldosterone-induced renal profibrotic responses in a murine model underwent uninephrectomy. Erlotinib-treated rats exhibited relieved structural lesion comparing with rats treated with aldosterone alone, as characterized by glomerular hypertrophy, mesangial cell proliferation and expansion. Also, erlotinib inhibited the expression of TGF-β, α-SMA and mesangial matrix proteins such as collagen Ⅳ and fibronectin. In cultured mesangial cells, inhibition of EGFR also abrogated aldosterone-induced expression of extracellular matrix proteins, cell proliferation and migration. We also demonstrated that aldosterone induced the phosphorylation of EGFR through generation of ROS. And the activation of EGFR resulted in the phosphorylation of ERK1/2, leading to the activation of profibrotic pathways. Taken together, we concluded that aldosterone-mediated tissue fibrosis relies on ROS induced EGFR/ERK activation, highlighting EGFR as a potential therapeutic target for modulating renal fibrosis. - Highlights: • EGFR was involved in aldosterone-induced renal profibrotic responses. • Aldosterone-induced EGFR activation was mediated by MR-dependent ROS generation. • EGFR activated the MAPK/ERK1/2 signaling to promote renal fibrosis.

Macrophage Inducible C-Type Lectin As a Multifunctional Player in Immunity

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Emmanuel C. Patin

2017-07-01

Full Text Available The macrophage-inducible C-type lectin (Mincle is an innate immune receptor on myeloid cells sensing diverse entities including pathogens and damaged cells. Mincle was first described as a receptor for the mycobacterial cell wall glycolipid, trehalose-6,6′-dimycolate, or cord factor, and the mammalian necrotic cell-derived alarmin histone deacetylase complex unit Sin3-associated protein 130. Upon engagement by its ligands, Mincle induces secretion of innate cytokines and other immune mediators modulating inflammation and immunity. Since its discovery more than 25 years ago, the understanding of Mincle’s immune function has made significant advances in recent years. In addition to mediating immune responses to infectious agents, Mincle has been linked to promote tumor progression, autoimmunity, and sterile inflammation; however, further studies are required to completely unravel the complex role of Mincle in these distinct host responses. In this review, we discuss recent findings on Mincle’s biology with an emphasis on its diverse functions in immunity.

Chitin stimulates expression of acidic mammalian chitinase and eotaxin-3 by human sinonasal epithelial cells in vitro.

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Lalaker, Ashley; Nkrumah, Louis; Lee, Won-Kyung; Ramanathan, Murugappan; Lane, Andrew P

2009-01-01

Sinonasal epithelial cells participate in host defense by initiating innate immune mechanisms against potential pathogens. Antimicrobial innate mechanisms have been shown to involve Th1-like inflammatory responses. Although epithelial cells can also be induced by Th2 cytokines to express proeosinophilic mediators, no environmental agents have been identified that promote this effect. Human sinonasal epithelial cells from patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) and controls were harvested and grown in primary culture. Cell cultures were exposed to a range of concentrations of chitin for 24 hours, and mRNA for acidic mammalian chitinase (AMCase), eotaxin-3, and thymic stromal-derived lymphopoietin (TSLP) were assessed. Other cultures were exposed to interleukin 4 (IL- 4) alone and in combination with dust-mite antigen (DMA) for 36 hours. Extracted mRNA and cell culture supernatant were analyzed for expression of AMCase and eotaxin-3. Chitin induced a dose-dependent expression of AMCase and eotaxin-3 mRNA but not TSLP. Patients with recalcitrant CRSwNPs showed lower baseline expression of AMCase when compared with treatment-responsive CRSwNP and less induction of AMCase expression by chitin. DMA did not directly induce expression of AMCase or eotaxin-3. Expression of eotaxin-3 was stimulated by IL-4 and further enhanced with the addition of DMA. Levels of AMCase were not significantly affected by either IL-4 or DMA exposure. In some cases, the combination of IL-4 and DMA was able to induce AMCase expression in cell cultures not producing AMCase at baseline. The abundant biopolymer chitin appears to be recognized by a yet uncharacterized receptor on sinonasal epithelial cells. Chitin stimulates production of AMCase and eotaxin-3, two pro-Th2 effector proteins. This finding suggests the existence of a novel innate immune pathway for local defense against chitin-containing organisms in the sinonasal tract. Dysregulation of this function could

Induced abortion and associated factors in health facilities of Guraghe zone, southern Ethiopia.

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Tesfaye, Gezahegn; Hambisa, Mitiku Teshome; Semahegn, Agumasie

2014-01-01

Unsafe abortion is one of the major medical and public health problems in developing countries including Ethiopia. However, there is a lack of up-to-date and reliable information on induced abortion distribution and its determinant factors in the country. This study was intended to assess induced abortion and associated factors in health facilities of Guraghe zone, Southern Ethiopia. Institution based cross-sectional study was conducted in eight health facilities in Guraghe zone. Client exit interview was conducted on 400 patients using a structured questionnaire. Bivariate and multivariate logistic regression analysis was performed to identify factors associated with induced abortion. Out of 400 women, 75.5% responded that the current pregnancy that ended in abortion is unwanted. However, only 12.3% of the respondents have admitted interference to the current pregnancy. Having more than four pregnancies (AOR = 4.28, CI: (1.24-14.71)), age of 30-34 years (AOR = 0.15, CI: (0.04-0.55)), primary education (AOR = 0.26, CI: (0.13-0.88)), and wanted pregnancy (AOR = 0.44, CI: (0.14-0.65)) were found to have association with induced abortion. The study revealed high level of induced abortion which is underpinned by high magnitude of unwanted pregnancy. There is requirement for widespread expansion of increased access to high quality family planning service and post-abortion care.

Placental Induced Growth Factor (PIGf) in Coronary Artery Disease

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Sundaresan, Alamelu; Carabello, Blaise; Mehta, Satish; Schlegel, Todd; Pellis, Neal; Ott, Mark; Pierson, Duane

2010-01-01

Our previous studies on normal human lymphocytes have shown a five-fold increase (p less than 0.001) in angiogenic inducers such as Placental Induced Growth Factor (PIGf) in physiologically stressful environments such as modeled microgravity, a space analog. This suggests de-regulation of cardiovascular signalling pathways indicated by upregulation of PIGf. In the current study, we measured PIGf in the plasma of 33 patients with and without coronary artery disease (CAD) to investigate whether such disease is associated with increased levels of PIGf. A control consisting of 31 sex matched apparently healthy subjects was also included in the study. We observed that the levels of PIGf in CAD patients were significantly increased compared to those in healthy control subjects (p less than 0.001) and usually increased beyond the clinical threshold level (greater than 27ng/L). The mechanisms leading to up-regulation of angiogenic factors and the adaptation of organisms to stressful environments such as isolation, high altitude, hypoxia, ischemia, microgravity, increased radiation, etc are presently unknown and require further investigation in spaceflight and these other physiologically stressed environments.

Bystander effects on mammalian cells induced by carbon ions

International Nuclear Information System (INIS)

Wang Jufang; Zhao Jing; Ma Qiufeng; Chinese Academy of Sciences, Beijing; Li Weijian; Zhou Guangming; Dang Bingrong; Mao Limin; Feng Yan

2004-01-01

Bystander effects on unirradiated V79 cells were observed by irradiated conditioned medium (ICM) method and co-cultured with carbon-ion-irradiated V79 cells. The results showed that the colony formation efficiency of unirradiated cells is obviously decreased by ICM. After co-culture with carbon-ion-irradiated cells for some time, the colony formation efficiency of co-cultured cells was lower than expected results assuming no bystander effects. The micronucleus frequency and hprt gene mutation rate was almost the same as expected results. Cytotoxic factor(s), which was effective for cell growth but not for micronucleus and mutation on unirradiated cells, might be released by irradiated cells. (authors)

An obligatory role of mind bomb-1 in notch signaling of mammalian development.

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Bon-Kyoung Koo

2007-11-01

Full Text Available The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur and Mind bomb (Mib, cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2(-/- mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.

A review of factors influencing the incidence and severity of plaque-induced gingivitis.

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Trombelli, L; Farina, R

2013-06-01

An individual variation in the gingival inflammatory response to the dental biofilm has been demonstrated. This variability can be observed between individuals with neither quantitative nor qualitative differences in plaque accumulation. The reported significant differences in gingival inflammatory response under quantitatively and/or qualitatively almost identical bacterial challenge suggest that the gingival response to plaque accumulation may be an individual trait, possibly genetic in origin. The most recent classification of periodontal diseases acknowledges that the clinical expression of plaque-induced gingival inflammation can be substantially modified by systemic factors, either inherent to the host or related to environmental influences. The aim of the present literature review is to describe (i) the factors influencing the development of plaque-induced gingivitis as well as (ii) those metabolic, environmental and systemic factors which have a direct impact on the etiopathogenetic pathway of plaque-induced gingivitis, thus altering the nature or course of the gingival inflammatory response to dental biofilm.

Involvement of growth factors and their receptors in radon-induced rat lung tumors

International Nuclear Information System (INIS)

Leung, F.C.; Dagle, G.E.; Cross, F.T.

1992-01-01

In this paper we examine the role of growth factors (GF) and their receptors (GFR) in radon-induced rat lung tumors. Inhalation exposure of radon and its daughters induced lung tumors in rats, but the molecule/cellular mechanisms are not known. Recent evidence suggests that GF/GFR play a critical role in the growth and development of lung cancer in humans and animals. We have developed immunocytochemical methods for identifying sites of production and action of GF/GFR at the cellular level; for example, the avidin-biotin horseradish peroxidase technique. In radon-induced rat epidermoid carcinomas, epidermal growth factor (EGF), EGF-receptors (EGF-R), transforming growth factor alpha (TGF-α), and bombesin were found to be abnormally expressed. These abnormal expressions, mainly associated with epidermoid carcinomas of the lung, were not found in any other lung tumor types. Our data suggest that EGF, EGF-R, TGF-α, and bombesin are involved in radon oncogenesis in rat lungs, especially in epidermoid carcinomas, possibly through the autocrine/paracrine pathway

Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

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Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

2014-06-03

Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

Femoral loading mechanics in the Virginia opossum, Didelphis virginiana: torsion and mediolateral bending in mammalian locomotion.

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Gosnell, W Casey; Butcher, Michael T; Maie, Takashi; Blob, Richard W

2011-10-15

Studies of limb bone loading in terrestrial mammals have typically found anteroposterior bending to be the primary loading regime, with torsion contributing minimally. However, previous studies have focused on large, cursorial eutherian species in which the limbs are held essentially upright. Recent in vivo strain data from the Virginia opossum (Didelphis virginiana), a marsupial that uses a crouched rather than an upright limb posture, have indicated that its femur experiences appreciable torsion during locomotion as well as strong mediolateral bending. The elevated femoral torsion and strong mediolateral bending observed in D. virginiana might result from external forces such as a medial inclination of the ground reaction force (GRF), internal forces deriving from a crouched limb posture, or a combination of these factors. To evaluate the mechanism underlying the loading regime of opossum femora, we filmed D. virginiana running over a force platform, allowing us to measure the magnitude of the GRF and its three-dimensional orientation relative to the limb, facilitating estimates of limb bone stresses. This three-dimensional analysis also allows evaluations of muscular forces, particularly those of hip adductor muscles, in the appropriate anatomical plane to a greater degree than previous two-dimensional analyses. At peak GRF and stress magnitudes, the GRF is oriented nearly vertically, inducing a strong abductor moment at the hip that is countered by adductor muscles on the medial aspect of the femur that place this surface in compression and induce mediolateral bending, corroborating and explaining loading patterns that were identified in strain analyses. The crouched orientation of the femur during stance in opossums also contributes to levels of femoral torsion as high as those seen in many reptilian taxa. Femoral safety factors were as high as those of non-avian reptiles and greater than those of upright, cursorial mammals, primarily because the load

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

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Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

2015-08-03

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells

International Nuclear Information System (INIS)

Zeeland, A.A. van; Filon, A.R.; Natarajan, A.T.; Bussmann, C.J.M.; Degrassi, F.; Kesteren-van Leeuwen, A.C. van; Palitti, F.; Rome Univ.

1982-01-01

The influence of aphidicolin, an inhibitor of polymerase α, on UV-induced repair replication in human skin fibroblasts, as well as in HeLa cells, was determined. In growing fibroblasts and in HeLa cells, aphidicolin had a potentiating effect on UV-induced repair replication, whereas in fibroblasts grown to confluency, aphidicolin had an inhibitory effect. This inhibitory effect was stronger when measured in the presence of hydroxyurea. In HeLa cells the presence of both aphidicolin and hydroxyurea also had an inhibitory effect, but in the presence of hydroxyurea alone, UV-induced repair replication was enhanced. The results of these studies can be explained on the basis of differences in deoxyribonucleotide triphosphate pool sizes in growing and confluent cells. Post-treatment of X-irradiated human lymphocytes in the G 0 and G 1 stages with aphidicolin increased the frequencies of X-ray-induced chromosomal aberrations. Such an increase was not observed in G 1 cells of CHO after similar treatment with X-rays and aphidicolin. However, treatment with aphidicolin, in the G 2 stage, increased the frequencies of induced chromatid breaks. The significance of these results is discussed. (orig.)

The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

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Angeliki Lyssimachou

Full Text Available Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT, which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR and peroxisome proliferator-activated receptor gamma (PPARγ. In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

Science.gov (United States)

Lyssimachou, Angeliki; Santos, Joana G; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C Marisa R; Teixeira, Catarina; Castro, L Filipe C; Santos, Miguel M

2015-01-01

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

International Nuclear Information System (INIS)

Estabrook, Ronald W.

2005-01-01

The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11β-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O 18 studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17α-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17α-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11β-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction

Molecular Markers for Interspecies Transmission of Avian Influenza Viruses in Mammalian Hosts

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Lee, Taehyung

2017-01-01

In the last decade, a wide range of avian influenza viruses (AIVs) have infected various mammalian hosts and continuously threaten both human and animal health. It is a result of overcoming the inter-species barrier which is mostly associated with gene reassortment and accumulation of mutations in their gene segments. Several recent studies have shed insights into the phenotypic and genetic changes that are involved in the interspecies transmission of AIVs. These studies have a major focus on transmission from avian to mammalian species due to the high zoonotic potential of the viruses. As more mammalian species have been infected with these viruses, there is higher risk of genetic evolution of these viruses that may lead to the next human pandemic which represents and raises public health concern. Thus, understanding the mechanism of interspecies transmission and molecular determinants through which the emerging AIVs can acquire the ability to transmit to humans and other mammals is an important key in evaluating the potential risk caused by AIVs among humans. Here, we summarize previous and recent studies on molecular markers that are specifically involved in the transmission of avian-derived influenza viruses to various mammalian hosts including humans, pigs, horses, dogs, and marine mammals. PMID:29236050

Leadership in Mammalian Societies: Emergence, Distribution, Power, and Payoff.

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Smith, Jennifer E; Gavrilets, Sergey; Mulder, Monique Borgerhoff; Hooper, Paul L; Mouden, Claire El; Nettle, Daniel; Hauert, Christoph; Hill, Kim; Perry, Susan; Pusey, Anne E; van Vugt, Mark; Smith, Eric Alden

2016-01-01

Leadership is an active area of research in both the biological and social sciences. This review provides a transdisciplinary synthesis of biological and social-science views of leadership from an evolutionary perspective, and examines patterns of leadership in a set of small-scale human and non-human mammalian societies. We review empirical and theoretical work on leadership in four domains: movement, food acquisition, within-group conflict mediation, and between-group interactions. We categorize patterns of variation in leadership in five dimensions: distribution (across individuals), emergence (achieved versus inherited), power, relative payoff to leadership, and generality (across domains). We find that human leadership exhibits commonalities with and differences from the broader mammalian pattern, raising interesting theoretical and empirical issues. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

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Johnson, Wesley; Dai, Changsheng; Liu, Jun; Wang, Xian; Luu, Devin K; Zhang, Zhuoran; Ru, Changhai; Zhou, Chao; Tan, Min; Pu, Huayan; Xie, Shaorong; Peng, Yan; Luo, Jun; Sun, Yu

2018-03-01

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Effect of mammalian kidney osmolytes on the folding pathway of sheep serum albumin.

Science.gov (United States)

Dar, Mohammad Aasif; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2017-04-01

Recently, we had published that urea-induced denaturation curves of optical properties of sheep serum albumin (SSA) are biphasic with a stable intermediate that has characteristics of molten globule (MG) state. In this study, we have extended the work by carrying out urea- and guanidinium chloride (GdmCl)-induced denaturations of SSA in the presence of naturally occurring mammalian kidney osmolytes, namely, sorbitol, myo-inositol and glycine betaine. We have observed that all these osmolytes (i) transform this biphasic transition into a co-operative, two-state transition and (ii) increase the stability of the protein in terms of midpoint of denaturation (C m ) and Gibbs free energy change in the absence of both denaturants (ΔG D 0 ). The relative effectiveness of different osmolytes on the stability of SSA follows the order: glycine betaine>myo-inositol>sorbitol. In this paper, we also report that kidney osmolytes destabilize MG state by shifting the equilibrium, native state↔MG state toward the left. This study will be helpful in understanding the existence of osmolytes in kidney and their role in folding of kidney proteins soaked with urea. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells.

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Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N; Wu, Zetang; Crary, Monica; Buckley, Kenneth J; Bisaro, David M; Parris, Deborah S

2012-03-01

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.

Fossilized Mammalian Erythrocytes Associated With a Tick Reveal Ancient Piroplasms.

Science.gov (United States)

Poinar, George

2017-07-01

Ticks transmit a variety of pathogenic organisms to vertebrates, especially mammals. The fossil record of such associations is extremely rare. An engorged nymphal tick of the genus Ambylomma in Dominican amber was surrounded by erythrocytes from its mammalian host. Some of the exposed erythrocytes contained developmental stages of a hemoprotozoan resembling members of the Order Piroplasmida. The fossil piroplasm is described, its stages compared with those of extant piroplasms, and reasons provided why the mammalian host could have been a primate. The parasites were also found in the gut epithelial cells and body cavity of the fossil tick. Aside from providing the first fossil mammalian red blood cells and the first fossil intraerythrocytic hemoparasites, the present discovery shows that tick-piroplasm associations were already well established in the Tertiary. This discovery provides a timescale that can be used in future studies on the evolution of the Piroplasmida. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com Version of Record, first published online March 20, 2017 with fixed content and layout in compliance with Art. 8.1.3.2 ICZN.

Optical sorting and photo-transfection of mammalian cells

CSIR Research Space (South Africa)

Mthunzi, P

2010-02-01

Full Text Available and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human...

Induction, regulation, degradation, and biological significance of mammalian metallothioneins.

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Miles, A T; Hawksworth, G M; Beattie, J H; Rodilla, V

2000-01-01

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

Structure of the Deactive State of Mammalian Respiratory Complex I.

Science.gov (United States)

Blaza, James N; Vinothkumar, Kutti R; Hirst, Judy

2018-02-06

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly active preparation of Bos taurus complex I into the biochemically defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. We show that the deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding to the NADH-reduced enzyme templates their reordering. Our structure both rationalizes biochemical data on the deactive state and offers new insights into its physiological and cellular roles. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

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Hao, Nan; Palmer, Adam C; Dodd, Ian B; Shearwin, Keith E

2017-03-15

Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat

Directory of Open Access Journals (Sweden)

Groneberg David A

2008-02-01

Full Text Available Abstract Background Platelet activating factor and tachykinins (substance P, neurokinin A, neurokinin B are important mediators contributing to increased airway secretion in the context of different types of respiratory diseases including acute and chronic asthma. Leukotriene receptor antagonists are recommended as add-on therapy for this disease. The cys-leukotriene-1 receptor antagonist montelukast has been used in clinical asthma therapy during the last years. Besides its inhibitory action on bronchoconstriction, only little is known about its effects on airway secretions. Therefore, the aim of this study was to evaluate the effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity. Methods The effects of montelukast on platelet activating factor- and tachykinin induced tracheal secretory activity in the rat were assessed by quantification of secreted 35SO4 labelled mucus macromolecules using the modified Ussing chamber technique. Results Platelet activating factor potently stimulated airway secretion, which was completely inhibited by the platelet activating factor receptor antagonist WEB 2086 and montelukast. In contrast, montelukast had no effect on tachykinin induced tracheal secretory activity. Conclusion Cys-leukotriene-1 receptor antagonism by montelukast reverses the secretagogue properties of platelet activating factor to the same degree as the specific platelet activating factor antagonist WEB 2086 but has no influence on treacheal secretion elicited by tachykinins. These results suggest a role of montelukast in the signal transduction pathway of platelet activating factor induced secretory activity of the airways and may further explain the beneficial properties of cys-leukotriene-1 receptor antagonists.

Role of tumor necrosis factor in flavone acetic acid-induced tumor vasculature shutdown

International Nuclear Information System (INIS)

Mahadevan, V.; Malik, S.T.; Meager, A.; Fiers, W.; Lewis, G.P.; Hart, I.R.

1990-01-01

Flavone acetic acid (FAA), a novel investigational antitumor agent, has been shown to cause early vascular shutdown in several experimental murine tumors, and this phenomenon is believed to be crucial to FAA's antitumor effects. However, the basis of this FAA-induced tumor vascular shutdown is unknown. In this study a radioactive tracer-clearance technique has been used as an objective indication of tumor blood flow to show that i.p. administered FAA induces a progressive and sustained reduction in blood flow in a colon 26 tumor growing s.c. in syngeneic mice. As early as 1 h after administration, there was a significant increase in the t1/2 clearance value for intratumorally injected 133Xe, reaching a peak at 3 h (117.3 +/- 36.4 versus 7.8 +/- 0.85 min for controls). Significant inhibition of blood flow was still apparent 48 h after a single injection of drug. This FAA-induced vascular shutdown was virtually abolished in tumor-bearing mice pretreated with an antiserum against tumor necrosis factor, while no such effect was observed in controls pretreated with nonimmune serum (t1/2 of 10.8 +/- 1.2 versus 65.6 +/- 8.0 min for controls). Furthermore, in vitro FAA was seen to induce tumor necrosis factor secretion from murine peritoneal cells and splenocytes. These studies suggest that FAA-induced tumor vascular shutdown in the colon 26 tumor is mediated by tumor necrosis factor

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

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Li, Ping; Noegel, Angelika A

2015-11-16

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation of iron.

Science.gov (United States)

Park, Sung-Soo; Bae, Insoo; Lee, Yong J

2008-04-15

Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF-1alpha is continuously degraded by the ubiquitin-proteasome pathway through pVHL (von Hippel-Lindau tumor suppressor protein). Under hypoxic conditions, HIF-1alpha is stabilized and induces the transcription of HIF-1 target genes. Quercetin, a flavonoid with anti-oxidant, anti-inflammatory, and kinase modulating properties, has been found to induce HIF-1alpha accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin-mediated HIF-1alpha accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF-1alpha can be induced through the phosphatidylinositol 3-kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant-type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin-induced HIF-1alpha accumulation. And we observed that HIF-1alpha accumulated by quercetin is not ubiquitinated and the interaction of HIF-1alpha with pVHL is reduced, compared with HIF-1alpha accumulated by the proteasome inhibitor MG132. The use of quercetin's analogues showed that only quercetin and galangin induce HIF-1/2alpha accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF-1/2alpha proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF-1/2alpha in normoxia by hindering PHD through chelating iron ions.

Growth Factors and Tension-Induced Skeletal Muscle Growth

Science.gov (United States)

Vandenburgh, Herman H.

1994-01-01

The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we

Down-regulation of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor by HEXIM1 attenuates myocardial angiogenesis in hypoxic mice.

Science.gov (United States)

Yoshikawa, Noritada; Shimizu, Noriaki; Ojima, Hidenori; Kobayashi, Hiroshi; Hosono, Osamu; Tanaka, Hirotoshi

2014-10-24

Pulmonary hypertension (PH) sustains elevation of pulmonary vascular resistance and ultimately leads to right ventricular (RV) hypertrophy and failure and death. Recently, proangiogenic factors hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) have been known to promote left ventricular myocardial angiogenesis and lead to cardiac hypertrophy, and this would be involved in RV hypertrophy of PH patients. Previously, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced cardiomyocyte hypertrophy and hypertrophic genes expression, and that cardiomyocyte-specific HEXIM1 transgenic mice ameliorates RV hypertrophy in hypoxia-induced PH model. Given these results, here we analyzed the effect of HEXIM1 on the expression of HIF-1α and VEGF and on myocardial angiogenesis of RV in PH. We revealed that overexpression of HEXIM1 prevented hypoxia-induced expression of HIF-1α protein and its target genes including VEGF in the cultured cardiac myocytes and fibroblasts, and that cardiomyocyte-specific HEXIM1 transgenic mice repressed RV myocardial angiogenesis in hypoxia-induced PH model. Thus, we conclude that HEXIM1 could prevent RV hypertrophy, at least in part, via suppression of myocardial angiogenesis through down-regulation of HIF-1α and VEGF in the myocardium under hypoxic condition. Copyright © 2014 Elsevier Inc. All rights reserved.

Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

NARCIS (Netherlands)

Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic

Monocytes can be induced by lipopolysaccharide-triggered T lymphocytes to express functional factor VII/VIIa protease activity

OpenAIRE

1984-01-01

In the present study we demonstrate that human monocytes can be induced by the model stimulus, lipopolysaccharide (LPS), to produce and assemble on their surface functional Factor VII/VIIa. This protease was not induced in relatively purified monocytes alone following exposure to LPS; but was induced in the presence of Leu-3a positive helper/inducer T cells. The Factor VII/VIIa protease activity represented 35-40% of the potential initiating activity for the extrinsic coagulation pathway and ...

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

Energy Technology Data Exchange (ETDEWEB)

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Hidalgo, Cecilia [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Lavandero, Sergio, E-mail: slavander@uchile.cl [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile)

2009-10-09

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

International Nuclear Information System (INIS)

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo; Hidalgo, Cecilia; Lavandero, Sergio

2009-01-01

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal α-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Yang, Bin [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Li, Wei [Department of Gerontology, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Zheng, Qichang [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Qin, Tao [Department of Hepatobiliary Pancreatic Surgery, People' s Hospital of Zhengzhou University, School of Medicine, Zhengzhou University, Zhengzhou 450003 (China); Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Liu, Sanguang, E-mail: sanguang1998@sina.com [Department of Hepatobiliary Surgery, The Second Hospital, Hebei Medical University, Shijiazhuang 050000 (China); Song, Zifang, E-mail: zsong@hust.edu.cn [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China)

2015-07-17

Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

International Nuclear Information System (INIS)

Yang, Bin; Li, Wei; Zheng, Qichang; Qin, Tao; Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen; Liu, Sanguang; Song, Zifang

2015-01-01

Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation

Evidence that platelet-derived growth factor may be a novel endogenous pyrogen in the central nervous system.

Science.gov (United States)

Pelá, I R; Ferreira, M E; Melo, M C; Silva, C A; Coelho, M M; Valenzuela, C F

2000-05-01

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory actions in the mammalian central nervous system (CNS). Like the cytokines, PDGF primarily signals through tyrosine phosphorylation-dependent pathways that activate multiple intracellular molecules including Janus family kinases. We previously showed that microinjection of PDGF-BB into the lateral ventricle induced a febrile response in rats that was reduced by pretreatment with Win 41662, a potent inhibitor of PDGF receptors (Pelá IR, Ferreira MES, Melo MCC, Silva CAA, and Valenzuela CF. Ann NY Acad Sci 856: 289-293, 1998). In this study, we further characterized the role of PDGF-BB in the febrile response in rats. Microinjection of PDGF-BB into the third ventricle produced a dose-dependent increase in colonic temperature that peaked 3-4 h postinjection. Win 41662 attenuated fever induced by intraperitoneal injection of bacterial lipopolysaccharide, suggesting that endogenous PDGF participates in the febrile response to this exogenous pyrogen. Importantly, febrile responses induced by tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were unchanged by Win 41662. Both indomethacin and dexamethasone blocked the PDGF-BB-induced increase in colonic temperature, and, therefore, we postulate that PDGF-BB may act via prostaglandin- and/or inducible enzyme-dependent pathways. Thus our findings suggest that PDGF-BB is an endogenous CNS mediator of the febrile response in rats.

Silver nanoparticle-enriched diamond-like carbon implant modification as a mammalian cell compatible surface with antimicrobial properties

Science.gov (United States)

Gorzelanny, Christian; Kmeth, Ralf; Obermeier, Andreas; Bauer, Alexander T.; Halter, Natalia; Kümpel, Katharina; Schneider, Matthias F.; Wixforth, Achim; Gollwitzer, Hans; Burgkart, Rainer; Stritzker, Bernd; Schneider, Stefan W.

2016-01-01

The implant-bone interface is the scene of competition between microorganisms and distinct types of tissue cells. In the past, various strategies have been followed to support bony integration and to prevent bacterial implant-associated infections. In the present study we investigated the biological properties of diamond-like carbon (DLC) surfaces containing silver nanoparticles. DLC is a promising material for the modification of medical implants providing high mechanical and chemical stability and a high degree of biocompatibility. DLC surface modifications with varying silver concentrations were generated on medical-grade titanium discs, using plasma immersion ion implantation-induced densification of silver nanoparticle-containing polyvinylpyrrolidone polymer solutions. Immersion of implants in aqueous liquids resulted in a rapid silver release reducing the growth of surface-bound and planktonic Staphylococcus aureus and Staphylococcus epidermidis. Due to the fast and transient release of silver ions from the modified implants, the surfaces became biocompatible, ensuring growth of mammalian cells. Human endothelial cells retained their cellular differentiation as indicated by the intracellular formation of Weibel-Palade bodies and a high responsiveness towards histamine. Our findings indicate that the integration of silver nanoparticles into DLC prevents bacterial colonization due to a fast initial release of silver ions, facilitating the growth of silver susceptible mammalian cells subsequently. PMID:26955791

Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1

Directory of Open Access Journals (Sweden)

Soham Chanda

2014-08-01

Full Text Available Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs into mature induced neuronal (iN cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

Science.gov (United States)

Xiong, Xianrong; Fu, Mei; Lan, Daoliang; Li, Jian; Zi, Xiangdong; Zhong, Jincheng

2015-01-01

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

The evolution of gene expression levels in mammalian organs

DEFF Research Database (Denmark)

Brawand, David; Soumillon, Magali; Necsulea, Anamaria

2011-01-01

and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped......Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across...... ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages...

The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

Science.gov (United States)

Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

2015-01-01

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

Stable isotope time-series in mammalian teeth: In situ δ18O from the innermost enamel layer

Science.gov (United States)

Blumenthal, Scott A.; Cerling, Thure E.; Chritz, Kendra L.; Bromage, Timothy G.; Kozdon, Reinhard; Valley, John W.

2014-01-01

Stable carbon and oxygen isotope ratios in mammalian tooth enamel are commonly used to understand the diets and environments of modern and fossil animals. Isotope variation during the period of enamel formation can be recovered by intra-tooth microsampling along the direction of growth. However, conventional sampling of the enamel surface provides highly time-averaged records in part due to amelogenesis. We use backscattered electron imaging in the scanning electron microscope (BSE-SEM) to evaluate enamel mineralization in developing teeth from one rodent and two ungulates. Gray levels from BSE-SEM images suggest that the innermost enamel layer, <20 μm from the enamel-dentine junction, is highly mineralized early in enamel maturation and therefore may record a less attenuated isotopic signal than other layers. We sampled the right maxillary incisor from a woodrat subjected to an experimentally induced water-switch during the period of tooth development, and demonstrate that secondary ion mass spectrometry (SIMS) can be used to obtain δ18O values with 4-5-μm spots from mammalian tooth enamel. We also demonstrate that SIMS can be used to discretely sample the innermost enamel layer, which is too narrow for conventional microdrilling or laser ablation. An abrupt δ18O switch of 16.0‰ was captured in breath CO2, a proxy for body water, while a laser ablation enamel surface intra-tooth profile of the left incisor captured a δ18O range of 12.1‰. The innermost enamel profile captured a δ18O range of 15.7‰, which approaches the full magnitude of δ18O variation in the input signal. This approach will likely be most beneficial in taxa such as large mammalian herbivores, whose teeth are characterized by less rapid mineralization and therefore greater attenuation of the enamel isotope signal.

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

International Nuclear Information System (INIS)

Lloyd, Richard E.

2015-01-01

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Lloyd, Richard E., E-mail: rlloyd@bcm.edu

2015-05-15

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells

International Nuclear Information System (INIS)

Protic-Sabljic, M.; Tuteja, N.; Munson, P.J.; Hauser, J.; Kraemer, K.H.; Dixon, K.

1986-01-01

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells

The Poxvirus C7L Host Range Factor Superfamily

OpenAIRE

Liu, Jia; Rothenburg, Stefan; McFadden, Grant

2012-01-01

Host range factors, expressed by the poxvirus family, determine the host tropism of species, tissue, and cell specificity. C7L family members exist in the genomes of most sequenced mammalian poxviruses, suggesting an evolutionarily conserved effort adapting to the hosts. In general, C7L orthologs influence the host tropism in mammalian cell culture, and for some poxviruses it is essential for the complete viral life cycle in vitro and in vivo. The C7L family members lack obvious sequence homo...

Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

Science.gov (United States)

Nally, Jarlath E; Grassmann, Andre A; Planchon, Sébastien; Sergeant, Kjell; Renaut, Jenny; Seshu, Janakiram; McBride, Alan J; Caimano, Melissa J

2017-01-01

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p 1.25 or expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with

Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

Directory of Open Access Journals (Sweden)

Jarlath E. Nally

2017-08-01

Full Text Available Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE. Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < −1.25 across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30

Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

International Nuclear Information System (INIS)

Jeong, Wooyoung; Bazer, Fuller W.; Song, Gwonhwa; Kim, Jinyoung

2016-01-01

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

Energy Technology Data Exchange (ETDEWEB)

Jeong, Wooyoung [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of); Bazer, Fuller W. [Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A& M University, College Station, TX (United States); Song, Gwonhwa, E-mail: ghsong@korea.ac.kr [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Jinyoung, E-mail: jinyoungkim@dankook.ac.kr [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of)

2016-01-08

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

International Nuclear Information System (INIS)

Permana, Paska A.; Menge, Christopher; Reaven, Peter D.

2006-01-01

Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-κB) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-κB inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity

Cardiorespiratory interactions previously identified as mammalian are present in the primitive lungfish.

Science.gov (United States)

Monteiro, Diana A; Taylor, Edwin W; Sartori, Marina R; Cruz, André L; Rantin, Francisco T; Leite, Cleo A C

2018-02-01

The present study has revealed that the lungfish has both structural and functional features of its system for physiological control of heart rate, previously considered solely mammalian, that together generate variability (HRV). Ultrastructural and electrophysiological investigation revealed that the nerves connecting the brain to the heart are myelinated, conferring rapid conduction velocities, comparable to mammalian fibers that generate instantaneous changes in heart rate at the onset of each air breath. These respiration-related changes in beat-to-beat cardiac intervals were detected by complex analysis of HRV and shown to maximize oxygen uptake per breath, a causal relationship never conclusively demonstrated in mammals. Cardiac vagal preganglionic neurons, responsible for controlling heart rate via the parasympathetic vagus nerve, were shown to have multiple locations, chiefly within the dorsal vagal motor nucleus that may enable interactive control of the circulatory and respiratory systems, similar to that described for tetrapods. The present illustration of an apparently highly evolved control system for HRV in a fish with a proven ancient lineage, based on paleontological, morphological, and recent genetic evidence, questions much of the anthropocentric thinking implied by some mammalian physiologists and encouraged by many psychobiologists. It is possible that some characteristics of mammalian respiratory sinus arrhythmia, for which functional roles have been sought, are evolutionary relics that had their physiological role defined in ancient representatives of the vertebrates with undivided circulatory systems.

Cardiorespiratory interactions previously identified as mammalian are present in the primitive lungfish

Science.gov (United States)

Monteiro, Diana A.; Taylor, Edwin W.; Sartori, Marina R.; Cruz, André L.; Rantin, Francisco T.; Leite, Cleo A. C.

2018-01-01

The present study has revealed that the lungfish has both structural and functional features of its system for physiological control of heart rate, previously considered solely mammalian, that together generate variability (HRV). Ultrastructural and electrophysiological investigation revealed that the nerves connecting the brain to the heart are myelinated, conferring rapid conduction velocities, comparable to mammalian fibers that generate instantaneous changes in heart rate at the onset of each air breath. These respiration-related changes in beat-to-beat cardiac intervals were detected by complex analysis of HRV and shown to maximize oxygen uptake per breath, a causal relationship never conclusively demonstrated in mammals. Cardiac vagal preganglionic neurons, responsible for controlling heart rate via the parasympathetic vagus nerve, were shown to have multiple locations, chiefly within the dorsal vagal motor nucleus that may enable interactive control of the circulatory and respiratory systems, similar to that described for tetrapods. The present illustration of an apparently highly evolved control system for HRV in a fish with a proven ancient lineage, based on paleontological, morphological, and recent genetic evidence, questions much of the anthropocentric thinking implied by some mammalian physiologists and encouraged by many psychobiologists. It is possible that some characteristics of mammalian respiratory sinus arrhythmia, for which functional roles have been sought, are evolutionary relics that had their physiological role defined in ancient representatives of the vertebrates with undivided circulatory systems. PMID:29507882

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

Science.gov (United States)

Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia

Measurement of Heme Synthesis Levels in Mammalian Cells.

Science.gov (United States)

Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

2015-07-09

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival

Directory of Open Access Journals (Sweden)

Joffrey ePelletier

2012-02-01

Full Text Available The hypoxia-inducible factor 1 (HIF-1, in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1, were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these hypoxia-preconditioned cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO2 acts as an alarm that prepares the cells to face subsequent nutrient depletion and to survive.

Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

Energy Technology Data Exchange (ETDEWEB)

Pelletier, Joffrey; Bellot, Grégory [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France); Gounon, Pierre; Lacas-Gervais, Sandra [Centre Commun de Microscopie Appliquée, University of Nice-Sophia Antipolis, Nice (France); Pouysségur, Jacques; Mazure, Nathalie M., E-mail: mazure@unice.fr [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France)

2012-02-28

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO{sub 2} acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

International Nuclear Information System (INIS)

Pelletier, Joffrey; Bellot, Grégory; Gounon, Pierre; Lacas-Gervais, Sandra; Pouysségur, Jacques; Mazure, Nathalie M.

2012-01-01

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO 2 acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

National Research Council Canada - National Science Library

Altman, Amy

2001-01-01

The main goal of the research proposal was to develop an assay system for studying the specific genetic elements, if any, involved in the initiation of DNA replication in mammalian cells as outlined in Task 1...

Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

Directory of Open Access Journals (Sweden)