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Sample records for cell mutants defective

  1. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

  2. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    Directory of Open Access Journals (Sweden)

    Sho W Suzuki

    2011-02-01

    Full Text Available Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA. We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  3. Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in Chinese hamster V79 cells by tritium suicide

    International Nuclear Information System (INIS)

    Bryant, R.E.; Schauer, I.E.; Hatcher, D.G.

    1981-01-01

    Tritium suicide was shown to be a highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of ( 3 H) hypoxanthine for 5 or 10 min, followed by storage of 3 H-labelled cells at -70 0 C for 4-10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of ( 3 H)hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent Ksub(m) for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine. (orig.)

  4. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

    KAUST Repository

    Hudik, Elodie

    2014-07-18

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  5. Cell lines derived from a Medaka radiation-sensitive mutant have defects in DNA double-strand break responses

    International Nuclear Information System (INIS)

    Hidaka, Masayuki; Oda, Shoji; Mitani, Hiroshi; Kuwahara, Yoshikazu; Fukumoto, Manabu

    2010-01-01

    It was reported that the radiation-sensitive Medaka mutant 'ric1' has a defect in the repair of DNA double-strand breaks (DSBs) induced by γ-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to γ-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after γ-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (γH2AX) foci were formed after γ-irradiation, however, the γH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of γH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro. (author)

  6. Cell-cycle kinetics and ultraviolet light survival in UV-1, a Chinese hamster ovary cell mutant defective in post-replication recovery

    International Nuclear Information System (INIS)

    Collins, A.

    1982-01-01

    UV-I, an ultraviolet-sensitive mutant of CHO-KI, is abnormally slow to recover from the inhibition of DNA synthesis caused by u.v. irradiation. When synchronized UV-I cells are irradiated in G 1 , their movement into S phase is unaltered, but thymidine incorporation is depressed. When irradiated in S phase, again incorporation is more depressed, and S phase suffers a greater delay in UV-I than in the parent cell. UV-I and its parent have similar capacities for excision repair of u.v.-induced damage inflicted in G 1 , and so enter S phase with similar amounts of unrepaired damage. The single-cell survival was measured after irradiation at different points in the cell cycle. The mutant and parent cells have similar values of D 0 (mean lethal dose) except in mitosis, when the parent cell shows markedly greater resistance to u.v. irradiation. Dsub(q) (quasi-threshold dose) is fairly constant for the parent cell, but in UV-I it falls to a minimum in S phase. The responses of UV-I to u.v. irradiation are generally consistent with its known defect in post-replication recovery, i.e. the ability to join up the abnormally small DNA fragments synthesized on a u.v.-damaged template. (author)

  7. Biofilm formation-defective mutants in Pseudomonas putida.

    Science.gov (United States)

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

    Science.gov (United States)

    Barbaro, Vanessa; Nasti, Annamaria A; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin E; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-06-01

    Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600. © 2016 AlphaMed Press.

  9. Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Umeki, Shigeko; Akiyama, Mitoshi

    1991-06-01

    The frequency of mutant T lymphocytes defective in T-cell receptor gene (α or β) expression was measured using the two-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of ≥ 1.5 Gy) and 125 of whom were distally exposed (DS86 doses of 228 Th formerly used for radiodiagnosis. In addition, thyroid disease patients treated with 131 I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for the measurement of recent exposures to genotoxic agents. (author)

  10. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

    Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

  11. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    Science.gov (United States)

    Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

    2009-01-01

    Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs. PMID:20161699

  12. Hair defects in Hoxc13 mutant mice.

    Science.gov (United States)

    Godwin, A R; Capecchi, M R

    1999-12-01

    Hox genes encode transcription factors that are important during normal embryonic development of diverse organisms including vertebrates. In mammals, Hox genes are responsible for conferring regional identity in embryonic tissues, including the limb bud, the neural tube, the presomitic mesoderm and the intestinal tract. Recent studies have demonstrated expression of Hox genes in skin and hair follicles, suggesting potential functions for these genes in epidermal appendages. These studies are reviewed here with emphasis on Hoxc13, as Hoxc13 mutants are the first Hox mutants to demonstrate overt hair defects. In addition, because Hoxc13 does not show regionally restricted expression in the skin, as demonstrated for other Hox genes, the potentially different roles of Hoxc13 versus other Hox genes in the skin are discussed.

  13. The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B

    International Nuclear Information System (INIS)

    Helbig, R.; Speit, G.; Zdzienicka, M.Z.

    1995-01-01

    The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible change in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS. 28 refs., 4 figs., 3 tabs

  14. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    OpenAIRE

    Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

    2010-01-01

    Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic ...

  15. The defect in the AT-like hamster cell mutants is complemented by mouse chromosome 9 but not by any of the human chromosomes

    NARCIS (Netherlands)

    W. Jongmans; G. Verhaegh (Gerald); N.G.J. Jaspers (Nicolaas); P. Demant (Peter); A.T. Natarajan; Y. Shiloh (Yosef); M. Oshimura (Mitsuo); E.J. Stanbridge (Eric); R.S. Athwal (Raghbir); A.P. Cuthbert (Andrew); R.F. Newbold (Robert); P.H.M. Lohmann (Paul); M.Z. Zdzienicka (Malgorzata)

    1996-01-01

    textabstractX-ray-sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following

  16. Isolation of a Defective Prion Mutant from Natural Scrapie.

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    Ilaria Vanni

    2016-11-01

    Full Text Available It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA. Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155-231 and ∼80-231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of "authentic" defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90-155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular

  17. Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress.

    Science.gov (United States)

    Sánchez-Corrionero, Álvaro; Sánchez-Vicente, Inmaculada; González-Pérez, Sergio; Corrales, Ascensión; Krieger-Liszkay, Anja; Lorenzo, Óscar; Arellano, Juan B

    2017-09-01

    The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen ( 1 O 2 ) and activating 1 O 2 -mediated cell death. Thylakoids of aba1 produced twice as much 1 O 2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1 O 2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1 O 2 generation in aba1. Up-regulation of the 1 O 2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1 O 2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1 O 2 -mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1 O 2 -overproducing mutants of Arabidopsis for investigating 1 O 2 -mediated cell death. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC.

    Science.gov (United States)

    Abiko, Yuki; Nagano, Keiji; Yoshida, Yasuo; Yoshimura, Fuminobu

    2014-01-01

    Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteins were Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties.

  19. Profiling of the toxicity mechanisms of coated and uncoated silver nanoparticles to yeast Saccharomyces cerevisiae BY4741 using a set of its 9 single-gene deletion mutants defective in oxidative stress response, cell wall or membrane integrity and endocytosis.

    Science.gov (United States)

    Käosaar, Sandra; Kahru, Anne; Mantecca, Paride; Kasemets, Kaja

    2016-09-01

    The widespread use of nanosilver in various antibacterial, antifungal, and antiviral products warrants the studies of the toxicity pathways of nanosilver-enabled materials toward microbes and viruses. We profiled the toxicity mechanisms of uncoated, casein-coated, and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) using Saccharomyces cerevisiae wild-type (wt) and its 9 single-gene deletion mutants defective in oxidative stress (OS) defense, cell wall/membrane integrity, and endocytosis. The 48-h growth inhibition assay in organic-rich growth medium and 24-h cell viability assay in deionized (DI) water were applied whereas AgNO3, H2O2, and SDS served as positive controls. Both coated AgNPs (primary size 8-12nm) were significantly more toxic than the uncoated (~85nm) AgNPs. All studied AgNPs were ~30 times more toxic if exposed to yeast cells in DI water than in the rich growth medium: the IC50 based on nominal concentration of AgNPs in the growth inhibition test ranged from 77 to 576mg Ag/L and in the cell viability test from 2.7 to 18.7mg Ag/L, respectively. Confocal microscopy showed that wt but not endocytosis mutant (end3Δ) internalized AgNPs. Comparison of toxicity patterns of wt and mutant strains defective in OS defense and membrane integrity revealed that the toxicity of the studied AgNPs to S. cerevisiae was not caused by the OS or cell wall/membrane permeabilization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

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    Christopher M Brennan

    Full Text Available The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA, the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

  1. Characterization of a Saccharomyces cerevisiae mutant defective in inositol sphingolipid synthesis

    International Nuclear Information System (INIS)

    Pinto, W.J.; Wells, G.B.; Williams, A.C.; Anderson, K.A.; Teater, E.C.; Lester, R.L.

    1986-01-01

    A mutant of S. cerevisiae, auxotrophic for the sphingolipid long chain bases sphinganine or 4-hydroxysphinganine has been further characterized. The mutant is defective in the first step in the synthesis of sphingolipid long chain bases, the formation of 3-keto-sphinganine from palmitoyl CoA and L-serine. Crude membranes from wild type cells exhibit a palmitoyl CoA dependent conversion of [ 3 H]serine to a labeled product with the properties of 3-ketosphinganine, whereas membranes from the mutant exhibit negligible activity. As expected from this result, they authors find that 3-ketosphinganine supports growth of the mutant as effectively as erythro-sphinganine. To study the sequelae of long chain base deprivation in the mutant required a method to effectively remove lipophilic long chain bases from the cultured cells. They have found that addition of α-cyclodextrin to the culture medium evidently binds sphinganine tightly enough such that within an hour they observe inhibition of growth (turbidity, cell count) and incorporation of [ 3 H]inositol into sphingolipids. Such treatment also results in a loss of cell viability, a long chain base less death possibly similar to the inositol-less death observed in inositol auxotrophs of yeast

  2. [Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

    Science.gov (United States)

    Sun, Dian Xing; Hu, Da Rong; Wu, Guang Hui; Hu, Xue Ling; Li, Juan; Fan, Gong Ren

    2002-08-01

    To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, PDNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual

  3. Mismatch Repair Mutants in Yeast Are Not Defective in Transcription-Coupled DNA Repair of Uv-Induced DNA Damage

    OpenAIRE

    Sweder, K. S.; Verhage, R. A.; Crowley, D. J.; Crouse, G. F.; Brouwer, J.; Hanawalt, P. C.

    1996-01-01

    Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively. Other factors probably are also uniquely involved in transcription-repair coupling. Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes. We examined removal of UV-induced ...

  4. AarF Domain Containing Kinase 3 (ADCK3 Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation.

    Directory of Open Access Journals (Sweden)

    Jason K Cullen

    Full Text Available Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016, arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3 is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10 biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.

  5. Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7.

    Science.gov (United States)

    Cha, Seung Bin; Lee, Won Jung; Shin, Min Kyoung; Jung, Myung Hwan; Shin, Seung Won; Yoo, An Na; Kim, Jong Wan; Yoo, Han Sang

    2013-06-27

    Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.

  6. A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

    Science.gov (United States)

    Yin, Guowei; Kistler, Samantha; George, Samuel D; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K; Lammers, Michael; Der, Channing J; Campbell, Sharon L

    2017-03-17

    The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Wing defects in Drosophila xenicid mutant clones are caused by C-terminal deletion of additional sex combs (Asx).

    Science.gov (United States)

    Bischoff, Kara; Ballew, Anna C; Simon, Michael A; O'Reilly, Alana M

    2009-12-01

    The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1]. Here, we demonstrate that expression of the betaPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells. Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed.

  8. Wing defects in Drosophila xenicid mutant clones are caused by C-terminal deletion of additional sex combs (Asx.

    Directory of Open Access Journals (Sweden)

    Kara Bischoff

    2009-12-01

    Full Text Available The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1].Here, we demonstrate that expression of the betaPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx is strongly increased in xenicid mutant cells.Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed.

  9. Positive selection of mutants with cell envelope defects of a Salmonella typhimurium strain hypersensitive to the products of genes hisF and hisH

    International Nuclear Information System (INIS)

    Anton, D.N.

    1979-01-01

    Strain SB564 and its derivative DA78 are hypersensitive to the inhibitory action of the proteins coded for by genes hisF and hisH on cell division. Transduction of hisO1242, a regulatory mutation that elicits a very high level of expression of the histidine operon, into these strains resulted in the production of long filamentous cells carrying large balloons and in growth failure. Forty-one hisO1242 derivatives that escaped inhibition were isolated. These strains showed a large variety of alterations, many of which were related to the cell envelope. The more-frequent alterations included: changes in cell shape, increased sensitivity to one or more of several drugs (deoxycholate, cycloserine, penicillin, novobiocin, acridine orange), increased autolytic activity in alkaline buffer, anomalous fermentation of maltose on eosin--methylene blue plates, and temperature-conditional cell division. The alterations are produced, in some of the strains, by pleiotropic mutations in gene envB. Strains affected in divC, divD, and rodA loci have also been identified. Genetic analaysis has shown that several strains carry more than one envelope mutation. It is assumed that envelope mutations are positively selected because they somehow alleviate the particularly severe inhibition of cell division caused, in strains SB564 and DA78, by the excessive synthesis of hisF and hisH gene products

  10. Dictyostelium LvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome

    Science.gov (United States)

    Harris, Edward; Wang, Ning; Wu, Wei-l; Weatherford, Alisha; De Lozanne, Arturo; Cardelli, James

    2002-01-01

    Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans (“lysosomal trafficking regulator”) or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal α-mannosidase were normal, although lvsB mutants inefficiently retained α-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis. PMID:11854420

  11. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    Directory of Open Access Journals (Sweden)

    Katherine Maringer

    2014-08-01

    Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  12. 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.)

  13. Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

    NARCIS (Netherlands)

    Johnson, M. A.; Waterham, H. R.; Ksheminska, G. P.; Fayura, L. R.; Cereghino, J. L.; Stasyk, O. V.; Veenhuis, M.; Kulachkovsky, A. R.; Sibirny, A. A.; Cregg, J. M.

    1999-01-01

    We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a

  14. Comparison of a coq7 deletion mutant with other respiration-defective mutants in fission yeast.

    Science.gov (United States)

    Miki, Risa; Saiki, Ryoichi; Ozoe, Yoshihisa; Kawamukai, Makoto

    2008-11-01

    Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more

  15. Defects in middle ear cavitation cause conductive hearing loss in the Tcof1 mutant mouse.

    Science.gov (United States)

    Richter, Carol A; Amin, Susan; Linden, Jennifer; Dixon, Jill; Dixon, Michael J; Tucker, Abigail S

    2010-04-15

    Conductive hearing loss (CHL) is one of the most common forms of human deafness. Despite this observation, a surprising gap in our understanding of the mechanisms underlying CHL remains, particularly with respect to the molecular mechanisms underlying middle ear development and disease. Treacher Collins syndrome (TCS) is an autosomal dominant disorder of facial development that results from mutations in the gene TCOF1. CHL is a common feature of TCS but the causes of the hearing defect have not been studied. In this study, we have utilized Tcof1 mutant mice to dissect the developmental mechanisms underlying CHL. Our results demonstrate that effective cavitation of the middle ear is intimately linked to growth of the auditory bulla, the neural crest cell-derived structure that encapsulates all middle ear components, and that defects in these processes have a profoundly detrimental effect on hearing. This research provides important insights into a poorly characterized cause of human deafness, and provides the first mouse model for the study of middle ear cavity defects, while also being of direct relevance to a human genetic disorder.

  16. Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant.

    Science.gov (United States)

    Finkel, J S; Yudanin, N; Nett, J E; Andes, D R; Mitchell, A P

    2011-11-01

    An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.

  17. Identification of a mutant α1 Na/K-ATPase that pumps but is defective in signal transduction.

    Science.gov (United States)

    Lai, Fangfang; Madan, Namrata; Ye, Qiqi; Duan, Qiming; Li, Zhichuan; Wang, Shaomeng; Si, Shuyi; Xie, Zijian

    2013-05-10

    It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. A420P mutant has normal pumping but not signaling function. Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.

  18. A novel Filamentous Flower mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels.

    Directory of Open Access Journals (Sweden)

    Scott J Douglas

    Full Text Available BREVIPEDICELLUS (BP encodes a class-I KNOTTED1-like homeobox (KNOX transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.

  19. Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice.

    Science.gov (United States)

    Chinn, Gregory A; Hirokawa, Karla E; Chuang, Tony M; Urbina, Cecilia; Patel, Fenil; Fong, Jeanette; Funatsu, Nobuo; Monuki, Edwin S

    2015-09-01

    Establishment of the corpus callosum involves coordination between callosal projection neurons and multiple midline structures, including the glial wedge (GW) rostrally and hippocampal commissure caudally. GW defects have been associated with agenesis of the corpus callosum (ACC). Here we show that conditional Lhx2 inactivation in cortical radial glia using Emx1-Cre or Nestin-Cre drivers results in ACC. The ACC phenotype was characterized by aberrant ventrally projecting callosal axons rather than Probst bundles, and was 100% penetrant on 2 different mouse strain backgrounds. Lhx2 inactivation in postmitotic cortical neurons using Nex-Cre mice did not result in ACC, suggesting that the mutant phenotype was not autonomous to the callosal projection neurons. Instead, ACC was associated with an absent hippocampal commissure and a markedly reduced to absent GW. Expression studies demonstrated strong Lhx2 expression in the normal GW and in its radial glial progenitors, with absence of Lhx2 resulting in normal Emx1 and Sox2 expression, but premature exit from the cell cycle based on EdU-Ki67 double labeling. These studies define essential roles for Lhx2 in GW, hippocampal commissure, and corpus callosum formation, and suggest that defects in radial GW progenitors can give rise to ACC. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

    International Nuclear Information System (INIS)

    LingNah Su; Little, J.B.

    1992-01-01

    A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

  1. Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity

    International Nuclear Information System (INIS)

    Morfiadakis, I.; Geissler, E.; Akademie der Wissenschaften der DDR, Berlin. Zentralinstitut fuer Molekularbiologie)

    1981-01-01

    A temperature-sensitive mutant of E. coli, WG24, was studied with respect to its sensitivity to photodynamic action, its capacity to perform host controlled reactivation, and its sensitivity to transduction at elevated temperatures. Mutant cells are much more sensitive than wild type cells to photodynamic action by thiopyronine and visible light at elevated temperatures. As well defined rec mutants, WG24 cells are less able to reactivate UV irradiated lambdac phages at elevated temperatures, while their ability to repair T1 phages is less impaired. Mutant cells cannot be transduced to T6 resistance at a detectable rate at elevated temperature. It is concluded, therefore, that some rec gene carries a ts mutation in this mutant. (author)

  2. The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells

    DEFF Research Database (Denmark)

    Jensen, Helle Lone; Norrild, Bodil

    2003-01-01

    Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus...

  3. Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

    Directory of Open Access Journals (Sweden)

    Khoa D Tran

    Full Text Available In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

  4. A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation.

    Science.gov (United States)

    Morandi, Dominique; le Signor, Christine; Gianinazzi-Pearson, Vivienne; Duc, Gérard

    2009-08-01

    One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc(++), Nod (-/+) phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.

  5. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    -utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5......-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate...

  6. A spontaneous and novel Pax3 mutant mouse that models Waardenburg syndrome and neural tube defects.

    Science.gov (United States)

    Ohnishi, Tetsuo; Miura, Ikuo; Ohba, Hisako; Shimamoto, Chie; Iwayama, Yoshimi; Wakana, Shigeharu; Yoshikawa, Takeo

    2017-04-05

    Genes responsible for reduced pigmentation phenotypes in rodents are associated with human developmental defects, such as Waardenburg syndrome, where patients display congenital deafness along with various abnormalities mostly related to neural crest development deficiency. In this study, we identified a spontaneous mutant mouse line Rwa, which displays variable white spots on mouse bellies and white digits and tail, on a C57BL/6N genetic background. Curly tail and spina bifida were also observed, although at a lower penetrance. These phenotypes were dominantly inherited by offspring. We searched for the genetic mechanism of the observed phenotypes. We harnessed a rapid mouse gene mapping system newly developed in our laboratories to identify a responsible gene. We detected a region within chromosome 1 as a probable locus for the causal mutation. Dense mapping using interval markers narrowed the locus down to a 670-kbp region, containing four genes including Pax3, a gene known to be implicated in the types I and III Waardenburg syndrome. Extensive mutation screening of Pax3 detected an 841-bp deletion, spanning the promoter region and intron 1 of the gene. The defective allele of Pax3, named Pax3 Rwa , lacked the first coding exon and co-segregated perfectly with the phenotypes, confirming its causal nature. The genetic background of Rwa mice is almost identical to that of inbred C57BL/6N. These results highlight Pax3 Rwa mice as a beneficial tool for analyzing biological processes involving Pax3, in particular the development and migration of neural crest cells and melanocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

    International Nuclear Information System (INIS)

    Zerler, B.R.; Wallace, S.S.

    1984-01-01

    The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detecttable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants

  8. Degradation routes of trafficking-defective VLDLR mutants associated with Dysequilibrium syndrome.

    Science.gov (United States)

    Kizhakkedath, Praseetha; John, Anne; Al-Gazali, Lihadh; Ali, Bassam R

    2018-01-25

    Low density lipoprotein receptor (LDLR) family members are involved in signaling in the developing brain. Previously we have reported that missense mutations in the Very Low Density Lipoprotein Receptor gene (VLDLR), causing Dysequilibrium syndrome (DES), disrupt ligand-binding, due to endoplasmic reticulum (ER) retention of the mutants. We explored the degradation routes of these VLDLR mutants in cultured cells. Our results indicate that VLDLR mutants are retained in the ER for prolonged periods which could be facilitated by association with the ER-resident chaperone calnexin. The mutants were prone to aggregation and capable of eliciting ER stress. The VLDLR mutants were found to be degraded predominantly by the proteasomal pathway, since ubiquitinated VLDLR was found to accumulate in response to proteasomal inhibition. Further, the mutants were found to interact with the ER degradation adaptor protein SEL1L. The degradation of VLDLR wild type and mutant were delayed in CRISPR/Cas9 edited SEL1L knock-out cells which was reversed by exogenous expression of SEL1L. In summary, ER retention of pathogenic VLDLR mutants involves binding to calnexin, elevated ER stress, and delayed degradation which is dependent on SEL1L. Since core LDLR family members share common structural domains, common mechanisms may be involved in their ER processing.

  9. uvsI mutants defective in UV mutagenesis define a fourth epistatic group of uvs genes in Aspergillus.

    Science.gov (United States)

    Chae, S K; Kafer, E

    1993-01-01

    Three UV-sensitive mutations of A. nidulans, uvsI, uvsJ and uvsA, were tested for epistatic relationships with members of the previously established groups, here called the "UvsF", "UvsC", and "UvsB" groups. uvsI mutants are defective for spontaneous and induced reversion of certain point mutations and differ also for other properties from previously analyzed uvs types. They are very sensitive to the killing effects of UV-light and 4-NQO (4-nitro-quinoline-N-oxide) but not to MMS (methylmethane sulfonate). When double- and single-mutant uvs strains were compared for sensitivity to these three agents, synergistic or additive effects were found for uvsI with all members of the three groups. The uvsI gene may therefore represent a fourth epistatic group, possibly involved in mutagenic repair. On the other hand, uvsJ was clearly epistatic with members of the UvsF group and fitted well into this group also by phenotype. The uvsA gene was tentatively assigned to the UvsC group. uvsA showed epistatic interactions with uvsC in all tests, and like UvsC-group mutants is UV-sensitive mainly in dividing cells. However, the uvsA mutation does not cause the defects in recombination and UV mutagenesis typical for this group.

  10. ups1, an Arabidopsis thaliana camalexin accumulation mutant defective in multiple defence signalling pathways.

    Science.gov (United States)

    Denby, Katherine J; Jason, Laure J M; Murray, Shane L; Last, Robert L

    2005-03-01

    We report the characterization of an Arabidopsis thaliana mutant, ups1, isolated on the basis of reduced expression of phosphoribosylanthranilate transferase, a tryptophan biosynthetic enzyme. ups1 also exhibits defects in a wide range of defence responses. After infection with Pseudomonas syringae or Botrytis cinerea, the expression of genes regulated by both the salicylic acid and jasmonic acid/ethylene pathways is reduced in ups1 compared with wild type. Camalexin accumulation in ups1 is greatly reduced after infection with these two pathogens, as well as after amino acid starvation or oxidative stress. Reactive oxygen species (ROS)-mediated gene expression is also compromised in ups1 indicating that this mutant is defective in signalling pathways activated in response to both biotic and abiotic stress. The fact that all three major defence signalling pathways are disrupted in ups1, together with the oxidative stress phenotype, leads us to suggest that UPS1 is involved in ROS signal transduction.

  11. Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection.

    Science.gov (United States)

    Righetti, S C; Perego, P; Corna, E; Pierotti, M A; Zunino, F

    1999-07-01

    carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.

  12. A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation.

    Science.gov (United States)

    Pislariu, Catalina I; Murray, Jeremy D; Wen, JiangQi; Cosson, Viviane; Muni, RajaSekhara Reddy Duvvuru; Wang, Mingyi; Benedito, Vagner A; Andriankaja, Andry; Cheng, Xiaofei; Jerez, Ivone Torres; Mondy, Samuel; Zhang, Shulan; Taylor, Mark E; Tadege, Million; Ratet, Pascal; Mysore, Kirankumar S; Chen, Rujin; Udvardi, Michael K

    2012-08-01

    A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

  13. Agrobacterium rhizogenes mutants that fail to bind to plant cells.

    OpenAIRE

    Crews, J L; Colby, S; Matthysse, A G

    1990-01-01

    Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria ...

  14. Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

    International Nuclear Information System (INIS)

    Kohno, Kenji; Hayes, H.; Mekada, Eisuke; Uchida, Tsuyoshi

    1987-01-01

    A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. 125 I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH 4 Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1,000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells

  15. Molecular defect of isovaleryl-CoA dehydrogenase in the skunk mutant of silkworm, Bombyx mori.

    Science.gov (United States)

    Urano, Kei; Daimon, Takaaki; Banno, Yutaka; Mita, Kazuei; Terada, Tohru; Shimizu, Kentaro; Katsuma, Susumu; Shimada, Toru

    2010-11-01

    The isovaleric acid-emanating silkworm mutant skunk (sku) was first studied over 30 years ago because of its unusual odour and prepupal lethality. Here, we report the identification and characterization of the gene responsible for the sku mutant. Because of its specific features and symptoms similar to human isovaleryl-CoA dehydrogenase (IVD) deficiency, also known as isovaleric acidaemia, IVD dysfunction in silkworms was predicted to be responsible for the phenotype of the sku mutant. Linkage analysis revealed that the silkworm IVD gene (BmIVD) was closely linked to the odorous phenotype as expected, and a single amino acid substitution (G376V) was found in BmIVD of the sku mutant. To investigate the effect of the G376V substitution on BmIVD function, wild-type and sku-type recombinants were constructed with a baculovirus expression system and the subsequent enzyme activity of sku-type BmIVD was shown to be significantly reduced compared with that of wild-type BmIVD. Molecular modelling suggested that this reduction in the enzyme activity may be due to negative effects of G376V mutation on FAD-binding or on monomer-monomer interactions. These observations strongly suggest that BmIVD is responsible for the sku locus and that the molecular defect in BmIVD causes the characteristic smell and prepupal lethality of the sku mutant. To our knowledge, this is, aside from humans, the first characterization of IVD deficiency in metazoa. Considering that IVD acts in the third step of leucine degradation and the sku mutant accumulates branched-chain amino acids in haemolymph, this mutant may be useful in the investigation of unique branched-chain amino acid catabolism in insects. © 2010 The Authors Journal compilation © 2010 FEBS.

  16. Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth

    DEFF Research Database (Denmark)

    Lombardo, Fabien; Heckmann, Anne Birgitte Lau; Miwa, Hiroki

    2006-01-01

    infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early...... symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itd1, itd3, and itd4 mutations...

  17. Characterization of harpy/Rca1/emi1 mutants: patterning in the absence of cell division.

    Science.gov (United States)

    Riley, Bruce B; Sweet, Elly M; Heck, Rebecca; Evans, Adrienne; McFarland, Karen N; Warga, Rachel M; Kane, Donald A

    2010-03-01

    We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce premature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number after approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromodeoxyuridine incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division. Copyright (c) 2010 Wiley-Liss, Inc.

  18. Carbon allocation and element composition in four Chlamydomonas mutants defective in genes related to the CO2 concentrating mechanism.

    Science.gov (United States)

    Memmola, Francesco; Mukherjee, Bratati; Moroney, James V; Giordano, Mario

    2014-09-01

    Four mutants of Chlamydomonas reinhardtii with defects in different components of the CO2 concentrating mechanism (CCM) or in Rubisco activase were grown autotrophically at high pCO2 and then transferred to low pCO2, in order to study the role of different components of the CCM on carbon allocation and elemental composition. To study carbon allocation, we measured the relative size of the main organic pools by Fourier Transform Infrared spectroscopy. Total reflection X-ray fluorescence was used to analyze the elemental composition of algal cells. Our data show that although the organic pools increased their size at high CO2 in all strains, their stoichiometry was highly homeostatic, i.e., the ratios between carbohydrates and proteins, lipid and proteins, and carbohydrates and lipids, did not change significantly. The only exception was the wild-type 137c, in which proteins decreased relative to carbohydrates and lipids, when the cells were transferred to low CO2. It is noticeable that the two wild types used in this study responded differently to the transition from high to low CO2. Malfunctions of the CCM influenced the concentration of several elements, somewhat altering cell elemental stoichiometry: especially the C/P and N/P ratios changed appreciably in almost all strains as a function of the growth CO2 concentration, except in 137c and the Rubisco activase mutant rca1. In strain cia3, defective in the lumenal carbonic anhydrase (CA), the cell quotas of P, S, Ca, Mn, Fe, and Zn were about 5-fold higher at low CO2 than at high CO2. A Principle Components Analysis showed that, mostly because of its elemental composition, cia3 behaved in a substantially different way from all other strains, at low CO2. The lumenal CA thus plays a crucial role, not only for the correct functioning of the CCM, but also for element utilization. Not surprisingly, growth at high CO2 attenuated differences among strains.

  19. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    International Nuclear Information System (INIS)

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B.

    1990-01-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli β-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses β-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system

  20. Streptococcus faecalis mutants defective in regulation of cytoplasmic pH.

    Science.gov (United States)

    Kobayashi, H; Unemoto, T

    1980-09-01

    We have isolated two acid-sensitive mutants of Streptococcus faecalis (ATCC 9790), designated AS13 and AS25, which grew at pH 7.5 but not at pH below 6.0. The ionophore gramicidin D, which collapsed the pH gradient between the cytoplasm and the medium, had little effect on the growth of these mutants, indicating that growing cells maintain only a small pH gradient. In the presence of gramicidin D the growth rates of the parent and mutant strains were identical over a range of pH values. When glucose was added to a cell suspension at pH 6.4, the parent strain generated a pH gradient of 1.0 unit, interior alkaline; AS13 generated a pH gradient of only 0.5 units, and AS25 generated no measurable pH gradient. The proton permeability of the mutant strains was the same as that of the parent strain. These results suggest that a cytoplasmic pH of around 7.5 is required for the growth of the cells and that the mutant strains are unable to establish a neutral cytoplasmic pH in acidic medium because of damage to the regulatory system of the cytoplasmic pH. Mutant strains also have a reduced capacity to extrude protons and take up potassium. Therefore, it is likely that these cation transport systems are involved in the regulation of cytoplasmic pH.

  1. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  2. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-01-01

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

  3. Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants.

    Science.gov (United States)

    Hebbar, Sarita; Sahoo, Ishtapran; Matysik, Artur; Argudo Garcia, Irene; Osborne, Kathleen Amy; Papan, Cyrus; Torta, Federico; Narayanaswamy, Pradeep; Fun, Xiu Hui; Wenk, Markus R; Shevchenko, Andrej; Schwudke, Dominik; Kraut, Rachel

    2015-12-07

    Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death.

  4. Homologous Recombination Defective Arabidopsis Mutants Exhibit Enhanced Sensitivity to Abscisic Acid.

    Directory of Open Access Journals (Sweden)

    Sujit Roy

    Full Text Available Abscisic acid (ABA acts as an important plant hormone in regulating various aspects of plant growth and developmental processes particularly under abiotic stress conditions. An increased ABA level in plant cells inhibits DNA replication and cell division, causing plant growth retardation. In this study, we have investigated the effects of ABA on the growth responses of some major loss-of-function mutants of DNA double-stand break (DSB repair genes in Arabidopsis during seed germination and early stages of seedling growth for understanding the role of ABA in the induction of genome instability in plants. A comparative analysis of ABA sensitivity of wild-type Arabidopsis and the knockout mutant lines related to DSB sensors, including atatm, atatr, the non-homologous end joining (NHEJ pathway genes, and mutants related to homologous recombination (HR pathway genes showed relatively enhanced sensitivity of atatr and HR-related mutants to ABA treatment. The expression levels of HR-related genes were increased in wild-type Arabidopsis (Col-0 during seed germination and early stages of seedling growth. Immunoblotting experiments detected phosphorylation of histone H2AX in wild-type (Col-0 and DSB repair gene mutants after ABA treatment, indicating the activation of DNA damage response due to ABA treatment. Analyses of DSB repair kinetics using comet assay under neutral condition have revealed comparatively slower DSB repair activity in HR mutants. Overall, our results have provided comprehensive information on the possible effect of ABA on DNA repair machinery in plants and also indicated potential functional involvement of HR pathway in repairing ABA induced DNA damage in Arabidopsis.

  5. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori, E-mail: aaohta@isc.chubu.ac.jp

    2014-03-07

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  6. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    International Nuclear Information System (INIS)

    Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1989-01-01

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well

  7. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y. (National Institute of Health, Tokyo (Japan))

    1989-11-25

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in (14C)ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of (14C)ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-(14C)ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.

  8. A pqr2 mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in Arabidopsis thaliana.

    Science.gov (United States)

    Dong, Shuchao; Hu, Huizhen; Wang, Youmei; Xu, Zhengdan; Zha, Yi; Cai, Xiwen; Peng, Liangcai; Feng, Shengqiu

    2016-09-01

    Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na2WO4, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.

  9. Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2.

    OpenAIRE

    De Mars Cody, J; Conway, T W

    1981-01-01

    A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RN...

  10. Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of hypoxia-inducible factor-alpha/Sima.

    Science.gov (United States)

    Centanin, Lázaro; Ratcliffe, Peter J; Wappner, Pablo

    2005-11-01

    Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-alpha/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima.

  11. Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-α/Sima

    Science.gov (United States)

    Centanin, Lázaro; Ratcliffe, Peter J; Wappner, Pablo

    2005-01-01

    Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-α polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-α/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima. PMID:16179946

  12. A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant.

    Science.gov (United States)

    Meyer, Régis E; Delaage, Michèle; Rosset, Roland; Capri, Michèle; Aït-Ahmed, Ounissa

    2010-11-16

    Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.

  13. A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant

    Science.gov (United States)

    2010-01-01

    Background Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes) and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. Results We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E), which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. Conclusions We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction. PMID:21080953

  14. A single mutation results in diploid gamete formation and parthenogenesis in a Drosophila yemanuclein-alpha meiosis I defective mutant

    Directory of Open Access Journals (Sweden)

    Capri Michèle

    2010-11-01

    Full Text Available Abstract Background Sexual reproduction relies on two key events: formation of cells with a haploid genome (the gametes and restoration of diploidy after fertilization. Therefore the underlying mechanisms must have been evolutionary linked and there is a need for evidence that could support such a model. Results We describe the identification and the characterization of yem1, the first yem-alpha mutant allele (V478E, which to some extent affects diploidy reduction and its restoration. Yem-alpha is a member of the Ubinuclein/HPC2 family of proteins that have recently been implicated in playing roles in chromatin remodeling in concert with HIRA histone chaperone. The yem1 mutant females exhibited disrupted chromosome behavior in the first meiotic division and produced very low numbers of viable progeny. Unexpectedly these progeny did not display paternal chromosome markers, suggesting that they developed from diploid gametes that underwent gynogenesis, a form of parthenogenesis that requires fertilization. Conclusions We focus here on the analysis of the meiotic defects exhibited by yem1 oocytes that could account for the formation of diploid gametes. Our results suggest that yem1 affects chromosome segregation presumably by affecting kinetochores function in the first meiotic division. This work paves the way to further investigations on the evolution of the mechanisms that support sexual reproduction.

  15. Pleurotus sajor-caju HSP100 complements a thermotolerance defect in hsp104 mutant Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Jin-Ohk; Jeong, Mi-Jeong; Kwon, Tack-Ryun; Lee, Seung-Kon; Byun, Myung-Ok; Chung, Ill-Min; Park, Soo-Chul

    2006-06-01

    A putative Hsp100 gene was cloned from the fungus Pleurotus sajor-caju. mRNA expression studies demonstrated that this gene (designated PsHsp100) is highly induced by high temperature,induced less strongly by exposure to ethanol, and not induced by drought or salinity. Heat shock induction is detectable at 37 degrees C and reaches a maximum level at 42 degrees C. PsHsp100 mRNA levels sharply increased within 15 min of exposure to high temperature, and reached a maximum expression level at 2 h that was maintained for several hours. These results indicate that PsHsp100 could work at an early step in thermotolerance. To examine its function, PsHsp100 was transformed into a temperature-sensitive hsp104 deletion mutant Saccharomycetes cerivisiae strain to test the hypothesis that PsHSP100 is an protein that functions in thermotolerance. Overexpression of PsHSP100 complemented the thermotolerance defect of the hsp104 mutant yeast, allowing them being survive even at 50 degree C for 4 h. These results indicate that PsHSP100 protein is functional as an HSP100 in yeast and could play and important role in thermotolerance in P. sajor-caju.

  16. Neurospora crassa ASM-1 complements the conidiation defect in a stuA mutant of Aspergillus nidulans.

    Science.gov (United States)

    Chung, Dawoon; Upadhyay, Srijana; Bomer, Brigitte; Wilkinson, Heather H; Ebbole, Daniel J; Shaw, Brian D

    2015-01-01

    Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development. © 2015 by The Mycological Society of America.

  17. Chinese hamster ovary mutant UV-1 is hypomutable and defective in a postreplication recovery process

    International Nuclear Information System (INIS)

    Stamato, T.D.; Hinkle, L.; Collins, A.R.; Waldren, C.A.

    1981-01-01

    CHO-UV-1 is a mutant of the Chinese hamster cell CHO-K1 hypersensitive to killing by ultraviolet light but with normal resistance to X-ray. It is also hypersensitive to killing by ethyl methane sulfonate. Hybrid clones formed bu fusing UV-1 and Chinese hamster lung cells display the normal ultraviolet resistance of the latter. The sensitive phenotype behaves, therefore, in a genetically recessive manner. Ultraviolet sensitivity of UV-1 is not associated with a deficiency in excision repair. Alkaline sucrose gradient sedimentation analysis of nascent DNA from ultraviolet-irradiated cells reveals that UV-1 is, however, markedly deficient in postreplication recovery. Furthermore, UV-1 has a lower rate of induced mutation to 6-thioguanine resistance than does the parental cell when treated with ultraviolet light or ethyl methane sulfonate. These results suggest that the phenotype of UV-1 is due to a mutation in a form of postreplication recovery which in normal cells is error prone

  18. Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; McKinney, Lea V; Pike, Helen

    2008-01-01

    The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11...... null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated...... site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death...

  19. A Mutant Isoform of ObgE Causes Cell Death by Interfering with Cell Division

    Directory of Open Access Journals (Sweden)

    Liselot Dewachter

    2017-06-01

    Full Text Available Cell division is a vital part of the cell cycle that is fundamental to all life. Despite decades of intense investigation, this process is still incompletely understood. Previously, the essential GTPase ObgE, which plays a role in a myriad of basic cellular processes (such as initiation of DNA replication, chromosome segregation, and ribosome assembly, was proposed to act as a cell cycle checkpoint in Escherichia coli by licensing chromosome segregation. We here describe the effect of a mutant isoform of ObgE (ObgE∗ that causes cell death by irreversible arrest of the cell cycle at the stage of cell division. Notably, chromosome segregation is allowed to proceed normally in the presence of ObgE∗, after which cell division is blocked. Under conditions of rapid growth, ongoing cell cycles are completed before cell cycle arrest by ObgE∗ becomes effective. However, cell division defects caused by ObgE∗ then elicit lysis through formation of membrane blebs at aberrant division sites. Based on our results, and because ObgE was previously implicated in cell cycle regulation, we hypothesize that the mutation in ObgE∗ disrupts the normal role of ObgE in cell division. We discuss how ObgE∗ could reveal more about the intricate role of wild-type ObgE in division and cell cycle control. Moreover, since Obg is widely conserved and essential for viability, also in eukaryotes, our findings might be applicable to other organisms as well.

  20. Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis

    Science.gov (United States)

    Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in OPG synthesis are unable to exhibit motility on moist surfaces (swarming) ...

  1. Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.

    Directory of Open Access Journals (Sweden)

    Haiyan Mao

    Full Text Available Treatment of LQT2 is inadequate. Many drugs which can pharmacologically rescue defective protein trafficking in LQT2 also result in potent blockade of HERG current, negating their therapeutic benefit. It is reported that PD-118057 and thapsigargin can rescue LQT2 without hERG channel blockade, but the precise mechanism of action is unknown. Furthermore, the effect of PD-118057 and thapsigargin on the dominant negative E637K-hERG mutant has not been previously investigated.IN THIS STUDY, WE INVESTIGATED: (a the effect of PD-118057 and thapsigargin on the current amplitudes of WT-hERG and WT/E637K-hERG channels; (b the effect of PD-118057 and thapsigargin on the biophysical properties of WT-hERG and WT/E637K-hERG channels; (c whether drug treatment can rescue channel processing and trafficking defects of the WT/E637K-hERG mutant.The whole-cell Patch-clamp technique was used to assess the effect of PD-118057 and thapsigargin on the electrophysiological characteristics of the rapidly activating delayed rectifier K(+ current (Ikr of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.In our study, PD-118057 was shown to significantly enhance both the maximum current amplitude and tail current amplitude, but did not alter the gating and kinetic properties of the WT-hERG channel, with the exception of accelerating steady-state inactivation. Additionally, thapsigargin shows a similar result as PD-118057 for the WT-hERG channel, but with the exception of attenuating steady-state inactivation. However, for the WT/E637K-hERG channel, PD-118057 had no effect on either the current or on the gating and kinetic properties. Furthermore, thapsigargin treatment did not alter the current or the gating and kinetic properties of the WT/E637K-hERG channel, with the exception of opening at more positive voltages.Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting

  2. [Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation].

    Science.gov (United States)

    Kovtunov, E A; Shelud'ko, A V; Chernyshova, M P; Petrova, L P; Katsy, E I

    2013-11-01

    Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR_p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.

  3. Complementation of human immunodeficiency virus type 1 vif mutants in some CD4+ T-cell lines.

    Science.gov (United States)

    Hevey, M; Donehower, L A

    1994-09-01

    The viral infectivity factor gene, vif of human immunodeficiency virus type 1 (HIV-1), is required for full infectivity in most T-cell lines. The replication kinetics exhibited by these mutants has been shown to be cell type-dependent. In H9 cells as well as primary lymphocytes, vif mutants are incapable of establishing infection. This has led to classification of these cell types as non-permissive for vif mutant replication. The T-cell lines Sup T1 and C8166 are able to replicate the vif mutant virus, leading to their classification as permissive for vif mutant replication. In this study, four cell lines (Sup T1, C8166, Molt 4 Clone 8, and A3.01) were tested for their ability to replicate vif mutant virus derived from two different strains of HIV-1 (HXB2 and NL4-3) that had been passaged on various cell lines. Although the kinetics of initial infection was delayed in all cells, by the second passage of vif mutant virus on Sup T1 or Molt 4 cells the kinetics of replication were identical to wild type virus. In contrast, mutant virus displayed delayed replication kinetics in C8166 and A3.01 cells in both initial and subsequent passages. In addition, the levels of viral DNA in infected Sup T1 cells were similar for delta vif and wild type virus, but in C8166 cells delta vif virus DNA levels were reduced compared to wild type virus. These results argue that in Sup T1 and Molt 4 cells there is a factor present that is able to complement the defect in vif mutant viruses which is absent or inefficient in its activity in C8166 and A3.01 cells.

  4. Disparate peroxisome-related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization.

    Science.gov (United States)

    Gonzalez, Kim L; Fleming, Wendell A; Kao, Yun-Ting; Wright, Zachary J; Venkova, Savina V; Ventura, Meredith J; Bartel, Bonnie

    2017-10-01

    Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  5. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng [National Yang Ming Univ., Taipei, Taiwan (China). Inst. of Radiological Sciences] (and others)

    2001-12-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  6. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

  7. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

    International Nuclear Information System (INIS)

    Donnelly, C.E.; Walker, G.C.

    1989-01-01

    Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus

  8. Metabolic reprogramming in mutant IDH1 glioma cells.

    Directory of Open Access Journals (Sweden)

    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  9. p53-Independent cell cycle and erythroid differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan anemia-proteins: RPS19 versus RPL5.

    Directory of Open Access Journals (Sweden)

    Sharon A Singh

    Full Text Available Diamond Blackfan anemia (DBA is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

  10. Induced expression of nucleolin phosphorylation-deficient mutant confers dominant-negative effect on cell proliferation.

    Directory of Open Access Journals (Sweden)

    Shu Xiao

    Full Text Available Nucleolin (NCL is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2 and mitotic cyclin-dependent kinase 1 (CDK1. Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2 homology 3 (BH3-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.

  11. Mouse Dach2 mutants do not exhibit gross defects in eye development or brain function.

    Science.gov (United States)

    Davis, Richard J; Pesah, Yakov I; Harding, Mark; Paylor, Richard; Mardon, Graeme

    2006-02-01

    Drosophila dachshund is a critical regulator of eye, brain, and limb formation. Vertebrate homologs, Dach1 and Dach2, are expressed in the developing retina, brain, and limbs, suggesting functional conservation of the dachshund/Dach gene family. Dach1 mutants die postnatally, but exhibit grossly normal development. Here we report the generation of Dach2 mutant mice. Although deletion of Dach2 exon 1 results in abrogation of RNA expression, Dach2 mutants are viable and fertile. Histochemical analysis reveals grossly normal Dach2 mutant eye development. In addition, a battery of neurological assays failed to yield significant differences in behavior between Dach2 mutants and controls. We discuss these findings in the light of published observations of DACH2 mutations in the human population. Finally, to test the functional conservation hypothesis, we generated Dach2; Dach1 double mutant mice. Dach double mutants die after birth, similar to Dach1 homozygotes. However, unlike Drosophila dachshund mutants that lack eyes and exhibit leg truncations, the eyes and limbs of Dach double mutants are present, suggesting differences between Dach and dachshund gene function during embryonic eye and limb formation.

  12. Ku80-deleted cells are defective at base excision repair

    International Nuclear Information System (INIS)

    Li, Han; Marple, Teresa; Hasty, Paul

    2013-01-01

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H 2 O 2 and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs

  13. Ku80-deleted cells are defective at base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Li, Han [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain); Marple, Teresa [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain)

    2013-05-15

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H{sub 2}O{sub 2} and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

  14. Isolation of cyanobacterial mutants exhibiting growth defects under microoxic conditions by transposon tagging mutagenesis of Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Terauchi, Kazuki; Sobue, Riho; Furutani, Yuho; Aoki, Rina; Fujita, Yuichi

    2017-05-12

    Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis by extracting electrons from water, with the generation of oxygen as a byproduct. Cyanobacteria use oxygen not only for respiration to produce energy in the dark but also for biosynthesis of various metabolites, such as heme and chlorophyll. Oxygen levels dynamically fluctuate in the field environments, from hyperoxic at daytime to almost anaerobic at night. Thus, adaptation to anaerobiosis should be important for cyanobacteria to survive in low-oxygen and anaerobic environments. However, little is known about the molecular mechanisms of cyanobacterial anaerobiosis because cyanobacteria have been regarded as aerobic organisms. As a first step to elucidate cyanobacterial adaptation mechanisms to low-oxygen environments, we isolated five mutants, T-1-T-5, exhibiting growth defects under microoxic conditions. The mutants were obtained from a transposon-tagged mutant library of the cyanobacterium Synechocystis sp. PCC 6803, which was produced by in vitro transposon tagging of cyanobacterial genomic DNA. Southern blot analysis indicated that a kanamycin resistance gene was inserted in the genome as a single copy. We identified the chromosomal transposon-tagged locus in T-5. Two open reading frames (sll0577 and sll0578) were partially deleted by the insertion of the kanamycin resistance gene in T-5. A reverse transcription polymerase chain reaction suggested that these co-transcribed genes are constitutively expressed under both aerobic and microoxic conditions. Then, we isolated two mutants in which one of the two genes was individually disrupted. Only the mutants partially lacking an intact sll0578 gene showed growth defects under microoxic conditions, whereas it grew normally under aerobic conditions. sll0578 is annotated as purK encoding N 5 -carboxy-aminoimidazole ribonucleotide synthetase involved in purine metabolism. This result implies the unexpected physiological importance of Pur

  15. Characterizing visible and invisible cell wall mutant phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.; McCann, Maureen C.

    2015-04-06

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  16. Prion propagation in cells expressing PrP glycosylation mutants.

    Science.gov (United States)

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  17. Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy.

    Directory of Open Access Journals (Sweden)

    Xin Dai

    Full Text Available KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.

  18. Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

    International Nuclear Information System (INIS)

    Brown, T.C.; Boyd, J.B.

    1981-01-01

    Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant. (orig./AJ) [de

  19. Isolation and characterisation of a dwarf rice mutant exhibiting defective gibberellins biosynthesis.

    Science.gov (United States)

    Ji, S H; Gururani, M A; Lee, J W; Ahn, B-O; Chun, S-C

    2014-03-01

    We have isolated a severe dwarf mutant derived from a Ds (Dissociation) insertion mutant rice (Oryza sativa var. japonica c.v. Dongjin). This severe dwarf phenotype, has short and dark green leaves, reduced shoot growth early in the seedling stage, and later severe dwarfism with failure to initiate flowering. When treated with bioactive GA3 , mutants are restored to the normal wild-type phenotype. Reverse transcription PCR analyses of 22 candidate genes related to the gibberellin (GA) biosynthesis pathway revealed that among 22 candidate genes tested, a dwarf mutant transcript was not expressed only in one OsKS2 gene. Genetic analysis revealed that the severe dwarf phenotype was controlled by recessive mutation of a single nuclear gene. The putative OsKS2 gene was a chromosome 4-located ent-kaurene synthase (KS), encoding the enzyme that catalyses an early step of the GA biosynthesis pathway. Sequence analysis revealed that osks2 carried a 1-bp deletion in the ORF region of OsKS2, which led to a loss-of-function mutation. The expression pattern of OsKS2 in wild-type cv Dongjin, showed that it is expressed in all organs, most prominently in the stem and floral organs. Morphological characteristics of the dwarf mutant showed dramatic modifications in internal structure and external morphology. We propose that dwarfism in this mutant is caused by a point mutation in OsKS2, which plays a significant role in growth and development of higher plants. Further investigation on OsKS2 and other OsKS-like proteins is underway and may yield better understanding of the putative role of OsKS in severe dwarf mutants. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  20. Defects of mutant DNMT1 are linked to a spectrum of neurological disorders.

    Science.gov (United States)

    Baets, Jonathan; Duan, Xiaohui; Wu, Yanhong; Smith, Gordon; Seeley, William W; Mademan, Inès; McGrath, Nicole M; Beadell, Noah C; Khoury, Julie; Botuyan, Maria-Victoria; Mer, Georges; Worrell, Gregory A; Hojo, Kaori; DeLeon, Jessica; Laura, Matilde; Liu, Yo-Tsen; Senderek, Jan; Weis, Joachim; Van den Bergh, Peter; Merrill, Shana L; Reilly, Mary M; Houlden, Henry; Grossman, Murray; Scherer, Steven S; De Jonghe, Peter; Dyck, Peter J; Klein, Christopher J

    2015-04-01

    significant cognitive deficit by the age of 45 years. Cognitive function decline often started with personality changes and psychiatric manifestations. A triad of hearing loss, sensory neuropathy and cognitive decline remains as the stereotypic presentation of HSAN1E. Moreover, we show that mutant DNMT1 proteins translocate to the cytoplasm and are prone to form aggresomes while losing their binding ability to heterochromatin during the G2 cell cycle. Our results suggest mutations in DNMT1 result in imbalanced protein homeostasis through aggresome-induced autophagy. This mechanism may explain why mutations in the sole DNA maintenance methyltransferase lead to selective central and peripheral neurodegeneration. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Arabidopsis thaliana cdd1 mutant uncouples the constitutive activation of salicylic acid signalling from growth defects

    NARCIS (Netherlands)

    Swain, S.; Roy, S.; Shah, J.; Wees, S.C.M. van; Pieterse, C.M.J.; Nandi, A.K.

    2011-01-01

    Arabidopsis genotypes with a hyperactive salicylic acidmediated signalling pathway exhibit enhanced disease resistance, which is often coupled with growth and developmental defects, such as dwarfing and spontaneous necrotic lesions on the leaves, resulting in reduced biomass yield. In this article,

  2. Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

    Science.gov (United States)

    Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

    2005-01-01

    Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

  3. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  4. Molecular Imaging of Metabolic Reprograming in Mutant IDH Cells.

    Science.gov (United States)

    Viswanath, Pavithra; Chaumeil, Myriam M; Ronen, Sabrina M

    2016-01-01

    Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  5. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  6. The phenotype of FancB-mutant mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States)

    2011-07-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  7. The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2

    Science.gov (United States)

    talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the e...

  8. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne U.; Hove-Jensen, Bjarne; Garber, Bruce B.

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine...

  9. A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana.

    Science.gov (United States)

    Boavida, Leonor C; Shuai, Bin; Yu, Hee-Ju; Pagnussat, Gabriela C; Sundaresan, Venkatesan; McCormick, Sheila

    2009-04-01

    Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since approximately 10% of the genes are expressed in the male gametophyte and approximately 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 and 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

  10. The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

    DEFF Research Database (Denmark)

    von Malek, Bernadette; van der Graaff, Eric; Schneitz, Kay

    2002-01-01

    The Arabidopsis thaliana (L.) Heynh. mutant delayed-dehiscence2-2 (dde2-2) was identified in an En1/Spm1 transposon-induced mutant population screened for plants showing defects in fertility. The dde2-2 mutant allele is defective in the anther dehiscence process and filament elongation and thus e...

  11. Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant.

    OpenAIRE

    Bauer, K A; Ben-Bassat, A; Dawson, M; de la Puente, V T; Neway, J O

    1990-01-01

    A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL...

  12. Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants.

    Directory of Open Access Journals (Sweden)

    Changchun Chen

    2009-07-01

    Full Text Available Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5'methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development.

  13. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

    Directory of Open Access Journals (Sweden)

    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  14. A shift to organismal stress resistance in programmed cell death mutants.

    Directory of Open Access Journals (Sweden)

    Meredith E Judy

    Full Text Available Animals have many ways of protecting themselves against stress; for example, they can induce animal-wide, stress-protective pathways and they can kill damaged cells via apoptosis. We have discovered an unexpected regulatory relationship between these two types of stress responses. We find that C. elegans mutations blocking the normal course of programmed cell death and clearance confer animal-wide resistance to a specific set of environmental stressors; namely, ER, heat and osmotic stress. Remarkably, this pattern of stress resistance is induced by mutations that affect cell death in different ways, including ced-3 (cell death defective mutations, which block programmed cell death, ced-1 and ced-2 mutations, which prevent the engulfment of dying cells, and progranulin (pgrn-1 mutations, which accelerate the clearance of apoptotic cells. Stress resistance conferred by ced and pgrn-1 mutations is not additive and these mutants share altered patterns of gene expression, suggesting that they may act within the same pathway to achieve stress resistance. Together, our findings demonstrate that programmed cell death effectors influence the degree to which C. elegans tolerates environmental stress. While the mechanism is not entirely clear, it is intriguing that animals lacking the ability to efficiently and correctly remove dying cells should switch to a more global animal-wide system of stress resistance.

  15. Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Meggio, F

    1996-01-01

    Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition...... downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis....

  16. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Science.gov (United States)

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  17. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.

    2003-01-01

    or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression......Background Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...

  18. Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.

    Science.gov (United States)

    Glerum, D M; Tzagoloff, A

    1997-08-04

    The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

  19. Isolating human DNA repair genes using rodent-cell mutants

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  20. Topological defects in epithelia govern cell death and extrusion

    Science.gov (United States)

    Saw, Thuan Beng; Doostmohammadi, Amin; Nier, Vincent; Kocgozlu, Leyla; Thampi, Sumesh; Toyama, Yusuke; Marcq, Philippe; Lim, Chwee Teck; Yeomans, Julia M.; Ladoux, Benoit

    2017-04-01

    Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting

  1. Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress.

    Science.gov (United States)

    Chaturvedi, V; Bartiss, A; Wong, B

    1997-01-01

    Polyols, or polyhydroxy alcohols, are produced by many fungi. Saccharomyces cerevisiae produces large amounts of glycerol, and several fungi that cause serious human infections produce D-arabinitol and mannitol. Glycerol functions as an intracellular osmolyte in S. cerevisiae, but the functions of D-arabinitol and mannitol in pathogenic fungi are not yet known. To investigate the functions of mannitol, we constructed a new mannitol biosynthetic pathway in S. cerevisiae. S. cerevisiae transformed with multicopy plasmids encoding the mannitol-1-phosphate dehydrogenase of Escherichia coli produced mannitol, whereas S. cerevisiae transformed with control plasmids did not. Although mannitol production had no obvious phenotypic effects in wild-type S. cerevisiae, it restored the ability of a glycerol-defective, osmosensitive osg1-1 mutant to grow in the presence of high NaCl concentrations. Moreover, osg1-1 mutants producing mannitol were more resistant to killing by oxidants produced by a cell-free H2O2-FeSO4-NaI system than were controls. These results indicate that mannitol can (i) function as an intracellular osmolyte in S. cerevisiae, (ii) substitute for glycerol as the principal intracellular osmolyte in S. cerevisiae, and (iii) protect S. cerevisiae from oxidative damage by scavenging toxic oxygen intermediates. PMID:8981993

  2. Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice.

    Directory of Open Access Journals (Sweden)

    Anaïs Noblanc

    Full Text Available We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4 and the epididymal glutathione peroxidase 5 (GPx5 activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2O(2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

  3. Identification and molecular characterization of a naturally occurring RNA virus mutant defective in the initiation of host recovery

    International Nuclear Information System (INIS)

    Xin Hongwu; Ding Shouwei

    2003-01-01

    The host recovery response is characterized by the disappearance of disease symptoms and activation of the RNA silencing virus resistance in the new growth following an initial symptomatic infection. However, it is not clear what triggers the initiation of recovery, which occurs naturally only in some virus-host interactions. Here we report the identification and characterization of a spontaneous mutant of Tobacco streak virus (TSV) that became defective in triggering recovery in tobacco plants. Infectious full-length cDNA clones corresponding to the tripartite RNA genome were constructed from both the wild-type and the nonrecovery mutant of TSV (TSVnr), the first sets of infectious cDNA clones from an Ilarvirus. Genetic and molecular analyses identified an A → G mutation in the TSVnr genome that was sufficient to confer nonrecovery when introduced into TSV. The mutation was located in the intergenic region of RNA 3 upstream of the mapped transcriptional start site of the coat protein mRNA. Intriguingly, induction of recovery by TSV was not accompanied by virus clearance and TSV consistently accumulated to significantly higher levels than TSVnr did even though TSVnr-infected plants displayed severe symptoms throughout the course of infection. Thus, our findings indicate that recovery of host can be initiated by minimal genetic changes in a viral genome and may occur in the absence of virus clearance. Mechanisms possibly involved in the initiation of host recovery are discussed

  4. Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

    Science.gov (United States)

    Noblanc, Anaïs; Peltier, Manon; Damon-Soubeyrand, Christelle; Kerchkove, Nicolas; Chabory, Eléonore; Vernet, Patrick; Saez, Fabrice; Cadet, Rémi; Janny, Laurent; Pons-Rejraji, Hanae; Conrad, Marcus; Drevet, Joël R.; Kocer, Ayhan

    2012-01-01

    We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice. PMID:22719900

  5. Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation.

    Science.gov (United States)

    Bruystens, Jessica Gh; Wu, Jian; Fortezzo, Audrey; Del Rio, Jason; Nielsen, Cole; Blumenthal, Donald K; Rock, Ruth; Stefan, Eduard; Taylor, Susan S

    2016-12-04

    Most disease-related mutations that impair cAMP protein kinase A (PKA) signaling are present within the regulatory (R) PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex (CNC) disease and, more recently, to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP-binding (CNB) domains (CNB-B), and here, we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the catalytic (C)-subunit reveals the dysfunctional regions of the RIα subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure, and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted, thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB domains is necessary for the functional regulation of PKA activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    Directory of Open Access Journals (Sweden)

    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  7. Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity.

    Science.gov (United States)

    Durrett, Timothy P; Connolly, Erin L; Rogers, Elizabeth E

    2006-08-01

    All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.

  8. Modeling the induced mutation process in bacterial cells with defects in excision repair system

    Science.gov (United States)

    Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2015-12-01

    A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.

  9. [Courtship behavior, communicative sound production and resistance to stress in Drosophila mutants with defective agnostic gene, coding for LIMK1].

    Science.gov (United States)

    Popov, A V; Kaminskaia, A N; Savvateeva-Popova, E V

    2009-01-01

    -motor circuits of the singing center) decreases, and the rate and stability of pacemakers of the pulse and the sine songs increases. So, the sharply elevated LIMK1 and p-cofilin concentrations in the cells of agn(ts3) mutants, in comparison to wild type males, leading to derangement of learning and memory (Medvedeva et al., 2008) are accompanied by decline of motivation of males but do not seriously influence the neuro-motor coordination during singing. The higher resistance of characterictics of their behavior and communicative sound signals to heat shock is in agreement with the fact that the extremely high LIMK1 and P-cofilin concentrations in their cells go down to normal values after HS. At the same time the amyloid aggregations disappear and normally defective learning and memory are restore.

  10. Repair of defects in photoactive layer of organic solar cells

    NARCIS (Netherlands)

    Oostra, A.J.; Blom, P.W.M.; Michels, J.J.

    2015-01-01

    Defects occurring during printing of the photoactive layer in organic solar cells lead to short-circuits due to direct contact between the PEDOT:PSS anode and metallic cathode. We provide a highly effective repair method where the defected zone with bare PEDOT:PSS is treated with aqueous sodium

  11. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  12. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  13. A comparative study of Purkinje cells in two RORalpha gene mutant mice: staggerer and RORalpha(-/-).

    Science.gov (United States)

    Doulazmi, M; Frédéric, F; Capone, F; Becker-André, M; Delhaye-Bouchaud, N; Mariani, J

    2001-04-30

    The staggerer (Rora(sg/sg)) mutation is a deletion in the RORalpha gene, one member of a family of nuclear receptor genes related to the retinoic acid receptor. Recently Steinmayr et al. (Proc. Natl. Acad. Sci. USA 95 (1998) 3960) generated a RORalpha null-mutant mouse (Rora(-/-)) by using a targeting vector in which a beta-Gal gene replaces the second finger of the DNA-binding domain of RORalpha. The Rora(-/-) cerebellum is qualitatively a phenocopy of the Rora(sg/sg) one, but the two strains differ slightly in their motor skills. To address the question whether the morphological defects in the Rora(-/-) cerebellum are identical to the Rora(sg/sg) one, we compared number and size of Purkinje cells in both staggerer and RORalpha null-mutant mice, using calbindin (CaBP) immunohistochemistry and revelation of beta-Gal activity. Compared to control cerebella the Rora(sg/sg) cerebellum has 82% fewer CaBP-positive cells. In Rora(-/-) mouse, all the the beta-Gal-positive Purkinje cells also expressed CaBP, but the cerebellum contained 78% less CaBP-positive cells than control, a deficit not different from the one observed in Rora(sg/sg). We show similar mediolateral compartments in Purkinje cell number and cytological abnormality in Rora(sg/sg) and Rora(-/-) mice. These results provide quantitative support for the hypothesis that the cerebellar phenotype in the homozygous Rora(sg/sg) is due to the lack of function of the RORalpha gene.

  14. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Jiou Wang

    2009-01-01

    Full Text Available The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.

  15. Cell size and cell number in dwarf mutants of barley (Hordeum vulgare)

    International Nuclear Information System (INIS)

    Blonstein, A.D.; Gale, M.D.

    1984-01-01

    Sixteen height mutants, induced by sodium azide treatment of the two-rowed barley variety Proctor, have been used to investigate the relationship between the extent and nature of stem shortening with alterations in cell size and cell number, and the pleiotropic effects of dwarfing genes on vegetative development and agronomic performance. The studies on epidermal cell number and cell length in the developmentally earliest and latest elongated vegetative tissues - the coleoptile and peduncle resprectively - suggest that cell number may be the primary determinant of plant height. One semi-prostrate and one erectoides mutant are used to illustrate different cell number/cell size strategies and their relationships with gibberellin sensitivity, growth rate and lodging resistance are discussed. (author)

  16. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    International Nuclear Information System (INIS)

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-01-01

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  17. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    Energy Technology Data Exchange (ETDEWEB)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  18. Brainstem respiratory oscillators develop independently of neuronal migration defects in the Wnt/PCP mouse mutant looptail.

    Directory of Open Access Journals (Sweden)

    Muriel Thoby-Brisson

    Full Text Available The proper development and maturation of neuronal circuits require precise migration of component neurons from their birthplace (germinal zone to their final positions. Little is known about the effects of aberrant neuronal position on the functioning of organized neuronal groups, especially in mammals. Here, we investigated the formation and properties of brainstem respiratory neurons in looptail (Lp mutant mice in which facial motor neurons closely apposed to some respiratory neurons fail to migrate due to loss of function of the Wnt/Planar Cell Polarity (PCP protein Vangl2. Using calcium imaging and immunostaining on embryonic hindbrain preparations, we found that respiratory neurons constituting the embryonic parafacial oscillator (e-pF settled at the ventral surface of the medulla in Vangl2(Lp/+ and Vangl2(Lp/Lp embryos despite the failure of tangential migration of its normally adjacent facial motor nucleus. Anatomically, the e-pF neurons were displaced medially in Lp/+ embryos and rostro-medially Lp/Lp embryos. Pharmacological treatments showed that the e-pF oscillator exhibited characteristic network properties in both Lp/+ and Lp/Lp embryos. Furthermore, using hindbrain slices, we found that the other respiratory oscillator, the preBötzinger complex, was also anatomically and functionally established in Lp mutants. Importantly, the displaced e-pF oscillator established functional connections with the preBötC oscillator in Lp/+ mutants. Our data highlight the robustness of the developmental processes that assemble the neuronal networks mediating an essential physiological function.

  19. Calpain and PARP activation during photoreceptor cell death in P23H and S334ter rhodopsin mutant rats.

    Directory of Open Access Journals (Sweden)

    Jasvir Kaur

    Full Text Available Retinitis pigmentosa (RP is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose polymerase (PARP in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage-key events in apoptotic cell death--were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.

  20. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis.

    Science.gov (United States)

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2014-03-07

    A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of (13)C-labeled diC8PC ((methyl-(13)C)3-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-(13)C)3-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

    Science.gov (United States)

    Jones, Sara E; Whitehead, Kristi; Saulnier, Delphine; Thomas, Carissa M; Versalovic, James; Britton, Robert A

    2011-01-01

    Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

  2. Defective planar cell polarity in polycystic kidney disease.

    Science.gov (United States)

    Fischer, Evelyne; Legue, Emilie; Doyen, Antonia; Nato, Faridabano; Nicolas, Jean-François; Torres, Vicente; Yaniv, Moshe; Pontoglio, Marco

    2006-01-01

    Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

  3. Recombination models for defects in silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Steingrube, Silke

    2011-07-06

    Rocombination of charge carriers via defects is a substantial loss mechanism in solar cells. In this work, recombination models for three defect types in crystalline silicon (c-Si) solar cells are developed and analyzed. First, a model is developed to describe the injection dependence of the effective surface recombination velocity S{sub eff} of both SiN{sub x} and Al{sub 2}O{sub 3} passivated c-Si surfaces. This model relies on a damaged layer in the silicon close to the interface. A suitable parametrization is given that allows to reproduce the measured effective surface recombination velocity S{sub eff} of the investigated interfaces for all relevant injection densities and dopant densities. With the help of this model, we discuss possible reasons for the damage on a microscopic scale. Second, the interface between amorphous and crystalline silicon is investigated. A Shockley-Read-Hall (SRF) model is suggested to approximate the amphoteric properties of the defects at the interface. In contrast to the exact model, the approximate model has a closed-form-solution and is therefore easily integrated into device simulators. Physically motivated error bounds are derived which can help to decide in which cases the simplified model may be applied. For typical injection densities at interfaces, the error of the SRH model is small if the correlation energy of the donor- und acceptor-like defect distribution is positive and if the properties of charged defects are described by asymmetric capture cross sections for electrons and holes. In addition, the defect distribution must lie in between the quasi-Fermi levels for traps. In low-injection, e.g. when applied to the p-n junction of a solar cell or at low illumination levels, it may fail dramatically. Further, dark current-voltage curves (I-V curves) of c-Si solar cells having diode-ideality factors n{sub D} > 2 in forward direction, i.e. increase sub-exponentially in certain voltage ranges, are analyzed. These &apos

  4. PTEN Redundancy: Overexpressing lpten, a Homolog of Dictyostelium discoideum ptenA, the Ortholog of Human PTEN, Rescues All Behavioral Defects of the Mutant ptenA−

    OpenAIRE

    Lusche, Daniel F.; Wessels, Deborah; Richardson, Nicole A.; Russell, Kanoe B.; Hanson, Brett M.; Soll, Benjamin A.; Lin, Benjamin H.; Soll, David R.

    2014-01-01

    Mutations in the tumor suppressor gene PTEN are associated with a significant proportion of human cancers. Because the human genome also contains several homologs of PTEN, we considered the hypothesis that if a homolog, functionally redundant with PTEN, can be overexpressed, it may rescue the defects of a PTEN mutant. We have performed an initial test of this hypothesis in the model system Dictyostelium discoideum, which contains an ortholog of human PTEN, ptenA. Deletion of ptenA results in ...

  5. X-ray-sensitive mutants of Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Jeggo, P.A.; Kemp, L.M.

    1983-01-01

    A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D 10 values 5-10-fold of wild-type D 10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D 10 values less than 2-fold of wild-type D 10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks. (Auth.)

  6. Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants.

    Science.gov (United States)

    Liu, Ruikang; Moss, Bernard

    2016-09-01

    Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent double-stranded RNA (dsRNA) accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type VACV and that they coprecipitated with antibody. An E3 deletion mutant induced protein kinase R (PKR) and eukaryotic translation initiation factor alpha (eIF2α) phosphorylation earlier and more strongly than a decapping enzyme mutant even though less dsRNA was made, leading to more profound effects on viral gene expression. Human HAP1 and A549 cells were genetically modified by clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knockout (KO) HAP1 cells in which RNase L is intrinsically inactive but only with a double knockout (DKO) of PKR and RNase L in A549 cells, indicating that both pathways decreased replication equivalently and that no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than that of wild-type virus) in DKO A549 cells but not in DKO HAP1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but nonpermissive for VACV; however, wild-type and mutant viruses replicated in triple-KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L was sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant, particularly in HAP1 DKO, cells indicated additional translational defects. Viruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded RNA (dsRNA) triggers in host cells. We showed that the dsRNA produced in

  7. Cell-extrinsic defective lymphocyte development in Lmna(-/- mice.

    Directory of Open Access Journals (Sweden)

    J Scott Hale

    2010-04-01

    Full Text Available Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna(-/- mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(-/- mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development.Lmna(-/- mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna(-/- bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4(+ and CD8(+ T cells. Transplantation of Lmna(-/- neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development.Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(-/- mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(-/- mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.

  8. Defective Natural Killer cell antiviral capacity in paediatric HBV infection

    DEFF Research Database (Denmark)

    Heiberg, Ida Louise; Laura J., Pallett; Winther, Thilde Nordmann

    2015-01-01

    Natural Killer (NK) cells exhibit dysregulated effector function in adult chronic HBV infection (CHB), which may contribute to virus persistence. The role of NK cells in children infected perinatally with HBV is less studied. Access to a unique cohort enabled the cross-sectional evaluation of NK...... cell frequency, phenotype and function in HBV-infected children relative to uninfected children. We observed a selective defect in NK cell IFN-γ production, with conserved cytolytic function, mirroring the functional dichotomy observed in adult infection. Reduced expression of NKp30 on NK cells...

  9. Molecular Characterization of Prostate Cancer Cell Oncolysis by Herpes Simplex Virus ICP0 Mutants

    National Research Council Canada - National Science Library

    Mossman, Karen

    2005-01-01

    .... Briefly, the goals of the proposal were to characterize the oncolytic capacity of Herpes simplex virus type 1 ICPO mutants in prostate cancer cells given the relationship between ICPO and two tumor...

  10. Molecular Characterization of Prostate Cancer Cell Oncolysis by Herpes Simplex Virus ICP0 Mutants

    National Research Council Canada - National Science Library

    Mossman, Karen

    2006-01-01

    .... Briefly, the goals of the proposal were to characterize the oncolytic capacity of Herpes simplex virus type 1 ICP0 mutants in prostate cancer cells given the relationship between ICP0 and two tumor...

  11. Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens.

    Science.gov (United States)

    Liu, Guang; Wang, Xue; Liu, Yufang; Zhang, Meili; Cai, Tao; Shen, Zhirong; Jia, Yuyan; Huang, Yue

    2017-12-15

    Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-of-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some 'missing' mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double-strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

    International Nuclear Information System (INIS)

    Rocchi, P.; Ferreri, A.M.; Capucci, A.; Prodi, G.

    1981-01-01

    Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (less than 8 x 10(-6)). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTr) mutants up to 1000-fold. The maximum recovery of DTr mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages

  13. Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light

    International Nuclear Information System (INIS)

    Rocchi, P.; Ferreri, A.M.; Capucci, A.; Prodi, G.

    1981-01-01

    Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency ( -6 ). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTsup(r)) mutants up to 1000-fold. The maximum recovery of DTsup(r) mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages. (author)

  14. Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

    Science.gov (United States)

    Chailakhyan, R K; Shekhter, A B; Ivannikov, S V; Tel'pukhov, V I; Suslin, D S; Gerasimov, Yu V; Tonenkov, A M; Grosheva, A G; Panyushkin, P V; Moskvina, I L; Vorob'eva, N N; Bagratashvili, V N

    2017-02-01

    We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

  15. A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells

    Directory of Open Access Journals (Sweden)

    Williams Briana

    2003-10-01

    Full Text Available Abstract Background In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-Like Kinase (TLK1B and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line. Results Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects. Conclusions TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible.

  16. Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

    Science.gov (United States)

    Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

    2016-01-01

    The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

  17. Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects

    Science.gov (United States)

    Ruegger, M.; Dewey, E.; Hobbie, L.; Brown, D.; Bernasconi, P.; Turner, J.; Muday, G.; Estelle, M.

    1997-01-01

    Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

  18. ATM mutants

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. ATM mutants. ATM (Ataxia Telangiectasia Mutated). AT2BE and AT5B1 cells – fibroblast cell lines from Ataxia telangiectasia patients. Deletion mutants expressing truncated ATM protein which is inactive. Have been used in studies looking at the role of ATM in DNA damage ...

  19. Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

    Science.gov (United States)

    Langlands, Alistair J; Carroll, Thomas D; Chen, Yu; Näthke, Inke

    2018-02-15

    More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

  20. Aspirin exerts high anti-cancer activity in PIK3CA-mutant colon cancer cells.

    Science.gov (United States)

    Gu, Mancang; Nishihara, Reiko; Chen, Yang; Li, Wanwan; Shi, Yan; Masugi, Yohei; Hamada, Tsuyoshi; Kosumi, Keisuke; Liu, Li; da Silva, Annacarolina; Nowak, Jonathan A; Twombly, Tyler; Du, Chunxia; Koh, Hideo; Li, Wenbin; Meyerhardt, Jeffrey A; Wolpin, Brian M; Giannakis, Marios; Aguirre, Andrew J; Bass, Adam J; Drew, David A; Chan, Andrew T; Fuchs, Charles S; Qian, Zhi Rong; Ogino, Shuji

    2017-10-20

    Evidence suggests that nonsteroidal anti-inflammatory drug aspirin (acetylsalicylic acid) may improve patient survival in PIK3CA -mutant colorectal carcinoma, but not in PIK3CA -wild-type carcinoma. However, whether aspirin directly influences the viability of PIK3CA -mutant colon cancer cells is poorly understood. We conducted in vitro experiments to test our hypothesis that the anti-proliferative activity of aspirin might be stronger for PIK3CA -mutant colon cancer cells than for PIK3CA -wild-type colon cancer cells. We measured the anti-proliferative effect of aspirin at physiologic concentrations in seven PIK3CA -mutant and six PIK3CA -wild-type human colon cancer cell lines. After exposure to aspirin, the apoptotic index and cell cycle phase of colon cancer cells were assessed. In addition, the effect of aspirin was examined in parental SW48 cells and SW48 cell clones with individual knock-in PIK3CA mutations of either c.3140A>G (p.H1047R) or c.1633G>A (p.E545K). Aspirin induced greater dose-dependent loss of cell viability in PIK3CA -mutant cells than in PIK3CA -wild-type cells after treatment for 48 and 72 hours. Aspirin treatment also led to higher proportions of apoptotic cells and G0/G1 phase arrest in PIK3CA -mutant cells than in PIK3CA -wild-type cells. Aspirin treatment of isogenic SW48 cells carrying a PIK3CA mutation, either c.3140A>G (p.H1047R) or c.1633G>A (p. E545K), resulted in a more significant loss of cell viability compared to wild-type controls. Our findings indicate that aspirin causes cell cycle arrest, induces apoptosis, and leads to loss of cell viability more profoundly in PIK3CA -mutated colon cancer cells than in PIK3CA -wild-type colon cancer cells. These findings support the use of aspirin to treat patients with PIK3CA -mutant colon cancer.

  1. Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

    Science.gov (United States)

    Jain, Roshan A; Bell, Hannah; Lim, Amy; Chien, Chi-Bin; Granato, Michael

    2014-02-19

    Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

  2. The respiration of cells and mitochondria of porin deficient yeast mutants is coupled.

    Science.gov (United States)

    Michejda, J; Guo, X J; Lauquin, G J

    1990-08-31

    Several mutants of yeast lacking the porin gene have been found stable and viable on glucose or glycerol media. Ethanol-supported respiration of porin-free mutant and wild cells appeared equally coupled in vivo being similarly depressed by inhibitors of ADP/ATP translocase or of ATP synthase and stimulated by the uncoupler FCCP. The absence of porin in isolated mutant mitochondria hardly impaired the electron flux but increased the requirement for Mg2+ (or Ca2+) and for ADP and carboxyatractylate concentrations necessary to drive effectively state 3 - state 4 and state 4 - state 3 transitions, respectively. The existence of another porin species, possibly controlled by bivalent cations, is postulated.

  3. Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

    International Nuclear Information System (INIS)

    Vikhanskaya, F.L.; Khrebtukova, I.A.; Manuilova, E.S.

    1985-01-01

    A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m 2 ) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2 ) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair. (orig.)

  4. Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos.

    Science.gov (United States)

    Concepcion, Daniel; Washkowitz, Andrew J; DeSantis, Akiko; Ogea, Phillip; Yang, Jason I; Douglas, Nataki C; Papaioannou, Virginia E

    2017-07-15

    Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6 -expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos. © 2017. Published by The Company of Biologists Ltd.

  5. Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos

    Directory of Open Access Journals (Sweden)

    Daniel Concepcion

    2017-07-01

    Full Text Available Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos.

  6. Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; Li, Ning

    2016-04-22

    The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatin immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.

  7. Mitochondrial mutant cells are hypersensitive to ionizing radiation, phleomycin and mitomycin C

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Rohan; Reither, Adrian; Thomas, Robert A. [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States); Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States)

    2009-04-26

    Mitochondrial DNA (mtDNA) is an important contributor to the ATP-generating oxidative phosphorylation complex. Single nucleotide mutations in mitochondrial genes involved in ATP synthesis result in a broad range of diseases. Leber optic atrophy and Leigh's syndrome are two such diseases arising from point mutations in the mitochondrial genome. Here, ionizing radiation, phleomycin and mitomycin C (MMC) were used to induce structural chromosomal aberrations in Leber's and Leigh's cells to investigate how these mitochondrial mutations affect the cell's DNA repair processes. Because of the energy deprivation that results from mitochondrial mutations, we hypothesized that these mutant cells would demonstrate hypersensitivity when exposed to oxidative and genotoxic stress and we also expected that these cells would not be able to repair nuclear DNA damage as efficiently as normal cells. As a consequence, these mutant cells are expected to show increased levels of DNA damage, longer cell cycle delays and increased levels of cell death. Following acute radiation exposure these mutant cells showed an increase in the number of chromosomal aberrations and decreased mitotic indices when compared with normal human lymphoblastoid cells with wild-type mtDNA. When exposed to phleomycin or MMC, the mitochondrial mutant cells again showed hypersensitivity and decreased mitotic indices compared to normal cells. These results suggest that Leber's and Leigh's cells have an impaired ability to cope with oxidative and genotoxic stress. These observations may help explain the role of ATP generation in understanding the enhanced sensitivity of mitochondrial mutant cells to cancer therapeutic agents and to adverse environmental exposure, suggesting that individuals with mtDNA mutations may be at a greater risk for cancer and other diseases that result from an accumulation of nuclear DNA damage.

  8. Mesenchymal Stem Cells for Treating Articular Cartilage Defects and Osteoarthritis.

    Science.gov (United States)

    Wang, Yu; Yuan, Mei; Guo, Quan-yi; Lu, Shi-bi; Peng, Jiang

    2015-01-01

    Articular cartilage damage and osteoarthritis are the most common joint diseases. Joints are prone to damage caused by sports injuries or aging, and such damage regularly progresses to more serious joint disorders, including osteoarthritis, which is a degenerative disease characterized by the thinning and eventual wearing out of articular cartilage, ultimately leading to joint destruction. Osteoarthritis affects millions of people worldwide. Current approaches to repair of articular cartilage damage include mosaicplasty, microfracture, and injection of autologous chondrocytes. These treatments relieve pain and improve joint function, but the long-term results are unsatisfactory. The long-term success of cartilage repair depends on development of regenerative methodologies that restore articular cartilage to a near-native state. Two promising approaches are (i) implantation of engineered constructs of mesenchymal stem cell (MSC)-seeded scaffolds, and (ii) delivery of an appropriate population of MSCs by direct intra-articular injection. MSCs may be used as trophic producers of bioactive factors initiating regenerative activities in a defective joint. Current challenges in MSC therapy are the need to overcome current limitations in cartilage cell purity and to in vitro engineer tissue structures exhibiting the required biomechanical properties. This review outlines the current status of MSCs used in cartilage tissue engineering and in cell therapy seeking to repair articular cartilage defects and related problems. MSC-based technologies show promise when used to repair cartilage defects in joints.

  9. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

    Directory of Open Access Journals (Sweden)

    Robert Ruggiero

    Full Text Available The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  10. DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation.

    Science.gov (United States)

    Wu, Yu-Yi; Hou, Bo-Han; Lee, Wen-Chi; Lu, Shin-Hua; Yang, Chen-Jui; Vaucheret, Hervé; Chen, Ho-Ming

    2017-06-01

    DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  11. Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome

    Directory of Open Access Journals (Sweden)

    Semina Elena V

    2006-07-01

    Full Text Available Abstract Background Axenfeld-Rieger syndrome (ARS is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. Methods We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss junction associated with exon 4 (IVS4-1G>T, a G>C mutation at position +5 of the 5' (ss of exon 4 (IVS4+5G>C, and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G. Results Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. Conclusion This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

  12. Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

    Directory of Open Access Journals (Sweden)

    C.R.C. Franco

    2001-08-01

    Full Text Available We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO cells. We used both wild-type (CHO-K1 cells and a mutant (CHO-745 which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.

  13. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    Science.gov (United States)

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

  14. Urea enhances cell lysis of Schizosaccharomyces pombe ura4 mutants.

    Science.gov (United States)

    Nishino, Kohei; Kushima, Misaki; Kaino, Tomohiro; Matsuo, Yasuhiro; Kawamukai, Makoto

    2017-07-01

    Cell lysis is induced in Schizosaccharomyces pombe ∆ura4 cells grown in YPD medium, which contains yeast extract, polypeptone, and glucose. To identify the medium components that induce cell lysis, we first tested various kinds of yeast extracts from different suppliers. Cell lysis of ∆ura4 cells on YE medium was observed when yeast extracts from OXOID, BD, Oriental, and Difco were used, but not when using yeast extract from Kyokuto. To determine which compounds induced cell lysis, we subjected yeast extract and polypeptone to GC-MS analysis. Ten kinds of compounds were detected in OXOID and BD yeast extracts, but not in Kyokuto yeast extract. Among them was urea, which was also present in polypeptone, and it clearly induced cell lysis. Deletion of the ure2 gene, which is responsible for utilizing urea, abolished the lytic effect of urea. The effect of urea was suppressed by deletion of pub1, and a similar phenotype was observed in the presence of polypeptone. Thus, urea is an inducer of cell lysis in S. pombe ∆ura4 cells.

  15. Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

    OpenAIRE

    Manos, M M; Gluzman, Y

    1984-01-01

    The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV...

  16. Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2.

    Science.gov (United States)

    De Mars Cody, J; Conway, T W

    1981-01-01

    A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein. Images PMID:6783768

  17. chy1, an Arabidopsis mutant with impaired beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase.

    Science.gov (United States)

    Zolman, B K; Monroe-Augustus, M; Thompson, B; Hawes, J W; Krukenberg, K A; Matsuda, S P; Bartel, B

    2001-08-17

    The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal beta-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes beta-hydroxyisobutyryl-CoA. We demonstrated that a human beta-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes beta-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered beta-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.

  18. Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and oxidative stress.

    Science.gov (United States)

    Polajnar, Mira; Zavašnik-Bergant, Tina; Kopitar-Jerala, Nataša; Tušek-Žnidarič, Magda; Zerovnik, Eva

    2014-09-01

    EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed. Copyright © 2014. Published by Elsevier B.V.

  19. Induction and selection of mutants from in vitro cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yung Il; Kim, Jae Sung; Shin, In Chul; Lee, Sang Jae [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1994-07-01

    Mutant cell lines are useful for biochemical, physiological and genetical material for marker in various genetic manipulation experiments and for the direct use in crop plant improvement. Mutant selection may lead to the production of plants showing resistance or tolerance to specific environmental stress, such as solinity, drought, toxed metals, herbicides, pathogens and low temperature. In this review, these included the production of the somatic variation, the selection process itself and stability of the selected characters in cell culture and regenerated plant. Which would seem to be useful for improving plants and securring genetic resources. 45 refs. (Author).

  20. Defective nucleolar localization and dominant interfering properties of a parafibromin L95P missense mutant causing the hyperparathyroidism-jaw tumor syndrome

    Science.gov (United States)

    Panicker, Leelamma M.; Zhang, Jian-Hua; Dagur, Pradeep K.; Gastinger, Matthew J.; Simonds, William F.

    2011-01-01

    The hyperparathyroidism-jaw tumor syndrome (HPT-JT) is a familial cancer syndrome that can result from germline inactivation of HRPT2/CDC73, a putative tumor suppressor gene that encodes parafibromin, a component of the transcriptional regulatory PAF1 complex with homology to the yeast protein Cdc73p. The vast majority of HRPT2/CDC73 germline mutations identified have been truncation or frameshift mutations, and loss-of-function due to missense mutation is rare. We report here a kindred with HPT-JT due to a germline L95P missense mutation in parafibromin. The mutant parafibromin was studied in vitro to understand the basis of its presumed loss-of-function. When transfected in cultured cells the L95P mutant was expressed to a lower level than wild-type parafibromin, a difference that was not overcome by inhibition of the proteasome degradation pathway. The L95P mutant parafibromin retained the ability to assemble with endogenous PAF1 complex components as evidenced by co-immunoprecipitation. Analysis of subcellular localization showed that the L95P mutant was markedly deficient in nucleolar localization compared to the wild-type, an impairment likely resulting from disruption of a putative nucleolar localization signal immediately upstream of the L95P mutation. Transfection of the L95P parafibromin mutant, but not the wild type, enhanced cell-cycle progression and increased cell survival in NIH-3T3 and HEK 293 cells, resulting apparently from dominant interference with endogenous parafibromin action. The simultaneous loss of nucleolar localization and acquisition of a growth stimulatory phenotype with the L95P mutation raise the possibility that parafibromin must interact with targets in the nucleolus to fully execute its tumor suppressor functions. PMID:20304979

  1. Defective immunoregulatory T-cell function in chronic lymphocytic leukemia

    International Nuclear Information System (INIS)

    Han, T.; Ozer, H.; Henderson, E.S.; Dadey, B.; Nussbaum-Blumenson, A.; Barcos, M.

    1981-01-01

    Chronic lymphocytic leukemia (CLL) of B-cell origin results in the malignant proliferation of small immunoglobulin-bearing lymphocytes. There is currently a controversy in the literature regarding both the ability of this leukemic population to differentiate into mature plasma cells, as well as the ability of apparently normal T cells from these patients to regulate allogeneic B-cell differentiation. In the present study we have examined the lymphocytes of CLL patients in various clinical stages of their disease and with different surface phenotypes of their leukemic B-cell population. Our results show that leukemic CLL B cells from all 20 patients (including one patient with a monoclonal IgM paraprotein and another with a monoclonal IgG paraprotein) are incapable of further differentiation even in the absence of suppressor T cells and the presence of helper T lymphocytes. This lack of capacity to differentiate is unaffected by clinical stage, by therapy, or by the phenotype of the malignant population. Since the leukemic B population did not suppress normal allogeneic B-cell differentiation, the maturation deficit is evidently intrinsic to the leukemic clone rather than a result of activity of non-T suppressor cells. T helper function was also variably depressed in the blood of some patients with CLL, and this depression did not correlate with clinical stage, with therapy, or with the degree of lymphocytosis. Dysfunction of radiosensitive T suppressor cells was found to be the most consistent regulatory deficit of CLL T cells. Each of 11 patients whose leukemic cell population was of the μdelta, μα, or μ phenotype had both helper and suppressor cell defects

  2. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    International Nuclear Information System (INIS)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-01-01

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  3. Acidithiobacillus caldus sulfur oxidation model based on transcriptome analysis between the wild type and sulfur oxygenase reductase defective mutant.

    Directory of Open Access Journals (Sweden)

    Linxu Chen

    Full Text Available Acidithiobacillus caldus (A. caldus is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox system (omitting SoxCD, non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR. The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system.An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor was created and its growth abilities were measured in media using elemental sulfur (S(0 and tetrathionate (K(2S(4O(6 as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR of the wild type and the Δsor mutant in S(0 and K(2S(4O(6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO and heterodisulfide reductase (HDR, the truncated Sox pathway, and the S(4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media.An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

  4. Acidithiobacillus caldus sulfur oxidation model based on transcriptome analysis between the wild type and sulfur oxygenase reductase defective mutant.

    Science.gov (United States)

    Chen, Linxu; Ren, Yilin; Lin, Jianqun; Liu, Xiangmei; Pang, Xin; Lin, Jianqiang

    2012-01-01

    Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system. An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S(0)) and tetrathionate (K(2)S(4)O(6)) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S(0) and K(2)S(4)O(6) media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S(4)I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media. An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

  5. Application of the systematic “DAmP” approach to create a partially defective C. albicans mutant

    OpenAIRE

    Finkel, JS; Yudanin, N; Nett, JE; Andes, DR; Mitchell, AP

    2011-01-01

    An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a “Decreased Abundance by mRNA Perturbation” (DAmP) allele (Yan et al., 2008). DAmP alleles...

  6. Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant.

    Science.gov (United States)

    Jastrab, Jordan B; Samanovic, Marie I; Copin, Richard; Shopsin, Bo; Darwin, K Heran

    2017-04-01

    Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis p roteasome a ccessory f actor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR ( h eat s hock p rotein r epressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress. Copyright © 2017 American Society for Microbiology.

  7. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sofie eVan Rossom

    2012-07-01

    Full Text Available DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine-nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2 to 6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human HEK293T cells. Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  8. Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

    International Nuclear Information System (INIS)

    Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

    2004-01-01

    Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

  9. Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization

    DEFF Research Database (Denmark)

    Bjerregaard, Annette; Mogensen, Helle Smidt; Hack, N

    1997-01-01

    We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G prote...

  10. A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

    Science.gov (United States)

    Moleleki, Lucy Novungayo; Pretorius, Rudolph Gustav; Tanui, Collins Kipngetich; Mosina, Gabolwelwe; Theron, Jacques

    2017-01-01

    Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain. © 2016 BSPP and John Wiley & Sons Ltd.

  11. Transfection of rat embryo cells with mutant p53 increases the intrinsic radiation resistance

    International Nuclear Information System (INIS)

    Pardo, F.S.; Su, M.; Gerweck, L.; Schmidt, E.V.; Borek, C.; Preffer, F.; Dombkowski, D.

    1994-01-01

    Dominant oncogenic sequences have been shown to modulate the intrinsic radiation sensitivity of cells of both human and murine tumor cell lines. Whether transfection with candidate tumor-suppressor genes can modulate intrinsic radiation sensitivity is unknown. The data presented here demonstrate that transfection of rat embryo cells with a mutant p53 allele can increase the intrinsic radiation resistance of cells in vitro. First, transfection with mutant p53 resulted in transformed cellular morphology. Second, the transfected clone and the corresponding pooled population of transfected clones were more resistant to ionizing radiation in vitro. Last, analyses of the parameters of cell kinetics suggested that the radiobiological effects were unlikely to be due to altered parameters of cell kinetics at the time of irradiation, suggesting that mutant p53 altered the intrinsic radiation resistance of transfected cells by a more direct mechanism. Further experimentation will be necessary to develop a mechanistic approach for the study of these alterations. 29 refs., 3 figs., 2 tabs

  12. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  13. bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

    Science.gov (United States)

    Kar, Ritika; Nangpal, Prachi; Mathur, Shubhita; Singh, Swati

    2017-01-01

    Owing to the devastation caused by tuberculosis along with the unsatisfactory performance of the Bacillus Calmette–Guérin (BCG) vaccine, a more efficient vaccine than BCG is required for the global control of tuberculosis. A number of studies have demonstrated an essential role of biotin biosynthesis in the growth and survival of several microorganisms, including mycobacteria, through deletion of the genes involved in de novo biotin biosynthesis. In this study, we demonstrate that a bioA mutant of Mycobacterium tuberculosis (MtbΔbioA) is highly attenuated in the guinea pig model of tuberculosis when administered aerogenically as well as intradermally. Immunization with MtbΔbioA conferred significant protection in guinea pigs against an aerosol challenge with virulent M. tuberculosis, when compared with the unvaccinated animals. Booster immunization with MtbΔbioA offered no advantage over a single immunization. These experiments demonstrate the vaccinogenic potential of the attenuated M. tuberculosis bioA mutant against tuberculosis. PMID:28658275

  14. Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

    Science.gov (United States)

    Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2010-01-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991

  15. Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein.

    Science.gov (United States)

    Rochon, D M

    1991-01-01

    It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptom-attenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general. Images PMID:1722320

  16. Mutant torsinA interacts with tyrosine hydroxylase in cultured cells.

    Science.gov (United States)

    O'Farrell, C A; Martin, K L; Hutton, M; Delatycki, M B; Cookson, M R; Lockhart, P J

    2009-12-15

    A specific mutation (DeltaE302/303) in the torsinA gene underlies most cases of dominantly inherited early-onset torsion dystonia. This mutation causes the protein to aggregate and form intracellular inclusion bodies in cultured cells and animal models. Co-expression of the wildtype and mutant proteins resulted in the redistribution of the wildtype protein from the endoplasmic reticulum to inclusion bodies in cultured HEK293 cells, and this was associated with increased interaction between the two proteins. Expression of DeltaE302/303 but not wildtype torsinA in primary postnatal midbrain neurons resulted in the formation of intracellular inclusion bodies, predominantly in dopaminergic neurons. Tyrosine hydroxylase was sequestered in these inclusions and this process was mediated by increased protein-protein interaction between mutant torsinA and tyrosine hydroxylase. Analysis in an inducible neuroblastoma cell culture model demonstrated altered tyrosine hydroxylase activity in the presence of the mutant but not wildtype torsinA protein. Our results suggest that the interaction of tyrosine hydroxylase and mutant torsinA may contribute to the phenotype and reported dopaminergic dysfunction in torsinA-mediated dystonia.

  17. Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila.

    Directory of Open Access Journals (Sweden)

    Vanessa Nieratschker

    2009-10-01

    Full Text Available Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP protein is associated with T-shaped ribbons ("T-bars" at presynaptic active zones (AZs. BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D which shows high homology to mammalian genes for SR protein kinases (SRPKs. SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins. Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the

  18. The zebrafish flotte lotte mutant reveals that the local retinal environment promotes the differentiation of proliferating precursors emerging from their stem cell niche.

    Science.gov (United States)

    Cerveny, Kara L; Cavodeassi, Florencia; Turner, Katherine J; de Jong-Curtain, Tanya A; Heath, Joan K; Wilson, Stephen W

    2010-07-01

    It is currently unclear how intrinsic and extrinsic mechanisms cooperate to control the progression from self-renewing to neurogenic divisions in retinal precursor cells. Here, we use the zebrafish flotte lotte (flo) mutant, which carries a mutation in the elys (ahctf1) gene, to study the relationship between cell cycle progression and neuronal differentiation by investigating how proliferating progenitor cells transition towards differentiation in a retinal stem cell niche termed the ciliary marginal zone (CMZ). In zebrafish embryos without Elys, CMZ cells retain the capacity to proliferate but lose the ability to enter their final neurogenic divisions to differentiate as neurons. However, mosaic retinae composed of wild-type and flo cells show that despite inherent cell cycle defects, flo mutant cells progress from proliferation to differentiation when in the vicinity of wild-type retinal neurons. We propose that the differentiated retinal environment limits the proliferation of precursors emerging from the CMZ in a manner that explains the spatial organisation of cells in the CMZ and ensures that proliferative retinal progenitors are driven towards differentiation.

  19. Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4

    Science.gov (United States)

    Yamamoto, M.; Yamamoto, K. T.

    1999-01-01

    The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.

  20. A rhamnose-deficient lipopolysaccharide mutant of Rhizobium sp. IRBG74 is defective in root colonization and beneficial interactions with its flooding-tolerant hosts Sesbania cannabina and wetland rice.

    Science.gov (United States)

    Mitra, Shubhajit; Mukherjee, Arijit; Wiley-Kalil, Audrey; Das, Seema; Owen, Heather; Reddy, Pallavolu M; Ané, Jean-Michel; James, Euan K; Gyaneshwar, Prasad

    2016-10-01

    Rhizobium sp. IRBG74 develops a classical nitrogen-fixing symbiosis with the aquatic legume Sesbania cannabina (Retz.). It also promotes the growth of wetland rice (Oryza sativa L.), but little is known about the rhizobial determinants important for these interactions. In this study, we analyzed the colonization of S. cannabina and rice using a strain of Rhizobium sp. IRBG74 dually marked with β-glucuronidase and the green fluorescent protein. This bacterium colonized S. cannabina by crack entry and through root hair infection under flooded and non-flooded conditions, respectively. Rhizobium sp. IRBG74 colonized the surfaces of wetland rice roots, but also entered them at the base of lateral roots. It became endophytically established within intercellular spaces in the rice cortex, and intracellularly within epidermal and hypodermal cells. A mutant of Rhizobium sp. IRBG74 altered in the synthesis of the rhamnose-containing O-antigen exhibited significant defects, not only in nodulation and symbiotic nitrogen fixation with S. cannabina, but also in rice colonization and plant growth promotion. Supplementation with purified lipopolysaccharides from the wild-type strain, but not from the mutant, restored the beneficial colonization of rice roots, but not fully effective nodulation of S. cannabina Commonalities and differences in the rhizobial colonization of the roots of wetland legume and rice hosts are discussed. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression.

    Science.gov (United States)

    Zhang, Xiaoran; Rao, Aparana; Sette, Paola; Deibert, Christopher; Pomerantz, Alexander; Kim, Wi Jin; Kohanbash, Gary; Chang, Yigang; Park, Yongseok; Engh, Johnathan; Choi, Jaehyuk; Chan, Timothy; Okada, Hideho; Lotze, Michael; Grandi, Paola; Amankulor, Nduka

    2016-10-01

    Diffuse gliomas are poorly immunogenic, fatal brain tumors. The basis for insufficient antitumor immunity in diffuse gliomas is unknown. Gain-of-function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. We analyzed the database of The Cancer Genome Atlas for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligand expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza-2'deoxycytodine (decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. IDH mutant glioma stemlike cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes are resistant to NK cell-mediated lysis. Decitabine increases NKG2D ligand expression and restores NK-mediated lysis of IDH mutant cells in an NKG2D-dependent manner. IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

    Directory of Open Access Journals (Sweden)

    Chun Fu

    Full Text Available After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance protein. Fanconi anemia (FA proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

  3. Identification of a previously undetected metabolic defect in the Complex II Caenorhabditis elegans mev-1 mutant strain using respiratory control analysis.

    Science.gov (United States)

    Fong, Sheng; Ng, Li Fang; Ng, Li Theng; Moore, Philip K; Halliwell, Barry; Gruber, Jan

    2017-04-01

    Hypometabolism may play an important role in the pathogenesis of ageing and ageing-related diseases. The nematode Caenorhabditis elegans offers the opportunity to study "living mitochondria" in a small (~1 mm) animal replete with a highly stereotypical, yet complex, anatomy and physiology. Basal oxygen consumption rate is often employed as a proxy for energy metabolism in this context. This parameter is traditionally measured using single-chamber Clark electrodes without the addition of metabolic modulators. Recently, multi-well oxygen electrodes, facilitating addition of metabolic modulators and hence study of respiratory control during different mitochondrial respiration states, have been developed. However, only limited official protocols exist for C. elegans, and key limitations of these techniques are therefore unclear. Following modification and testing of some of the existing protocols, we used these methods to explore mitochondrial bioenergetics in live nematodes of an electron transfer chain Complex II mutant strain, mev-1, and identified a previously undetected metabolic defect. We find that mev-1 mutants cannot respond adequately to increased energy demands, suggesting that oxidative phosphorylation is more severely impaired in these animals than has previously been appreciated.

  4. Anatomical distributional defects in mutant genes associated with dominant intermediate Charcot-Marie-Tooth disease type C in an adenovirus-mediated mouse model

    Directory of Open Access Journals (Sweden)

    SeoJin Lee

    2017-01-01

    Full Text Available Dominant intermediate Charcot-Marie-Tooth disease type C (DI-CMTC is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features. DI-CMTC is linked to tyrosyl-tRNA synthetase (YARS-associated neuropathies, which are caused by E196K and G41R missense mutations and a single de novo deletion (153-156delVKQV. It is well-established that these YARS mutations induce neuronal dysfunction, morphological symptoms involving axonal degeneration, and impaired motor performance. The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria. An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord, peripheral axons, and dorsal root ganglia. Following transfection of YARS-expressing viruses, the distributions of wild-type (WT YARS and E196K mutant proteins were compared in all expressed regions; G41R was not expressed. The proportion of Flag/green fluorescent protein (GFP double-positive signaling in the E196K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons. All adenovirus genes, and even the empty vector without the YARS gene, exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter. The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes. Thus, use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons, the DI-CMTC phenotype, and potential treatment approaches for this disease.

  5. Loss-of-function mutants and overexpression lines of the Arabidopsis cyclin CYCA1;2/Tardy Asynchronous Meiosis exhibit different defects in prophase-i meiocytes but produce the same meiotic products.

    Directory of Open Access Journals (Sweden)

    Yixing Wang

    Full Text Available In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/Tardy Asynchronous Meiosis (TAM gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products.

  6. Proteomics Characterization of the Molecular Mechanisms of Mutant P53 Reactivation with PRIMA-1 in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Daoud, Sayed S

    2006-01-01

    The main purpose of the study is to identify novel protein-protein interactions in various locations of cells to establish the molecular mechanisms of mutant p53 reactivation with PRIMA-1 in breast cancer cells...

  7. Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae.

    Science.gov (United States)

    Bhalla, Tek Chand; Prashant; Kumari, Nisha; Kumar, Vijay; Kumar, Virender; Savitri

    2016-01-01

    The resting cells of Gordonia terrae mutant E9 having enhanced nitrilase activity were used for biotransformation of 4-hydroxy-3-methoxybenzonitrile into vanillic acid. The maximum conversion was observed in 0.1 M phosphate buffer (pH 8.0), using 60 mM substrate and 0.75 mgDCW resting cells in 1 mL reaction at 40 °C. Km of the whole cell nitrilase of wild and mutant strains of G. terrae for this substrate were 20 and 16.6 mM, and Vmax were 0.19 and 0.95 Umg(-1)(DCW), respectively. Fed batch reaction for transformation of 4-hydroxy-3-methoxybenzonitrile using whole cell nitrilase of wild G. terrae resulted in 2.36 g of vanillic acid in 5 h with a catalytic and volumetric productivity of 0.78 gg(-1)(DCW) h(-1) and 4.72 gL(-1)h(-1), respectively. The whole cell nitrilase of G. terrae mutant E9 resulted in higher catalytic and volumetric productivity, i.e., 1.68 gg(-1)DCW h(-1) and 10 gL(-1)h(-1). A total 5.04 g of vanillic acid with 99% purity were accumulated in 100 mL of reaction after 5 h.

  8. Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Buyck, N.; Thomas, S.

    2001-01-01

    Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) at the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.

  9. Tumorigenic Properties of Drosophila Epithelial Cells Mutant for lethal giant larvae.

    Science.gov (United States)

    Calleja, Manuel; Morata, Ginés; Casanova, Jordi

    2016-08-01

    Mutations in Drosophila tumor suppressor genes (TSGs) lead to the formation of invasive tumors in the brain and imaginal discs. Here we studied the tumorigenic properties of imaginal discs mutant for the TSG gene lethal giant larvae (lgl). lgl mutant cells display the characteristic features of mammalian tumor cells: they can proliferate indefinitely, induce additional tracheogenesis (an insect counterpart of vasculogenesis) and invade neighboring tissues. Lgl mutant tissues exhibit high apoptotic levels, which lead to the activation of the Jun-N-Terminal Kinase (JNK) pathway. We propose that JNK is a key factor in the acquisition of these tumorigenic properties; it promotes cell proliferation and induces high levels of Mmp1 and confers tumor cells capacity to invade wild-type tissue. Noteworthy, lgl RNAi-mediated down-regulation does not produce similar transformations in the central nervous system (CNS), thereby indicating a fundamental difference between the cells of developing imaginal discs and those of differentiated organs. We discuss these results in the light of the "single big-hit origin" of some human pediatric or developmental cancers. Down-regulation of lgl in imaginal discs is sufficient to enhance tracheogenesis and to promote invasion and colonization of other larval structures including the CNS. Developmental Dynamics 245:834-843, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Prion Propagation in Cells Expressing PrP Glycosylation Mutants

    Science.gov (United States)

    Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-01-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

  11. RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

    Directory of Open Access Journals (Sweden)

    Simona Lamba

    2014-09-01

    Full Text Available KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

  12. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    Science.gov (United States)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  13. Two types of albino mutants in desert and migratory locusts are caused by gene defects in the same signaling pathway.

    Science.gov (United States)

    Sugahara, Ryohei; Tanaka, Seiji; Jouraku, Akiya; Shiotsuki, Takahiro

    2017-04-15

    Albinism is caused by mutations in the genes involved in melanin production. Albino nymphs of Locusta migratoria and Schistocerca gregaria reared under crowded conditions are uniformly creamy-white in color. However, nothing is known about the molecular mechanisms underlying this phenomenon in locusts. The albino strain of L. migratoria is known to lack the dark-color-inducing neuropeptide corazonin (Crz). In this study, we report that this albino strain has a 10-base-pair deletion in the gene LmCRZ, which encodes Crz. This mutation was found to cause a frame-shift, resulting in a null mutation in Crz. On the other hand, the albino strain of S. gregaria is known to have an intact Crz. This strain was found to possess a single-nucleotide substitution in the middle of the Crz receptor-encoding gene, SgCRZR, which caused a nonsense mutation, resulting in a truncated receptor. Silencing of SgCRZR in wild-type S. gregaria nymphs greatly reduced the area and intensity of their black patterning, suggesting that the functional defect of SgCRZR likely causes the albinism. The expression level of SgCRZR in the albino S. gregaria was comparable to that in the wild type. Unlike the wild type, the albino strain of this locust did not show a phase-dependent shift in a morphometric trait controlled by Crz. From these results, we conclude that the mutations in LmCRZ and SgCRZR are responsible for the albinism in L. migratoria and S. gregaria, respectively, indicating that the two types of albinism are caused by different genetic defects in the same Crz signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro.

    Science.gov (United States)

    Ivan, M; Ludgate, M; Gire, V; Bond, J A; Wynford-Thomas, D

    1997-08-01

    Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

  15. Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    International Nuclear Information System (INIS)

    Miyazaki, Nobuyoshi; Nagai, Kazuo; Tamura, Gakuzo

    1976-01-01

    A thermosensitive mutant ts 42, of Bacillus subtilis Marburg 168 thy trp2 which requires uracil, was examined as to the colony-forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in modified woese's medium. However, the cells retained the viability when sodium succinate or potassium chloride was added to the medium at that temperature, although uranil deficiency was unchanged. A little but significant incorporation of adenine-8- 14 C into RNA still continued even after the incorporation of N-acetyl- 3 H-D-glucosamine into the acid-insoluble fraction of the cells terminated in the modified Woese's medium at 48 0 C. Both incorporations as well as the increase of absorbance were slowed down in the presence of sodium succinate at 48 0 C. This mutant, ts42, was more sensitive to deoxycholate than the parent wild strain. The resoration of the colony-forming ability after the temperature shifted back from 48 0 to 37 0 C was suppressed by the addition of deoxycholate to the medium. However, the cells became resistant to deoxycholate when uracil had been added to the medium prior to the temperature shift. (Kobatake, H.)

  16. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    Science.gov (United States)

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  17. Gene expression profiling of oxidative stress response of C. elegans aging defective AMPK mutants using massively parallel transcriptome sequencing

    Directory of Open Access Journals (Sweden)

    Baillie David L

    2011-02-01

    Full Text Available Abstract Background A strong association between stress resistance and longevity in multicellular organisms has been established as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the C. elegans homolog of an alpha subunit of AMP-activated protein kinase (AMPK is an intracellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and lifespan extension. Findings Here, we investigated global transcriptional responses of aak-2 mutants to oxidative stress and in turn identified potential downstream targets of AAK-2 involved in stress resistance in C. elegans. We employed massively parallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis. Specifically, we compared the transcriptomes of aak-2 and wild type animals under normal conditions and conditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptional activities that take place in C. elegans in response to oxidative stress both in the presence and absence of AAK-2. Conclusions The analysis presented in this study has enabled us to identify potential genes involved in stress resistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended our current knowledge of general defense responses of C. elegans against oxidative stress supporting the function for AAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptional regulation of genes involved in reproductive processes.

  18. A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens Mutante de Salmonella enterica serovar Gallinarum duplo defectivo na biossíntese de cobalamina é avirulento para aves

    Directory of Open Access Journals (Sweden)

    Jacqueline Boldrin de Paiva

    2009-09-01

    Full Text Available Salmonella enterica serovar Gallinarum (SG is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.Salmonella enterica serovar Gallinarum (SG é o agente do tifo aviário, doença severa que provoca mortalidade em até 80% do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir

  19. Stem-cells used in treatment of periodontal bone defects

    International Nuclear Information System (INIS)

    Perez Borrego, Amparo; Dominguez Rodriguez, Libia; Ilisastigui Ortueta, Zaida Teresa; Hernandez Ramirez, Porfirio

    2009-01-01

    The aggressive periodontitis might to provoke the tooth loss, of its function and to affect the patient's aesthetics. The techniques used for the lost bone regeneration, not always are successful and in occasions are very expensive. For years it is working in tissues regeneration by stem-cells implantation. Periodontium could be a potential for this task. This is a study of a female patient aged 26 with an apparent health status and aggressive periodontitis backgrounds treated from 10 years ago, seen in our service due to dental mobility producing mastication nuisances. At clinical examination we noted systemic chronic inflammation of gums, grade II and III dental mobility in incisives and molars teeth, 4-8 mm systemic periodontal sacs and furcation lesions in inferior molars. At radiographs advanced bone losses and a decrease of systemic bone density are noted. After written consent and the initial preparation, we carried out a periodontal flap in the 35 and 37 teeth zone, where the stem-cells concentrate was placed, in bone defects of superior molars (16-17) and previous radicular scraping and isolation, treatment consisted in stem-cells perfusion without flap. There were not postoperative side effects. At 7 days there was a normal coloration, at three months on noted at radiograph a bone neoformation, and at six months gum remained healthy, with a decrease of dental mobility in segment treated and in the evolutionary radiograph it was evidenced the formation and increase of density

  20. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Science.gov (United States)

    Bueno, Carlos; Tabares-Seisdedos, Rafael; Moraleda, Jose M; Martinez, Salvador

    2016-01-01

    Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may

  1. Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

    Directory of Open Access Journals (Sweden)

    Carlos Bueno

    Full Text Available Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the

  2. Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

    Science.gov (United States)

    A seed-specific maize mutant, defective endosperm18 (de18), accumulates approximately 40% less dry mass and 10- to 15- fold less auxin (IAA) as compared to the De18; however, a causal basis of these changes is not known. Cellular analyses here showed that the de18 developing endosperm had lower tota...

  3. Molecular description of eye defects in the zebrafish Pax6b mutant, sunrise, reveals a Pax6b-dependent genetic network in the developing anterior chamber.

    Directory of Open Access Journals (Sweden)

    Masanari Takamiya

    Full Text Available The cornea is a central component of the camera eye of vertebrates and even slight corneal disturbances severely affect vision. The transcription factor PAX6 is required for normal eye development, namely the proper separation of the lens from the developing cornea and the formation of the iris and anterior chamber. Human PAX6 mutations are associated with severe ocular disorders such as aniridia, Peters anomaly and chronic limbal stem cell insufficiency. To develop the zebrafish as a model for corneal disease, we first performed transcriptome and in situ expression analysis to identify marker genes to characterise the cornea in normal and pathological conditions. We show that, at 7 days post fertilisation (dpf, the zebrafish cornea expresses the majority of marker genes (67/84 tested genes found also expressed in the cornea of juvenile and adult stages. We also characterised homozygous pax6b mutants. Mutant embryos have a thick cornea, iris hypoplasia, a shallow anterior chamber and a small lens. Ultrastructure analysis revealed a disrupted corneal endothelium. pax6b mutants show loss of corneal epithelial gene expression including regulatory genes (sox3, tfap2a, foxc1a and pitx2. In contrast, several genes (pitx2, ctnnb2, dcn and fabp7a were ectopically expressed in the malformed corneal endothelium. Lack of pax6b function leads to severe disturbance of the corneal gene regulatory programme.

  4. Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling.

    Science.gov (United States)

    Rahnamoun, Homa; Lu, Hanbin; Duttke, Sascha H; Benner, Christopher; Glass, Christopher K; Lauberth, Shannon M

    2017-09-29

    Inflammation influences cancer development, progression, and the efficacy of cancer treatments, yet the mechanisms by which immune signaling drives alterations in the cancer cell transcriptome remain unclear. Using ChIP-seq, RNA-seq, and GRO-seq, here we demonstrate a global overlap in the binding of tumor-promoting p53 mutants and the master proinflammatory regulator NFκB that drives alterations in enhancer and gene activation in response to chronic TNF-α signaling. We show that p53 mutants interact directly with NFκB and that both factors impact the other's binding at diverse sets of active enhancers. In turn, the simultaneous and cooperative binding of these factors is required to regulate RNAPII recruitment, the synthesis of enhancer RNAs, and the activation of tumor-promoting genes. Collectively, these findings establish a mechanism by which chronic TNF-α signaling orchestrates a functional interplay between mutant p53 and NFκB that underlies altered patterns of cancer-promoting gene expression.Inflammation is known to affect cancer development, yet the mechanisms by which immune signaling drives transformation remain unclear. Here, the authors provide evidence that chronic TNF-α signaling promotes the enhancer binding and transcriptional interplay between mutant p53 and NFκB.

  5. Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression.

    Science.gov (United States)

    Durbarry, Anjusha; Vizir, Igor; Twell, David

    2005-01-01

    Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants.

  6. Male Germ Line Development in Arabidopsis. duo pollen Mutants Reveal Gametophytic Regulators of Generative Cell Cycle Progression1[w

    Science.gov (United States)

    Durbarry, Anjusha; Vizir, Igor; Twell, David

    2005-01-01

    Male germ line development in flowering plants is initiated with the formation of the generative cell that is the progenitor of the two sperm cells. While structural features of the generative cell are well documented, genetic programs required for generative cell cycle progression are unknown. We describe two novel Arabidopsis (Arabidopsis thaliana) mutants, duo pollen1 (duo1) and duo pollen2 (duo2), in which generative cell division is blocked, resulting in the formation of bicellular pollen grains at anthesis. duo1 and duo2 map to different chromosomes and act gametophytically in a male-specific manner. Both duo mutants progress normally through the first haploid division at pollen mitosis I (PMI) but fail at distinct stages of the generative cell cycle. Mutant generative cells in duo1 pollen fail to enter mitosis at G2-M transition, whereas mutant generative cells in duo2 enter PMII but arrest at prometaphase. In wild-type plants, generative and sperm nuclei enter S phase soon after inception, implying that male gametic cells follow a simple S to M cycle. Mutant generative nuclei in duo1 complete DNA synthesis but bypass PMII and enter an endocycle during pollen maturation. However, mutant generative nuclei in duo2 arrest in prometaphase of PMII with a 2C DNA content. Our results identify two essential gametophytic loci required for progression through different phases of the generative cell cycle, providing the first evidence to our knowledge for genetic regulators of male germ line development in flowering plants. PMID:15618418

  7. Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

    DEFF Research Database (Denmark)

    Jensen, T G; Bross, P; Andresen, B S

    1995-01-01

    ." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

  8. Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

    Directory of Open Access Journals (Sweden)

    Divya Bhagirath

    Full Text Available Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent

  9. Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

    Science.gov (United States)

    Bhagirath, Divya; Zhao, Xiangshan; Mirza, Sameer; West, William W; Band, Hamid; Band, Vimla

    2016-01-01

    Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not

  10. Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

    Directory of Open Access Journals (Sweden)

    Nichole Goodsmith

    2015-02-01

    Full Text Available The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955 resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

  11. Hippocampal Mutant APP and Amyloid Beta Induced Cognitive Decline, Dendritic Spine Loss, Defective Autophagy, Mitophagy and Mitochondrial Abnormalities in a Mouse Model of Alzheimer's Disease.

    Science.gov (United States)

    Manczak, Maria; Kandimalla, Ramesh; Yin, Xiangling; Reddy, P Hemachandra

    2018-02-01

    The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (Aβ) in 12-month-old APP transgenic mice. Using rotarod and Morris Water Maze tests, immunoblotting & immunofluorescence, Golgi-cox staining and transmission electron microscopy, we assessed cognitive behavior, protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2, and quantified dendritic spines and mitochondrial number and length in 12-month-old APP mice that express swedish mutation. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial ATP. Morris Water Maze and rotarod tests revealed that hippocampal learning and memory and motor learning & coordination were impaired in APP mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 & TFAM), autophagy (ATG5 & LC3BI, LC3BII), Mitophagy (PINK1 & TERT), synaptic (synaptophysin & PSD95) and dendritic (MAP2) proteins were found in 12-month-old APP mice relative to age-matched non-transgenic WT mice. Golgi-cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in APP mice. These findings suggest that hippocampal accumulation of mutant APP and Aβ is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins & reduced dendritic spines and hippocampal based learning and memory impairments, and mitochondrial structural and functional changes in 12-month-old APP mice. © The Author 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Michelle A Favila

    2015-12-01

    Full Text Available Leishmania major infection induces robust interleukin-12 (IL12 production in human dendritic cells (hDC, ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG and other phosphoglycan-containing molecules (PGs, making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS responsible for IL12 induction.Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-, or generally deficient for all PGs, (FV1 lpg2-. Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB and Interferon Regulatory Factor (IRF mediated transcription.These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

  13. A comparative proteome analysis of Escherichia coli DeltarelA mutant cells

    Directory of Open Access Journals (Sweden)

    Sónia Carneiro

    2016-10-01

    Full Text Available The bacterial RelA-dependent stringent response exerts a strong influence over a wide variety of processes. In this work, the impact of the relA gene mutation in E. coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labelling and high resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ∆relA mutant strains were performed at two dilution rates (0.1 and 0.2 h-1 to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression.

  14. Target-directed discovery and production of pharmaceuticals in transgenic mutant plant cells

    Science.gov (United States)

    Brown, DP; Rogers, DT; Gunjan, SK; Gerhardt, GA; Littleton, JM

    2016-01-01

    Plants are a source of complex bioactive compounds, with value as pharmaceuticals, or leads for synthetic modification. Many of these secondary metabolites have evolved as defenses against competing organisms. and their pharmaceutical value is “accidental”, resulting from homology between target proteins in these competitors, and human molecular therapeutic targets. Here we show that it is possible to use mutation and selection of plant cells to re-direct their “evolution” toward metabolites that interact with the therapeutic target proteins themselves. This is achieved by expressing the human target protein in plant cells, and selecting mutants for survival based on the interaction of their metabolome with this target. This report describes the successful evolution of hairy root cultures of a Lobelia species toward increased biosynthesis of metabolites that inhibit the human dopamine transporter protein. Many of the resulting selected mutants are overproducing the active metabolite found in the wild-type plant, but others overproduce active metabolites that are not readily detectable in non-mutants. This technology can access the whole genomic capability of a plant species to biosynthesize metabolites with a specific target. It has potential value as a novel platform for plant drug discovery and production, or as a means of optimizing the therapeutic value of medicinal plant extracts. PMID:27637316

  15. Determining the role of inflammation in the selection of JAK2 mutant cells in myeloproliferative neoplasms.

    Science.gov (United States)

    Zhang, Jie; Fleischman, Angela G; Wodarz, Dominik; Komarova, Natalia L

    2017-07-21

    Myeloproliferative neoplasm (MPN) is a hematologic malignancy characterized by the clonal outgrowth of hematopoietic cells with a somatically acquired mutation most commonly in JAK2 (JAK2 V617F ). This mutation endows upon myeloid progenitors cytokine independent growth and consequently leads to excessive production of myeloid lineage cells. It has been previously suggested that inflammation may play a role in the clonal evolution of JAK2 V617F mutants. In particular, it is possible that one or more cellular kinetic parameters of hematopoietic stem cells (HSCs) are affected by inflammation, such as division or death rates of cells, and the probability of HSC differentiation. This suggests a mechanism that can steer the outcome of the cellular competition in favor of the mutants, initiating the disease. In this paper we create a number of mathematical evolutionary models, from very abstract to more concrete, that describe cellular competition in the context of inflammation. It is possible to build a model axiomatically, where only very general assumptions are imposed on the modeling components and no arbitrary (and generally unknown) functional forms are used, and still generate a set of testable predictions. In particular, we show that, if HSC death is negligible, the evolutionary advantage of mutant cells can only be conferred by an increase in differentiation probability of HSCs in the presence of inflammation, and if death plays a significant role in the dynamics, an additional mechanism may be an increase of HSC's division-to-death ratio in the presence of inflammation. Further, we show that in the presence of inflammation, the wild type cell population is predicted to shrink under inflammation (even in the absence of mutants). Finally, it turns out that if only the differentiation probability is affected by the inflammation, then the resulting steady state population of wild type cells will contain a relatively smaller percentage of HSCs under inflammation. If

  16. SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae.

    OpenAIRE

    Neigeborn, L; Celenza, J L; Carlson, M

    1987-01-01

    Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression. All ssn20 alleles cause recessive, temperature-sensitive lethality. In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell. Genetic mapping of SSN20 to a locus on chromosome VII 1...

  17. Perturbations of microRNA function in mouse dicer mutants produce retinal defects and lead to aberrant axon pathfinding at the optic chiasm.

    Directory of Open Access Journals (Sweden)

    Rita Pinter

    2010-04-01

    Full Text Available During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.

  18. Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro

    International Nuclear Information System (INIS)

    Ronen, A.; Gingerich, J.D.; Duncan, A.M.V.; Heddle, J.A.

    1984-01-01

    Diptheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. Resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [ 3 H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [ 3 H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium. 23 references, 6 figures

  19. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

    Directory of Open Access Journals (Sweden)

    Diana J M van den Wollenberg

    Full Text Available Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

  20. Distinct CCR2(+) Gr1(+) cells control growth of the Yersinia pestis ΔyopM mutant in liver and spleen during systemic plague.

    Science.gov (United States)

    Ye, Zhan; Uittenbogaard, Annette M; Cohen, Donald A; Kaplan, Alan M; Ambati, Jayakrishna; Straley, Susan C

    2011-02-01

    We are using a systemic plague model to identify the cells and pathways that are undermined by the virulence protein YopM of the plague bacterium Yersinia pestis. In this study, we pursued previous findings that Gr1(+) cells are required to selectively limit growth of ΔyopM Y. pestis and that CD11b(+) cells other than polymorphonuclear leukocytes (PMNs) are selectively lost in spleens infected with parent Y. pestis. When PMNs were ablated from mice, ΔyopM Y. pestis grew as well as the parent strain in liver but not in spleen, showing that these cells are critical for controlling growth of the mutant in liver but not spleen. In mice lacking expression of the chemokine receptor CCR2, wild-type growth was restored to ΔyopM Y. pestis in both organs. In spleen, the Gr1(+) cells differentially recruited by parent and ΔyopM Y. pestis infections were CCR2(+) Gr1(+) CD11b(+) CD11c(Lo-Int) MAC3(+) iNOS(+) (inducible nitric oxide synthase-positive) inflammatory dendritic cells (iDCs), and their recruitment to spleen from blood was blocked when YopM was present in the infecting strain. Consistent with influx of iDCs being affected by YopM in spleen, the growth defect of the ΔyopM mutant was relieved by the parent Y. pestis strain in a coinfection assay in which the parent strain could affect the fate of the mutant in trans. In a mouse model of bubonic plague, CCR2 also was shown to be required for ΔyopM Y. pestis to show wild-type growth in skin. The data imply that YopM's pathogenic effect indirectly undermines signaling through CCR2. We propose a model for how YopM exerts its different effects in liver and spleen.

  1. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

    Science.gov (United States)

    Yun, Jihye; Mullarky, Edouard; Lu, Changyuan; Bosch, Kaitlyn N; Kavalier, Adam; Rivera, Keith; Roper, Jatin; Chio, Iok In Christine; Giannopoulou, Eugenia G; Rago, Carlo; Muley, Ashlesha; Asara, John M; Paik, Jihye; Elemento, Olivier; Chen, Zhengming; Pappin, Darryl J; Dow, Lukas E; Papadopoulos, Nickolas; Gross, Steven S; Cantley, Lewis C

    2015-12-11

    More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations. Copyright © 2015, American Association for the Advancement of Science.

  2. Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

    Directory of Open Access Journals (Sweden)

    Jeffery Glen

    2008-05-01

    Full Text Available Abstract Background The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs. At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+ showed that (1 the total number of RGC axons projected by the retina and (2 the proportions that are sorted into the ipsilateral and

  3. Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior.

    Science.gov (United States)

    Giacometti, Romina; Souto, Guadalupe; Silberstein, Susana; Giasson, Luc; Cantore, María Leonor; Passeron, Susana

    2006-01-01

    We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

  4. The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis

    DEFF Research Database (Denmark)

    Post, David A.; Switzer, Robert L.; Hove-Jensen, Bjarne

    1996-01-01

    An Escherichia coli strain which is temperature-sensitive for growth due to a mutation (prs-2) causing a defective phosphoribosyl diphosphate (PRPP) synthase has been characterized. The temperature-sensitive mutation was mapped to a 276 bp HindIII-BssHII DNA fragment located within the open reading...... temperature shift to 42 degrees C. The other mutation was a C -> T transition located 39 bp upstream of the G -> A mutation, i.e. outside the coding sequence and close to the Shine-Dalgarno sequence. Cells harbouring only the C -> T mutation in a plasmid contained approximately three times as much PRPP...

  5. DNA sequence analysis of HPRT- mutants induced in human lymphoblastoid cells adapted to ionizing radiation

    International Nuclear Information System (INIS)

    Rigaud, O.; Laquerbe, A.; Moustacchi, E.

    1995-01-01

    Radioadaptation to the mutagenic effect of ionizing radiation by pre-exposure of human cells to a low dose has been shown to decrease the proportion of HPRT - mutants of the deletion type. To determine whether point mutations would be affected by the adaptive treatment, the molecular nature of mutations induced after exposure to low, high or low plus high doses was established. DNA sequencing of 38 point mutants which still expressed mRNA was performed using reverse transcription/polymerase chain reaction amplification. Under all conditions, base substitutions were the most common mutational event (range 72-80%), the remainder being frameshift and small deletions. The types and proportions of base changes did not appear to be differentially modified. A clustering of mutations was observed in exon 8, independently of the radiation protocol. About 40% of the mutants exhibited incorrect splicing of mRNA. The lack of striking modifications between the different molecular spectra of point mutations suggests that low-dose pre-exposure does not affect the production and/or the processing of lesions leading to point mutations. Thus the highly significant effect triggered by the low dose is the preferential reduction of deletion-type mutations. In view of the actual small data set, definitive conclusions will be drawn only when our observations are confirmed or can be generalized to human endogenous loci other than the HPRT locus, which is particularly prone to the recovery of deletion-type mutations. 37 refs., 1 fig., 5 tabs

  6. Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

    Directory of Open Access Journals (Sweden)

    Gaëlle Gendronneau

    Full Text Available The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo.We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury.The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis.These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

  7. Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

    Science.gov (United States)

    Gendronneau, Gaëlle; Sanii, Sadaf; Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

  8. Pu-erh Tea Inhibits Tumor Cell Growth by Down-Regulating Mutant p53

    Science.gov (United States)

    Zhao, Lanjun; Jia, Shuting; Tang, Wenru; Sheng, Jun; Luo, Ying

    2011-01-01

    Pu-erh tea is a kind of fermented tea with the incorporation of microorganisms’ metabolites. Unlike green tea, the chemical characteristics and bioactivities of Pu-erh tea are still not well understood. Using water extracts of Pu-erh tea, we analyzed the tumor cell growth inhibition activities on several genetically engineered mouse tumor cell lines. We found that at the concentration that did not affect wild type mouse embryo fibroblasts (MEFs) growth, Pu-erh tea extracts could inhibit tumor cell growth by down-regulated S phase and cause G1 or G2 arrest. Further study showed that Pu-erh tea extracts down-regulated the expression of mutant p53 in tumor cells at the protein level as well as mRNA level. The same concentration of Pu-erh tea solution did not cause p53 stabilization or activation of its downstream pathways in wild type cells. We also found that Pu-erh tea treatment could slightly down-regulate both HSP70 and HSP90 protein levels in tumor cells. These data revealed the action of Pu-erh tea on tumor cells and provided the possible mechanism for Pu-erh tea action, which explained its selectivity in inhibiting tumor cells without affecting wild type cells. Our data sheds light on the application of Pu-erh tea as an anti-tumor agent with low side effects. PMID:22174618

  9. Progressive Muscle Cell Delivery as a Solution for Volumetric Muscle Defect Repair

    OpenAIRE

    Ji Hyun Kim; In Kap Ko; Anthony Atala; James J. Yoo

    2016-01-01

    Reconstructing functional volumetric tissue in vivo following implantation remains a critical challenge facing cell-based approaches. Several pre-vascularization approaches have been developed to increase cell viability following implantation. Structural and functional restoration was achieved in a preclinical rodent tissue defect; however, the approach used in this model fails to repair larger (>mm) defects as observed in a clinical setting. We propose an effective cell delivery system utili...

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

    DEFF Research Database (Denmark)

    Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna

    2017-01-01

    -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...... 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....

  11. Mutation induction in γ-irradiated primary human bronchial epithelial cells and molecular analysis of the HPRT- mutants

    International Nuclear Information System (INIS)

    Suzuki, Keiji; Hei, Tom K.

    1996-01-01

    We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in γ-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137 Cs γ-rays, indicates that the D 0 , D q , and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT - mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/20), respectively, while 30.0% (6/20) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by γ-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 γ-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that γ-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides a useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells

  12. Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.

    Science.gov (United States)

    Fujiwara, Yuya; Hiraoka, Yuri; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2015-06-30

    To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

  13. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  14. ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

    International Nuclear Information System (INIS)

    Okumura, Yasuko; Sugiyama, Noriyuki; Tanimura, Susumu; Nishida, Masashi; Hamaoka, Kenji; Kohno, Michiaki; Yokoyama, Takahiko

    2009-01-01

    Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants

  15. Defect generation/passivation by low energy hydrogen implant for silicon solar cells

    International Nuclear Information System (INIS)

    Sopori, B.L.; Zhou, T.Q.; Rozgonyi, G.A.

    1990-01-01

    Low energy ion implant is shown to produce defects in silicon. These defects include surface damage, hydrogen agglomeration, formation of platelets with (111) habit plane and decoration of dislocations. Hydrogen also produces an inversion type of surface on boron doped silicon. These effects indicate that a preferred approach for passivation is to incorporate hydrogen from the back side of the cell. A backside H + implant technique is described. The results show that degree of passivation differs for various devices. A comparison of the defect structures of hydrogenated devices indicates that the structure and the distribution of defects in the bulk of the material plays a significant role in determining the degree of passivation

  16. Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23.

    Science.gov (United States)

    Smeets, Cleo J L M; Jezierska, Justyna; Watanabe, Hiroyuki; Duarri, Anna; Fokkens, Michiel R; Meijer, Michel; Zhou, Qin; Yakovleva, Tania; Boddeke, Erik; den Dunnen, Wilfred; van Deursen, Jan; Bakalkin, Georgy; Kampinga, Harm H; van de Sluis, Bart; Verbeek, Dineke S

    2015-09-01

    Spinocerebellar ataxia type 23 is caused by mutations in PDYN, which encodes the opioid neuropeptide precursor protein, prodynorphin. Prodynorphin is processed into the opioid peptides, α-neoendorphin, and dynorphins A and B, that normally exhibit opioid-receptor mediated actions in pain signalling and addiction. Dynorphin A is likely a mutational hotspot for spinocerebellar ataxia type 23 mutations, and in vitro data suggested that dynorphin A mutations lead to persistently elevated mutant peptide levels that are cytotoxic and may thus play a crucial role in the pathogenesis of spinocerebellar ataxia type 23. To further test this and study spinocerebellar ataxia type 23 in more detail, we generated a mouse carrying the spinocerebellar ataxia type 23 mutation R212W in PDYN. Analysis of peptide levels using a radioimmunoassay shows that these PDYN(R212W) mice display markedly elevated levels of mutant dynorphin A, which are associated with climber fibre retraction and Purkinje cell loss, visualized with immunohistochemical stainings. The PDYN(R212W) mice reproduced many of the clinical features of spinocerebellar ataxia type 23, with gait deficits starting at 3 months of age revealed by footprint pattern analysis, and progressive loss of motor coordination and balance at the age of 12 months demonstrated by declining performances on the accelerating Rotarod. The pathologically elevated mutant dynorphin A levels in the cerebellum coincided with transcriptionally dysregulated ionotropic and metabotropic glutamate receptors and glutamate transporters, and altered neuronal excitability. In conclusion, the PDYN(R212W) mouse is the first animal model of spinocerebellar ataxia type 23 and our work indicates that the elevated mutant dynorphin A peptide levels are likely responsible for the initiation and progression of the disease, affecting glutamatergic signalling, neuronal excitability, and motor performance. Our novel mouse model defines a critical role for opioid

  17. Evidence for mutagenic repair in V79 cell mutant with aphidicolin-resistant DNA polymerce-alpha

    International Nuclear Information System (INIS)

    Liu, P.K.; Chang, C.C.; Trosko, J.E.

    1984-01-01

    An aphidicolin-resistant (aphr) mutant of Chinese hamster V79 cells, aphr -4-2, is shown to be slow-growing, sensitive to ultraviolet (UV) irradiation, hypermutable for spontaneous and UV-induced mutations, and known to contain an aphr mutant DNA polymerase-alpha, with a 10-fold reduction in the Km for dCTP but not for dATP. We show here that the mutant had a normal repair replication measured by unscheduled DNA synthesis assay. The mutant was specifically sensitive and hypermutable to UV and N-methyl-N'-nitro-N-nitrosoguanidine, but it had normal sensitivity to ionizing radiation and dimethyl sulfate. Unlike the V79 (wt) cells, the mutant exhibited further enhancement in the already elevated mutability following UV and conditioned medium treatment. The mutant characteristic is explained by the presence of an error-prone long-patch excision repair synthesis. The association in the mutant properties--an aphr DNA polymerase-alpha, UV sensitivity, and hypermutability to UV-induced mutation--provides the genetic evidence that DNA polymerase-alpha is likely to be involved in UV-induced DNA repair synthesis

  18. Legionella dumoffii Tex-KL Mutated in an Operon Homologous to traC-traD is Defective in Epithelial Cell Invasion.

    Science.gov (United States)

    Qin, Tian; Ken-Ichiro, Iida; Ren, Hong Yu; Zhou, Hai Jian; Yoshida, Shin-Ichi

    2016-06-01

    To understand the mechanism of invasion by Legionella dumoffii. The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. The transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain.. Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  19. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

    Directory of Open Access Journals (Sweden)

    Nicolas Lecland

    2013-01-01

    In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

  20. Satellite spot defect reduction on 193-nm contact hole lithography using photo cell monitor methodology

    Science.gov (United States)

    Boulenger, Caroline; Caze, Jean-Luc; Mihet, Mihaela

    2006-03-01

    The goal of overall process and yield improvement requires a litho defect management and reduction strategy, which includes several layers of tactical methods. Defects may be identified through a number of schemes, including After-Develop Inspection (ADI), which was the primary tool in this study in our 0,13μ fab. Defects on 193nm contact hole lithography were identified using a KLA-Tencor 2351 High Resolution Imaging Patterned Wafer Inspection System, coupled with in-line Automatic Defect Classification (iADC). The optimized inspection was used at the core of the Photo Cell Monitor (PCM) to isolate critical defect types. PCM uses the fab's standard production resist coat, exposure, develop, and rinse process, with the focus and exposure optimized for resist on silicon test wafers. Through Pareto analysis of 193nm defects, one defect type, called satellite spot, was targeted for immediate improvement and monitoring. This paper describes the work done in improving the litho defectivity. The work includes optimization of inspection and classification parameters and the Design of Experiments (DOE) to identify the source (including the interaction between the resist and developer) and contributing factors. Several process modifications were identified which resulted in lowered defectivity up to complete suppression of satellite spot defects, although at higher process complexity and cost. This work was also done in conjunction with resist suppliers, which used the same inspection to confirm the problem at their facilities. The work with the suppliers continues with the goal of identifying a less expensive permanent solution.

  1. Induction, selection and isolation of auxin heterotrophic and auxin-resistant mutants from cultured crown gall cells irradiated with gamma rays

    International Nuclear Information System (INIS)

    Atsumi, Shigeaki

    1980-01-01

    Cultured crown gall cells were irradiated with gamma rays to induce mutation in indoleacetic acid biosynthesis. The irradiated cells were plated on a selection medium which contained auxin. Mutant cells adapted to selection media were characterized as auxin-heterotrophic and auxin-resistant cell lines. The auxin-heterotrophic mutants contained little auxin, whereas the auxin-resistant and -autotrophic mutants contained large amounts of auxin even when cultured with 0.3 ppm of 2,4-dichlorophenoxyacetic acid. Each mutant cell line contained as much octopine as its parental cells. The mutation rate was calculated as in the order of 10 -8 . (author)

  2. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  3. Glycerol restores the p53 function in human lingual cancer cells bearing mutant p53

    International Nuclear Information System (INIS)

    Ota, Ichiro; Yane, Katsunari; Yuki, Kazue; Kanata, Hirokazu; Hosoi, Hiroshi; Miyahara, Hiroshi

    2001-01-01

    Mutations in p53, tumor suppressor gene, have recently been shown to have an impact on the clinical course of several human tumors, including head and neck cancers. The genetic status of the p53 gene has been focused on as the most important candidate among various cancer-related genes for prognosis-predictive assays of cancer therapy. We examined the restoration of radiation- or cisplatin (CDDP)-induced p53-dependent apoptosis in human lingual cancer cells. The results suggest that glycerol is effective in inducing a conformational change of p53 and restoring normal function of mutant p53, leading to enhanced radiosensitivity or chemosensitivity through the induction of apoptosis. We have also represented the same results in vivo as in vitro. Thus, this novel tool for enhancement of radiosensitivity or chemosensitivity in cancer cells bearing m p53 may be applicable for p53-targeted cancer therapy. (author)

  4. The spontaneous ataxic mouse mutant tippy is characterized by a novel Purkinje cell morphogenesis and degeneration phenotype

    Science.gov (United States)

    Shih, Evelyn K.; Sekerková, Gabriella; Ohtsuki, Gen; Aldinger, Kimberly A.; Chizhikov, Victor V.; Hansel, Christian; Mugnaini, Enrico; Millen, Kathleen J.

    2015-01-01

    This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function. PMID:25626522

  5. Isolation and partial characterisation of a mammalian cell mutant hypersensitive to topoisomerase II inhibitors and X-rays

    International Nuclear Information System (INIS)

    Davies, S.M.; Davies, S.L.; Hickson, I.D.; Hall, A.G.

    1990-01-01

    The authors have isolated, following one-step mutagenesis, a Chinese hamster ovary cell mutant hypersensitive to the intercalating agent, adriamycin. This agent exerts at least part of its cytotoxic action via inhibition of the nuclear enzyme, topoisomerase II. The mutant, designated ADR-3, showed hypersensitivity to all classes of topoisomerase II inhibitors, inlcuding actinomycin D, amsacrine (m-AMSA), etoposide (VP16) and mitoxantrone. ADR-3 cells also showed cross-sensitivity to ionizing radiation, but not no UV light. Topoisomerase II activity was elevated to a small but significant degree in ADR-3 cells, and this was reflected in a 1.5-fold higher level of topoisomerase II protein in ADR-3 than in CHO-K1 cells, as judged by Western blotting. ADR-3 cells were hypersensitive to cumene hydroperoxide but cross-resistant to hydrogen peroxide, suggesting possible abnormality in the detoxification of peroxides by glutathione peroxidase or catalase. Glutathione peroxidase activity against hydroperoxide was elevated to a small but significant extent in mutant cells. Catalase levels were not significantly different in ADR-3 and CHO-K1 cells. ADR-3 cells were recessive in hybrids with parental CHO-K1 cells with respect to sensitivity to topoisomerase II inhibitors and X-rays, and represent a different genetic complementation group from the previously reported adriamycin-sensitive mutant, ADR-1. (author). 34 refs.; 5 figs.; 3 tabs

  6. Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

    OpenAIRE

    Wu, C-H; Chen, Y-F; Wang, J-Y; Hsieh, M-C; Yeh, C-S; Lian, S-T; Shin, S-J; Lin, S-R

    2002-01-01

    The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adre...

  7. A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Min Hwa Shin

    2016-02-01

    Full Text Available The inactivation of p53 creates a major challenge for inducing apoptosis in cancer cells. An attractive strategy is to identify and subsequently target the survival signals in p53 defective cancer cells. Here we uncover a RUNX2-mediated survival signal in p53 defective cancer cells. The inhibition of this signal induces apoptosis in cancer cells but not non-transformed cells. Using the CRISPR technology, we demonstrate that p53 loss enhances the apoptosis caused by RUNX2 knockdown. Mechanistically, RUNX2 provides the survival signal partially through inducing MYC transcription. Cancer cells have high levels of activating histone marks on the MYC locus and concomitant high MYC expression. RUNX2 knockdown decreases the levels of these histone modifications and the recruitment of the Menin/MLL1 (mixed lineage leukemia 1 complex to the MYC locus. Two inhibitors of the Menin/MLL1 complex induce apoptosis in p53 defective cancer cells. Together, we identify a RUNX2-mediated epigenetic mechanism of the survival of p53 defective cancer cells and provide a proof-of-principle that the inhibition of this epigenetic axis is a promising strategy to kill p53 defective cancer cells.

  8. A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells.

    Science.gov (United States)

    Shin, Min Hwa; He, Yunlong; Marrogi, Eryney; Piperdi, Sajida; Ren, Ling; Khanna, Chand; Gorlick, Richard; Liu, Chengyu; Huang, Jing

    2016-02-01

    The inactivation of p53 creates a major challenge for inducing apoptosis in cancer cells. An attractive strategy is to identify and subsequently target the survival signals in p53 defective cancer cells. Here we uncover a RUNX2-mediated survival signal in p53 defective cancer cells. The inhibition of this signal induces apoptosis in cancer cells but not non-transformed cells. Using the CRISPR technology, we demonstrate that p53 loss enhances the apoptosis caused by RUNX2 knockdown. Mechanistically, RUNX2 provides the survival signal partially through inducing MYC transcription. Cancer cells have high levels of activating histone marks on the MYC locus and concomitant high MYC expression. RUNX2 knockdown decreases the levels of these histone modifications and the recruitment of the Menin/MLL1 (mixed lineage leukemia 1) complex to the MYC locus. Two inhibitors of the Menin/MLL1 complex induce apoptosis in p53 defective cancer cells. Together, we identify a RUNX2-mediated epigenetic mechanism of the survival of p53 defective cancer cells and provide a proof-of-principle that the inhibition of this epigenetic axis is a promising strategy to kill p53 defective cancer cells.

  9. Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity▿†

    Science.gov (United States)

    Vanderlinde, Elizabeth M.; Magnus, Samantha A.; Tambalo, Dinah D.; Koval, Susan F.; Yost, Christopher K.

    2011-01-01

    The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA+ ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella. PMID:21357485

  10. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  11. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

    Science.gov (United States)

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

  12. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

    Science.gov (United States)

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

  13. Factors effecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation

    International Nuclear Information System (INIS)

    Thacker, J.; Stephens, M.A.; Stretch, A.

    1976-01-01

    In the Chines hamster cell line V79-4, the frequencies of cells selected for their resistance to purine analogues do not always reflect the true frequencies of resistant mutants. The frequency of cells resistant to 8-azaguanine varied widely, especially when different sources of serum were used in the selective medium. Even with the more efficient analogue, 6-thioguanine, small colonies arose in the selective medium at a frequency which was strongly dependent upon analogue concentration and viable cell seeding density. These colonies were shown to have a phenotype which was indistinguishable from wild type. Hence with irradiated cells, where the viability of the cell population is reduced to an extent varying with the dose and the interval allowed for mutant expression, the counting of all colonies arising in selective medium can lead to spuriously variable, and sometimes very high, 'mutation frequencies'. Although the frequency of wild type colonies selected in thioguanine was diminished by the use of high concentrations of the analogue, a loss of induced mutants also occurred at these concentrations. Further, the V79-4 line contained two distinct types of mutant with different levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity, and only one of these types (HGPRT-negative mutants) increased in frequency with radiation dose. These results can account for many of the anomalies encountered in previous studies with purine analogues as selective agents, and show that some care has to be taken to characterize the mutants selected by resistance to purine analogues before meaningful dose-response relationships can be established

  14. Mutant p53 transfection of astrocytic cells results in altered cell cycle control, radiation sensitivity, and tumorigenicity

    International Nuclear Information System (INIS)

    Kanady, Kirk E.; Mei Su; Proulx, Gary; Malkin, David M.; Pardo, Francisco S.

    1995-01-01

    Introduction: Alterations in the p53 tumor suppressor gene are one of the most frequent genetic alterations in malignant gliomas. An understanding of the molecular genetic events leading to glial tumor progression would aid in designing therapeutic vectors for controlling these challenging tumor types. We investigated whether mutations in coding exons of the p53 gene result in functional changes altering cell cycle 'checkpoint' control and the intrinsic radiation sensitivity of glial cells. Methods: An astrocytic cell line was derived from a low grade astrocytoma and characterized to be of human karyotype and GFAP positivity. Additionally, the cellular population has never formed tumors in immune-deficient mice. At early passage ( 2 as parameters. Cell kinetic analyses after 2, 5, and 10 Gy of ionizing radiation were conducted using propidium iodide FACS analyses. Results: Overall levels of p53 expression were increased 5-10 fold in the transfected cellular populations. Astrocytic cellular populations transfected with mutant p53 revealed a statistically significant increase in levels of resistance to ionizing radiation in vitro (2-tailed test, SF2, MID). Astrocytic cellular populations transfected with mutant p53, unlike the parental cells, were tumorigenic in SCID mice. Cell kinetic analyses indicated that the untransfected cell line demonstrated dose dependent G1 and G2 arrests. Following transfection, however, the resultant cellular population demonstrated a predominant G2 arrest. Conclusions: Astrocytic cellular populations derived from low grade astrocytomas, are relatively radiation sensitive, non-tumorigenic, and have intact cell cycle ''checkpoints.'' Cellular populations resulting upon transfection of parental cells with a dominant negative p53 mutation, are relatively radiation resistant, when compared to both parental and mock-transfected cells. Transfected cells demonstrate abnormalities of cell cycle control at the G1/S checkpoint, increases in levels

  15. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Bin Huang

    Full Text Available Huntingtin (Htt is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q and mutant (46Q and 128Q Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.

  16. L-Ascorbic acid can abrogate SVCT-2-dependent cetuximab resistance mediated by mutant KRAS in human colon cancer cells.

    Science.gov (United States)

    Jung, Soo-A; Lee, Dae-Hee; Moon, Jai-Hee; Hong, Seung-Woo; Shin, Jae-Sik; Hwang, Ih Yeon; Shin, Yu Jin; Kim, Jeong Hee; Gong, Eun-Yeung; Kim, Seung-Mi; Lee, Eun Young; Lee, Seul; Kim, Jeong Eun; Kim, Kyu-Pyo; Hong, Yong Sang; Lee, Jung Shin; Jin, Dong-Hoon; Kim, TaeWon; Lee, Wang Jae

    2016-06-01

    Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor, which is an effective clinical therapy for patients with wild-type KRAS. Numerous combinatorial therapies have been tested to overcome the resistance to cetuximab. However, no combinations have been found that can be used as effective therapeutic strategies. In this study, we demonstrate that L-ascorbic acid partners with cetuximab to induce killing effects, which are influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human colon cancer cells with a mutant KRAS. L-Ascorbic acid treatment of human colon cancer cells that express a mutant KRAS differentially and synergistically induced cell death with cetuximab in a SVCT-2-dependent manner. The ectopic expression of SVCT-2 induced sensitivity to L-ascorbic acid treatment in human colon cancer cells that do not express SVCT-2, whereas the knockdown of endogenous SVCT-2 induced resistance to L-ascorbic acid treatment in SVCT-2-positive cells. Moreover, tumor regression via the administration of L-ascorbic acid and cetuximab in mice bearing tumor cell xenografts corresponded to SVCT-2 protein levels. Interestingly, cell death induced by the combination of L-ascorbic acid and cetuximab resulted in both apoptotic and necrotic cell death. These cell death mechanisms were related to a disruption of the ERK pathway and were represented by the impaired activation of RAFs and the activation of the ASK-1-p38 pathway. Taken together, these results suggest that resistance to cetuximab in human colon cancer patients with a mutant KRAS can be bypassed by L-ascorbic acid in an SVCT-2-dependent manner. Furthermore, SVCT-2 in mutant KRAS colon cancer may act as a potent marker for potentiating L-ascorbic acid co-treatment with cetuximab. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Ethanol exposure disrupts extraembryonic microtubule cytoskeleton and embryonic blastomere cell adhesion, producing epiboly and gastrulation defects

    Directory of Open Access Journals (Sweden)

    Swapnalee Sarmah

    2013-08-01

    Fetal alcohol spectrum disorder (FASD occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell, prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented that characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression was not affected by ethanol exposure, but E-cadherin distribution, which controls epiboly and gastrulation, was changed. E-cadherin was redistributed into cytoplasmic aggregates in blastomeres and dramatically redistributed in the extraembryonic yolk cell. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a, which controls epiboly, was significantly reduced in ethanol exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression.

  18. Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion.

    Science.gov (United States)

    Hückelhoven, R; Fodor, J; Trujillo, M; Kogel, K H

    2000-12-01

    The pathogenesis-related accumulation of superoxide radical anions (O2*-) and hydrogen peroxide (H2O2) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes "required for Mla12-specified disease resistance" (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H2O2 accumulation was detected in dying cells, while O2*- emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H2O2 and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase.

  19. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    Science.gov (United States)

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  20. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Hee Jin Shim

    Full Text Available Ionizing radiation (IR treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

  1. Polymorphic human (CTAT)n microsatellite provides a conserved linkage marker for mouse mutants causing cleft palate, vestibular defects, obesity and ataxia

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, A.J.; Burgess, D.L.; Kohrman, D. [Univ. of MIchigan, Ann Arbor, MI (United States)] [and others

    1994-09-01

    The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA from two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.

  2. Expression of phage-transposons of Pseudomonas aeruginosa in cells of Pseudomonas putida PpGl. II. Zygotic induction, an essential condition for the emergence of defective lysogens

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunova, S.A.; Akhverdyan, V.Z.; Krylov, V.N.

    1986-06-01

    The presence of a large number of clones, which have lost the ability to produce phase, is a feature of PpGl exconjugants, which have acquired markers of the hybrid plasmid containing the genome of the PAO1 phage transposon. Analysis of these clones indicates that they contain plasmids with different defects in the phage genome (point mutations and different deletion lengths which may have an effect on both the phage genome and also plasmid RP4). Mutations of a particular region are selected, the region of early prophage genes. The process of zygotic induction is an essential condition for the arisal of mutants (including deletion mutants). The results of the experiment, which was based on the Luria-Delbruik test, showed that a significant proportion of the mutations, including deletion mutations, arise in the recipient cells PpGl.

  3. Screening for yeast mutants defective in recipient ability for transkingdom conjugation with Escherichia coli revealed importance of vacuolar ATPase activity in the horizontal DNA transfer phenomenon.

    Science.gov (United States)

    Mizuta, Mami; Satoh, Emi; Katoh, Chika; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2012-05-20

    Proteobacterium Escherichia coli strains harboring wide-transfer-range conjugative plasmids are able to transfer these plasmids to several yeast species. Whole plasmid DNA is mobilizable in the transkingdom conjugation phenomenon. Owing to the availability of various conjugative plasmids in bacteria, the horizontal DNA transfer has potential to occur between various bacteria and eukaryotes. In order to know host factor genes involved in such conjugation, we systematically tested the conjugability of strains among a yeast library comprising single-gene-knockout mutants in this study. This genome-wide screen identified 26 host chromosomal genes whose absence reduced the efficiency of the transkingdom conjugation. Among the 26 genes, 20 are responsible for vacuolar ATPase activity, while 5 genes (SHP1, CSG2, CCR4, NOT5, and HOF1) seem to play a role in maintaining the cell surface. Lack of either ZUO1 gene or SSZ1 gene, each of which encodes a component of the ribosome-associated cytoplasmic molecular chaperone, also strongly affected transkingdom conjugation. Copyright © 2011 Elsevier GmbH. All rights reserved.

  4. Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

    Science.gov (United States)

    Hauptmann, Peter; Lehle, Ludwig

    2008-07-04

    N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

  5. Spectroscopy of Technological Defects in Si Solar Cells by Analysis of Temperature Dependent Generation Currents

    Directory of Open Access Journals (Sweden)

    Jevgenij PAVLOV

    2014-09-01

    Full Text Available The efficiency of solar cells considerably depends on the technological defects introduced by the formation of junctions, passivation layers and electrodes. Identification of these defects present in the high conductivity base layer of modern solar cells by usage of the standard techniques, such as capacitance deep level spectroscopy, is restricted by extremely small size of samples with inherent enhanced leakage current on sample boundaries. Therefore, it is important to develop the alternative methods for the defect spectroscopy in the high conductivity junction structures, to directly control a relative low concentration of the technological defects. In this work, the spectroscopy of deep traps has been performed by combining the temperature scans of the thermal generation currents extracted from barrier capacitance charging transients and capacitance deep level transient spectroscopy techniques. The dominant carrier traps ascribed to the Cu and Ni impurities were revealed. DOI: http://dx.doi.org/10.5755/j01.ms.20.3.5194

  6. Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

    Directory of Open Access Journals (Sweden)

    Yannick Romero

    Full Text Available BACKGROUND: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs and endogenous small interfering RNAs (endo-siRNAs, but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice. PRINCIPAL FINDINGS: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

  7. Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

    1995-01-01

    This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

  8. Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin.

    Directory of Open Access Journals (Sweden)

    Yun Xia

    Full Text Available α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR. MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent.

  9. Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

    Science.gov (United States)

    Nie, Xuguang; Deng, Chu-xia; Wang, Qin; Jiao, Kai

    2008-01-01

    TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development. PMID:18334251

  10. Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Keisuke Tabara

    Full Text Available Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs. However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2 or BIBW2992 (pan-TKI of EGFR family proteins. Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

  11. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

    Science.gov (United States)

    Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

    2016-01-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  12. Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9.

    Science.gov (United States)

    Kumar, Vijay; Kumar, Virender; Thakur, Neerja; Bhalla, Tek Chand

    2015-07-01

    Mutants of Gordonia terrae were generated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50 mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1 M potassium phosphate buffer (pH 8.0) at 40 °C. Total six feedings each at an interval of 45 min resulted in accumulation of 360 mM (21.6 g) of p-HBA with a purity of 99%. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8 g h(-1) g DCW (-1) and 43.2 g L(-1), respectively, from 0.78 g h(-1) g DCW (-1) and 28.8 g L(-1) using resting cells of wild strain. K m and V max of purified nitrilase from mutant E9 were 55 U mg(-1) and 1.8 mM for p-HBN with a higher turnover number of 36 s(-1) × 10(-3).

  13. Calcium homeostasis in photoreceptor cells of Drosophila mutants inaC and trp studied with the pupil mechanism

    NARCIS (Netherlands)

    Hofstee, CA; Stavenga, DG

    1996-01-01

    The light-driven pupil mechanism, consisting of an assembly of mobile pigment granules inside the photoreceptor cells, has been investigated by in vivo reflection microspectrophotometry in wild type (WT) Drosophila and in the photoreceptor mutants inaC and trp. The pupillary response of a

  14. Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species

    NARCIS (Netherlands)

    Gechev, Tsanko S.; Ferwerda, MargFiet A.; Mehterov, Nikolay; Laloi, Christophe; Qureshi, Muhammad K.; Hille, Jacques

    2008-01-01

    The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased Susceptibility to AAL-toxin due to the knockout of a gene involved in

  15. Multicopy Fzf1 (Sul1) Suppresses the Sulfite Sensitivity but Not the Glucose Derepression or Aberrant Cell Morphology of a Grr1 Mutant of Saccharomyces Cerevisiae

    OpenAIRE

    Avram, D.; Bakalinsky, A. T.

    1996-01-01

    An ssu2 mutation in Sacccharomyces cerevisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants...

  16. Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261.

    Directory of Open Access Journals (Sweden)

    Taketo Yoshida

    Full Text Available The effects of spermidine analogues [norspermidine (NSPD, 33, spermidine (SPD, 34, homospermidine (HSPD, 44 and aminopropylcadaverine (APCAD, 35] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD. The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

  17. Rosiglitazone activation of PPARγ-dependent signaling is neuroprotective in mutant huntingtin expressing cells.

    Science.gov (United States)

    Chiang, Ming-Chang; Cheng, Yi-Chuan; Nicol, Christopher J; Lin, Kuan-Hung; Yen, Chia-Hui; Chen, Shiang-Jiuun; Huang, Rong-Nan

    2015-11-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a crucial transcription factor for neuroprotection in several brain diseases. Using a mouse model of Huntington's Disease (HD), we recently showed that PPARγ not only played a major function in preventing HD, but also oral intake of a PPARγ agonist (thiazolidinedione, TZD) significantly reduced the formation of mutant Huntingtin (mHtt) aggregates in the brain (e.g., cortex and striatum). The molecular mechanisms by which PPARγ exerts its HD neuroprotective effects remain unresolved. We investigated whether the PPARγ agonist (rosiglitazone) mediates neuroprotection in the mHtt expressing neuroblastoma cell line (N2A). Here we show that rosiglitazone upregulated the endogenous expression of PPARγ, its downstream target genes (including PGC1α, NRF-1 and Tfam) and mitochondrial function in mHtt expressing N2A cells. Rosiglitazone treatment also significantly reduced mHtt aggregates that included ubiquitin (Ub) and heat shock factor 1 (HSF1), as assessed by a filter-retardation assay, and increased the levels of the functional ubiquitin-proteasome system (UPS), HSF1 and heat shock protein 27/70 (HSP27/70) in N2A cells. Moreover, rosiglitazone treatment normalized endoplasmic reticulum (ER) stress sensors Bip, CHOP and ASK1, and significantly increased N2A cell survival. Taken together, these findings unveil new insights into the mechanisms by which activation of PPARγ signaling protects against the HD-mediated neuronal impairment. Further, our data also support the concept that PPARγ may be a novel therapeutic target for treating HD. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis

    Directory of Open Access Journals (Sweden)

    Sally Martin

    2014-10-01

    Full Text Available Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.

  19. Multivariate analysis of T-cell functional defects and circulating serum factors in Hodgkin's disease.

    Science.gov (United States)

    Schulof, R S; Bockman, R S; Garofalo, J A; Cirrincione, C; Cunningham-Rundles, S; Fernandes, G; Day, N K; Pinsky, C M; Incefy, G S; Thaler, H T; Good, R A; Gupta, S

    1981-08-15

    A comprehensive immunologic and serologic analysis was performed on 31 untreated patients with Hodgkin's disease. Immune evaluations stressed T-cell functional activity and included traditional parameters (PHA responsiveness and delayed hypersensitivity skin reactivity), as well as newer functional assays (T-cell colony formation, chemotaxis, spontaneous and antibody-dependent cytotoxicity, and concanavalin A-induced suppressor cell activity (CISA). Serum factors included ferritin, prostaglandins, zinc, copper, immune complexes, and thymic hormone activity. Every patient exhibited at least one T-cell or serum abnormality. The greatest percentage of patients exhibited T-cell defects in chemotaxis (85%), colony formation (81%). and PHA reactivity (64%). Immune defects were more common with advanced disease but were not related to absolute T-cell or monocyte count, skin test anergy, or abnormalities of T mu/T gamma cell proportions. Linear relationships were identified among abnormalities in the three assays employing mononuclear cells (PHA, colony formation, CISA) which may have reflected the inhibitory influence of monocytes present in the mononuclear cell preparations. Low serum zinc correlated with marked impairment of T-cell chemotaxis. Elevated prostaglandins were associated with high PHA reactivity and with depressed colony formation. Our results indicate that many complex factors, including intrinsic T-cell defects, contribute to the impaired immunity associated with Hodgkin's disease.

  20. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6 causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Directory of Open Access Journals (Sweden)

    Takao Sasado

    Full Text Available Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68. CPSF6 is a component of the Cleavage Factor Im complex (CFIm which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  1. The absence of phosphorylated tyrosine hydroxylase expression in the purkinje cells of the ataxic mutant pogo mouse.

    Science.gov (United States)

    Lee, N S; Kim, C T; Han, S Y; Kawk, J H; Sawada, K; Fukui, Y; Jeong, Y G

    2006-06-01

    The pogo mouse is a new ataxic autosomal recessive mutant that arose in Korean wild mice (KJR/Mskist). Its ataxic phenotype includes difficulty in maintaining a normal posture and the inability to walk in a straight line. Several studies have reported that tyrosine hydroxylase (TH) is persistently ectopically expressed in particular subsets of Purkinje cells in a parasagittal banding pattern in several ataxic mutant mice, e.g. tottering alleles and pogo mice. In this present study, we examined the expression of an enzymatically active form of TH and phosphorylated TH at Ser(40) (phospho-TH) by using immunohistochemistry and double immunofluorescence in the cerebellum of pogo mice. TH immunostaining appeared in some Purkinje cells in pogo, but in only a few of Purkinje cells of their heterozygous littermate controls. In all groups of mice, no phospho-TH immunoreactive Purkinje cells were observed in the cerebellum, although subsets of TH immunoreactive Purkinje cells were found in adjacent sections. This study suggests that TH expression in the Purkinje cells of pogo abnormally increases without activation of this enzyme by phosphorylation. This may mean that TH in the Purkinje cells of these mutants does not catalyse the conversion of tyrosine to l-DOPA, and is not related to catecholamine synthesis.

  2. Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells.

    Science.gov (United States)

    Hung, Sandy S C; Van Bergen, Nicole J; Jackson, Stacey; Liang, Helena; Mackey, David A; Hernández, Damián; Lim, Shiang Y; Hewitt, Alex W; Trounce, Ian; Pébay, Alice; Wong, Raymond C B

    2016-05-01

    Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.

  3. Defective monocyte function in Legionnaires' disease complicating hairy cell leukaemia

    DEFF Research Database (Denmark)

    Nielsen, H; Bangsborg, Jette Marie; Rechnitzer, C

    1986-01-01

    We describe a case of Legionnaires' disease in a 64-year-old man, in which hairy cell leukaemia was diagnosed after the onset of the infection. Immunological studies revealed a complete suppression of blood monocyte chemotactic and oxidative burst activities. We suggest that in hairy cell leukaem...

  4. dMyc functions downstream of Yorkie to promote the supercompetitive behavior of hippo pathway mutant cells.

    Directory of Open Access Journals (Sweden)

    Marcello Ziosi

    2010-09-01

    Full Text Available Genetic analyses in Drosophila epithelia have suggested that the phenomenon of "cell competition" could participate in organ homeostasis. It has been speculated that competition between different cell populations within a growing organ might play a role as either tumor promoter or tumor suppressor, depending on the cellular context. The evolutionarily conserved Hippo (Hpo signaling pathway regulates organ size and prevents hyperplastic disease from flies to humans by restricting the activity of the transcriptional cofactor Yorkie (yki. Recent data indicate also that mutations in several Hpo pathway members provide cells with a competitive advantage by unknown mechanisms. Here we provide insight into the mechanism by which the Hpo pathway is linked to cell competition, by identifying dMyc as a target gene of the Hpo pathway, transcriptionally upregulated by the activity of Yki with different binding partners. We show that the cell-autonomous upregulation of dMyc is required for the supercompetitive behavior of Yki-expressing cells and Hpo pathway mutant cells, whereas the relative levels of dMyc between Hpo pathway mutant cells and wild-type neighboring cells are critical for determining whether cell competition promotes a tumor-suppressing or tumor-inducing behavior. All together, these data provide a paradigmatic example of cooperation between tumor suppressor genes and oncogenes in tumorigenesis and suggest a dual role for cell competition during tumor progression depending on the output of the genetic interactions occurring between confronted cells.

  5. Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

    Science.gov (United States)

    Normanly, J; Grisafi, P; Fink, G R; Bartel, B

    1997-01-01

    Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo. PMID:9368415

  6. Influence of defects of nanostructured ZnO films on the photovoltaic characteristics of perovskite solar cells

    Science.gov (United States)

    Afanasyev, D. A.; Mirzoev, K. Yu; Ibrayev, N. Kh

    2018-01-01

    The influence of the defects in ZnO films on the electrical and photovoltaic properties of perovskite solar cells was investigated in the work. According to the results of the research it was established that the defects in ZnO films affects the concentration of defects of perovskite films synthesized on the ZnO surface. However, the difference in the defect concentration in perovskite films is about 30%, while the concentration of defects in ZnO differs by 1000 times. A less significant influence is the concentration of ZnO defects on the electrical and photovoltaic properties of perovskite solar cells. The magnitude of the short-circuit photocurrent and the open voltage of the cells are affected by the concentration of perovskite defects and the quality of the perovskite-ZnO interface.

  7. Establishment of screening technique for mutant cell and analysis of base sequence in the mutation

    International Nuclear Information System (INIS)

    Sofuni, Toshio; Nomi, Takehiko; Yamada, Masami; Masumura, Kenichi

    2000-01-01

    This research project aimed to establish an easy and quick detection method for radiation-induced mutation using molecular-biological techniques and an effective analyzing method for the molecular changes in base sequence. In this year, Spi mutants derived from γ-radiation exposed mouse were analyzed by PCR method and DNA sequence method. Male transgenic mice were exposed to γ-ray at 5,10, 50 Gy and the transgene was taken out from the genome DNA from the spleen in vivo packaging method. Spi mutant plaques were obtained by infecting the recovered phage to E. coli. Sequence analysis for the mutants was made using ALFred DNA sequencer and SequiTherm TM Long-Red Cycle sequencing kit. Sequence analysis was carried out for 41 of 50 independent Spi mutants obtained. The deletions were classified into 4 groups; Group 1 included 15 mutants that were characterized with a large deletion (43 bp-10 kb) with a short homologous sequence. Group 2 included 11 mutants of a large deletion having no homologous sequence at the connecting region. Group 3 included 11 mutants having a short deletion of less than 20 bp, which occurred in the non-repetitive sequence of gam gene and possibly caused by oxidative breakage of DNA or recombination of DNA fragment produced by the breakage. Group 4 included 4 mutants having deletions as short as 20 bp or less in the repetitive sequence of gam gene, resulting in an alteration of the reading frame. Thus, the synthesis of Gam protein was terminated by the appearance of TGA between code 13 and 14 of redB gene, leading to inactivation of gam gene and redBA gene. These results indicated that most of Spi mutants had a deletion in red/gam region and the deletions in more than half mutants occurred in homologous sequences as short as 8 bp. (M.N.)

  8. Expression of an amino-terminal BRCA1 deletion mutant causes a dominant growth inhibition in MCF10A cells.

    Science.gov (United States)

    You, Fanglei; Chiba, Natsuko; Ishioka, Chikashi; Parvin, Jeffrey D

    2004-07-29

    Expression of deletion mutants of the breast and ovarian cancer-specific tumor suppressor protein, BRCA1, in the mammary epithelial cell line MCF10A revealed a powerful growth suppressive effect by a mutant that has the amino-terminal 302 amino acids deleted (DeltaN-BRCA1). The growth suppression is associated with an increase in apoptosis and amplification in centrosome number. The growth inhibitory effect of DeltaN-BRCA1 was not observed in cervical epithelial HeLa cells, suggesting that the phenotypes of BRCA1 mutant proteins differ depending on the cell line being tested. An internal domain, including BRCA1 residues 303-1292, caused the suppression of MCF10A cell growth, and the amino terminus of BRCA1 autoinhibited the growth suppression. Single point mutations that disrupted the amino-terminal RING domain of BRCA1 caused significant suppression of growth in MCF10A cells. These results suggest that the proper function of the RING domain, likely to be ubiquitin ligase function, is important in regulating the growth of the mammary epithelial cell line and in autoregulating the powerful internal growth-inhibiting domain of the BRCA1 tumor suppressor.

  9. Heterodimerization of wild-type and mutant fibroblast growth factor receptors in cell-derived vesicles

    Science.gov (United States)

    Hristova, Kalina; Del Piccolo, Nuala; Sarabipour, Sarvenaz

    The activity of receptor tyrosine kinases (RTKs) is controlled through their lateral dimerization in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared to homodimers, due to limitations in current experimental methods. Here, we develop a Förster Resonance Energy Transfer (FRET)-based methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three Fibroblast Growth Factor Receptors - FGFR1, FGFR2, and FGFR3 - in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane..

  10. Investigation of Near-Surface Defects Induced by Spike Rapid Thermal Annealing in c-SILICON Solar Cells

    Science.gov (United States)

    Liu, Guodong; Ren, Pan; Zhang, Dayong; Wang, Weiping; Li, Jianfeng

    2016-01-01

    The defects induced by a spike rapid thermal annealing (RTA) process in crystalline silicon (c-Si) solar cells were investigated by the photoluminescence (PL) technique and the transmission electron microscopy (TEM), respectively. Dislocation defects were found to form in the near-surface junction region of the monocrystalline Si solar cell after a spike RTA process was performed at 1100∘C. Photo J-V characteristics were measured on the Si solar cell before and after the spike RTA treatments to reveal the effects of defects on the Si cell performances. In addition, the Silvaco device simulation program was used to study the effects of defects density on the cell performances by fitting the experimental data of RTA-treated cells. The results demonstrate that there was an obvious degradation in the Si solar cell performances when the defect density after the spike RTA treatment was above 1×1013cm-3.

  11. A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

    NARCIS (Netherlands)

    Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

    2008-01-01

    To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative a-glucan

  12. Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

    Directory of Open Access Journals (Sweden)

    Bianca Schmid

    2015-12-01

    Full Text Available Dengue virus (DENV is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

  13. ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells.

    Science.gov (United States)

    Takada, Naoto; Takatsu, Hiroyuki; Miyano, Rie; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-11-01

    Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

    Science.gov (United States)

    Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

    2017-06-01

    In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

  15. Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress.

    Science.gov (United States)

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Liu, Fen; Zhang, Yuan Yuan; Yari, Hamed; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-03-01

    Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy. ©2016 American Association for Cancer Research.

  16. Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity

    International Nuclear Information System (INIS)

    Moore, C.W.

    1982-01-01

    Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 0 C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +- 0.07 and 5.7 +- 0.6 single-strand breaks per 10 8 daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 0 C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +- 0.3 (at 23 0 C) or 0.21 +- 0.05 (at 37 0 C) per 10 8 daltons in parental cells, but remained at 2.1 +- 0.06 (at 23 0 C) or 2.3 +- 0.07 (at 37 0 C) per 10 8 daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 0 C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 0 C, DNAs in both strains were completely rejoined

  17. Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1

    DEFF Research Database (Denmark)

    Issinger, O G; Hausmann, R

    1973-01-01

    During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection...

  18. Analyzing Defects in the "Caenorhabditis Elegans" Nervous System Using Organismal and Cell Biological Approaches

    Science.gov (United States)

    Guziewicz, Megan; Vitullo, Toni; Simmons, Bethany; Kohn, Rebecca Eustance

    2002-01-01

    The goal of this laboratory exercise is to increase student understanding of the impact of nervous system function at both the organismal and cellular levels. This inquiry-based exercise is designed for an undergraduate course examining principles of cell biology. After observing the movement of "Caenorhabditis elegans" with defects in their…

  19. Delayed expression of apoptosis in X-irradiated human leukemic MOLT-4 cells transfected with mutant p53

    International Nuclear Information System (INIS)

    Nakano, Hisako; Yonekawa, Hiromichi; Shinohara, Kunio

    2003-01-01

    The effects of X-rays on cell survival, apoptosis, and long-term response in the development of cell death as measured by the dye exclusion test were studied in human leukemic MOLT-4 cells (p53 wild-type) stably transfected with a mutant p53 cDNA expression vector. Cell survival, as determined from colony-forming ability, was increased in an expression level dependent manner, but the increase was partial even with the highest-expressing clone (B3). This contrasts with the prior observation that cell death and apoptosis in B3 are completely inhibited at 24 h after irradiation with 1.8 Gy of X-rays. The examination of B3 cells incubated for longer than 24 h after X-irradiation showed a delay in the induction of cell death and apoptosis. Western blot analysis revealed that the time required to reach the highest level of wild-type p53 protein in B3 was longer than the time in MOLT-4 and that the p53 may be stabilized by the phosphorylation at Ser-15. These results suggest that the introduction of mutant p53 into MOLT-4 merely delays the development of apoptosis, during which the cells could repair the damage induced by X-rays, and results in the partial increase in cell survival. (author)

  20. Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

    Directory of Open Access Journals (Sweden)

    Chia Yin Lee

    2011-12-01

    Full Text Available Chikungunya virus (CHIKV is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

  1. [Effect of simvastatin on inducing endothelial progenitor cells homing and promoting bone defect repair].

    Science.gov (United States)

    Song, Quansheng; Wang, Lingying; Zhu, Jinglin; Han, Xiaoguang; Li, Xu; Yang, Yanlin; Sun, Yan; Song, Chunli

    2010-09-01

    To investigate the effect of simvastatin on inducing endothelial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 x 10(6) cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins (2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% +/- 4.07% in experimental group and 59.45% +/- 5.43% in control group, showing significant difference (P < 0.05). Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs

  2. Cholesterol pathways affected by small molecules that decrease sterol levels in Niemann-Pick type C mutant cells.

    Directory of Open Access Journals (Sweden)

    Madalina Rujoi

    2010-09-01

    Full Text Available Niemann-Pick type C (NPC disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165.

  3. Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells.

    Science.gov (United States)

    Pradhan, Shrikant; Mahajan, Divyank; Kaur, Prabhjot; Pandey, Namita; Sharma, Chandresh; Srivastava, Tapasya

    2016-11-01

    Non-small cell lung cancer (NSCLC), comprising 85% of lung cancer cases, has been associated with resistance to chemo/radiotherapy. The hypoxic tumor micro-environment, where insufficient vasculature results in poor drug penetrance and sub-optimal chemotherapy in the tumor interiors contributes heavily to this resistance. Additionally, epigenetic changes in tumorigenic cells also change their response to different forms of therapy. In our study, we have investigated the effectiveness of a combination of cisplatin with scriptaid [a pan-Histone Deacetylase inhibitor (HDACi)] in a model that mimics the tumor microenvironment of hypoxia and sub-lethal chemotherapy. Scriptaid synergistically increases the efficacy of cisplatin in normoxia as well as hypoxia, accompanied with reduced metastasis and enhanced DNA damage. Addition of scriptaid also overcomes the cisplatin resistance exhibited in lung cancer cells with stabilized hypoxia inducible factor 1 (HIF1)-α (mutant) and mutant p53. Molecular studies showed that the combination treatment increased apoptotic cell death in both normoxia and hypoxia with a dual role of p38MAPK. Together, our results suggest that the combination of low dose cisplatin and scriptaid is cytotoxic to NSCLC lines, can overcome hypoxia induced resistance and mutant p53- induced instability often associated with this cancer, and has the potential to be an effective therapeutic modality.

  4. Deletion of Pkd1 in renal stromal cells causes defects in the renal stromal compartment and progressive cystogenesis in the kidney.

    Science.gov (United States)

    Nie, Xuguang; Arend, Lois J

    2017-12-01

    Autosomal dominant polycystic kidney disease (ADPKD), caused by PKD1 and PKD2 gene mutations, is one of the most common genetic diseases, affecting up to 1 in 500 people. Mutations of PKD1 account for over 85% of ADPKD cases. However, mechanisms of disease progression and explanations for the wide range in disease phenotype remain to be elucidated. Moreover, functional roles of PKD1 in the renal stromal compartment are poorly understood. In this work, we tested if Pkd1 is essential for development and maintenance of the renal stromal compartment and if this role contributes to pathogenesis of polycystic kidney disease using a novel tissue-specific knockout mouse model. We demonstrate that deletion of Pkd1 from renal stromal cells using Foxd1-driven Cre causes a spectrum of defects in the stromal compartment, including excessive apoptosis/proliferation and extracellular matrix deficiency. Renal vasculature was also defective. Further, mutant mice showed epithelial changes and progressive cystogenesis in adulthood modeling human ADPKD. Altogether, we provide robust evidence to support indispensable roles for Pkd1 in development and maintenance of stromal cell derivatives by using a novel ADPKD model. Moreover, stromal compartment defects caused by Pkd1 deletion might serve as an important mechanism for pathogenesis of ADPKD.

  5. Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

    International Nuclear Information System (INIS)

    Steinborn, G.

    1978-01-01

    Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

  6. Evaluation of cell sheet application on one wall bone defect in Macaca nemestrina through periostin expression

    Science.gov (United States)

    Tamin, R. Y.; Soeroso, Y.; Amir, L.; Idrus, E.

    2017-08-01

    Chronic periodontitis is an oral disease in which the destruction of periodontal tissue leads to tooth loss. Regenerative therapy for attachment cannot be applied to one wall bone defects owing to the minimal existing healthy bone. Tissue engineering in the form of cell sheets has been developed to overcome this limitation. In a previous study, cell sheet application to a one wall bone defect in Macaca nemestrina showed good clinical results. To evaluate the effectiveness of cell sheet application histologically, the level of periostin expression in the gingival crevicular fluid (GCF) of M. nemestrina was determined. Periostin is a 90-kDa protein that regulates coordination and interaction for regeneration and tissue repair. A laboratory observation study was performed to see the differences in periostin levels in samples collected from M. nemestrina’s GCF, where a cell sheet was applied to the bone defect. Gel electrophoresis with SDS-PAGE was performed to detect periostin expression based on its molecular weight and to compare the expression band between the cell sheet and the control at 1, 2, and 3 weeks after treatment. The gel electrophoresis result shows different thicknesses of the protein band around the molecular weight of periostin between the cell sheet groups.

  7. Visual Field Defects and Retinal Ganglion Cell Losses in Human Glaucoma Patients

    Science.gov (United States)

    Harwerth, Ronald S.; Quigley, Harry A.

    2007-01-01

    Objective The depth of visual field defects are correlated with retinal ganglion cell densities in experimental glaucoma. This study was to determine whether a similar structure-function relationship holds for human glaucoma. Methods The study was based on retinal ganglion cell densities and visual thresholds of patients with documented glaucoma (Kerrigan-Baumrind, et al.) The data were analyzed by a model that predicted ganglion cell densities from standard clinical perimetry, which were then compared to histologic cell counts. Results The model, without free parameters, produced accurate and relatively precise quantification of ganglion cell densities associated with visual field defects. For 437 sets of data, the unity correlation for predicted vs. measured cell densities had a coefficient of determination of 0.39. The mean absolute deviation of the predicted vs. measured values was 2.59 dB, the mean and SD of the distribution of residual errors of prediction was -0.26 ± 3.22 dB. Conclusions Visual field defects by standard clinical perimetry are proportional to neural losses caused by glaucoma. Clinical Relevance The evidence for quantitative structure-function relationships provides a scientific basis of interpreting glaucomatous neuropathy from visual thresholds and supports the application of standard perimetry to establish the stage of the disease. PMID:16769839

  8. Mast cell deficiency attenuates acupuncture analgesia for mechanical pain using c-kit gene mutant rats

    Science.gov (United States)

    Xu, Dandan; Zhang, Youyou; He, Xun; Liu, Hao; Gao, Xinyan; Zhu, Bing

    2018-01-01

    Background Acupuncture therapy plays a pivotal role in pain relief, and increasing evidence demonstrates that mast cells (MCs) may mediate acupuncture analgesia. The present study aims to investigate the role of MCs in acupuncture analgesia using c-kit gene mutant–induced MC-deficient rats. Materials and methods WsRC-Ws/Ws rats and their wild-type (WT) littermates (WsRC-+/+) were used. The number of MCs in skin of ST36 area was compared in two rats after immunofluorescence labeling. Mechanical withdrawal latency (MWL), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were measured on bilateral plantar for pain threshold evaluation before and after each stimulus. Acupuncture- and moxibustion-like stimuli (43°C, 46°C heat, 1 mA electroacupuncture [EA], 3 mA EA, and manual acupuncture [MA]) were applied randomly on different days. Results Fewer MCs were observed in the skin of ST36 in mutant rats compared to WT rats (Ppain thresholds, MWL and MWT were higher in WsRC-Ws/Ws compared to WsRC-+/+ on bilateral paws (P0.05). Bilateral MWL and MWT in WsRC-+/+ rats increased significantly after each stimulus compared to baseline (Ppain (46°C, 3 mA EA, MA) (Ppain threshold. MCs participate in acupuncture analgesia in mechanical but not in thermal nociception, in that MC deficiency may attenuate the mechanical analgesia evoked by high-intensity stimuli and eliminate analgesia provoked by low-intensity stimuli. PMID:29551908

  9. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    Energy Technology Data Exchange (ETDEWEB)

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  10. Cross Talk between Cell Cell and Cell Matrix Adhesion Signaling Pathways during Heart Organogenesis: Implications for Cardiac Birth Defects

    Science.gov (United States)

    Linask, Kersti K.; Manisastry, Shyam; Han, Mingda

    2005-06-01

    The anterior posterior and dorsal ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule [beta]-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to [beta]-catenin stabilization. Lithium exposure results in an anterior posterior progression of severe cardiac defects.

  11. β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity.

    Science.gov (United States)

    Hasim, Sahar; Allison, David P; Retterer, Scott T; Hopke, Alex; Wheeler, Robert T; Doktycz, Mitchel J; Reynolds, Todd B

    2017-01-01

    Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system. Copyright © 2016 American Society for Microbiology.

  12. Reduced host cell invasiveness and oxidative stress tolerance in double and triple csp gene family deletion mutants of Listeria monocytogenes.

    Science.gov (United States)

    Loepfe, Chantal; Raimann, Eveline; Stephan, Roger; Tasara, Taurai

    2010-07-01

    The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and

  13. Defective bone repair in mast cell-deficient Cpa3Cre/+ mice.

    Directory of Open Access Journals (Sweden)

    Jose Luis Ramirez-GarciaLuna

    Full Text Available In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1 mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2 re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3 the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.

  14. Expression of ALS-linked TDP-43 mutant in astrocytes causes non-cell-autonomous motor neuron death in rats

    Science.gov (United States)

    Tong, Jianbin; Huang, Cao; Bi, Fangfang; Wu, Qinxue; Huang, Bo; Liu, Xionghao; Li, Fang; Zhou, Hongxia; Xia, Xu-Gang

    2013-01-01

    Mutation of Tar DNA-binding protein 43 (TDP-43) is linked to amyotrophic lateral sclerosis. Although astrocytes have important roles in neuron function and survival, their potential contribution to TDP-43 pathogenesis is unclear. Here, we created novel lines of transgenic rats that express a mutant form of human TDP-43 (M337V substitution) restricted to astrocytes. Selective expression of mutant TDP-43 in astrocytes caused a progressive loss of motor neurons and the denervation atrophy of skeletal muscles, resulting in progressive paralysis. The spinal cord of transgenic rats also exhibited a progressive depletion of the astroglial glutamate transporters GLT-1 and GLAST. Astrocytic expression of mutant TDP-43 led to activation of astrocytes and microglia, with an induction of the neurotoxic factor Lcn2 in reactive astrocytes that was independent of TDP-43 expression. These results indicate that mutant TDP-43 in astrocytes is sufficient to cause non-cell-autonomous death of motor neurons. This motor neuron death likely involves deficiency in neuroprotective genes and induction of neurotoxic genes in astrocytes. PMID:23714777

  15. Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.

    Science.gov (United States)

    Moda, Bruno S; Ferreira-Júnior, José Ribamar; Barros, Mario H

    2016-08-01

    Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.

  16. Deletion of the cardiolipin-specific phospholipase Cld1 rescues growth and life span defects in the tafazzin mutant: implications for Barth syndrome

    NARCIS (Netherlands)

    Ye, Cunqi; Lou, Wenjia; Li, Yiran; Chatzispyrou, Iliana A.; Hüttemann, Maik; Lee, Icksoo; Houtkooper, Riekelt H.; Vaz, Frédéric M.; Chen, Shuliang; Greenberg, Miriam L.

    2014-01-01

    Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects, and in humans, this results in the

  17. Cleft palate defect of Dlx1/2-/- mutant mice is caused by lack of vertical outgrowth in the posterior palate

    NARCIS (Netherlands)

    Jeong, J.; Cesario, J.; Zhao, Y.; Burns, L.; Westphal, H.; Rubenstein, J.L.

    2012-01-01

    BACKGROUND: Mice lacking the activities of Dlx1 and Dlx2 (Dlx1/2-/-) exhibit cleft palate, one of the most common human congenital defects, but the etiology behind this phenotype has been unknown. Therefore, we analyzed the morphological, cellular, and molecular changes caused by inactivation of

  18. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  19. Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy- l -fucose induces defects in root cell elongation

    Energy Technology Data Exchange (ETDEWEB)

    Dumont, Marie [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France; Lehner, Arnaud [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France; Bardor, Muriel [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France; Burel, Carole [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France; Vauzeilles, Boris [Institut de Chimie Moléculaire et des Matériaux d' Orsay (ICMMO) UMR CNRS 8182, Université de Paris Sud, 91405 Orsay France; Institut de Chimie des Substances Naturelles (ICSN) UPR CNRS 2301, 91198 Gif-sur-Yvette France; Click4Tag, Zone Luminy Biotech, Case 922, 163 Avenue de Luminy 13009 Marseille France; Lerouxel, Olivier [Centre de Recherches sur les Macromolécules Végétales (CERMAV) - CNRS BP 53, 38041 Grenoble Cedex 9 France; Anderson, Charles T. [Department of Biology and Center for Lignocellulose Structure and Formation, Pennsylvania State University, University Park Pennsylvania USA; Mollet, Jean-Claude [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France; Lerouge, Patrice [Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IRIB, VASI, Normandie Université, 76821 Mont-Saint-Aignan France

    2015-12-01

    Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism.

  20. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    Directory of Open Access Journals (Sweden)

    Jessica Lenzi

    2015-07-01

    Full Text Available Patient-derived induced pluripotent stem cells (iPSCs provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

  1. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    Science.gov (United States)

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  2. Antiproliferative and Apoptotic Effect of Dendrosomal Curcumin Nanoformulation in P53 Mutant and Wide-Type Cancer Cell Lines.

    Science.gov (United States)

    Montazeri, Maryam; Pilehvar-Soltanahmadi, Younes; Mohaghegh, Mina; Panahi, Alireza; Khodi, Samaneh; Zarghami, Nosratollah; Sadeghizadeh, Majid

    2017-01-01

    The aim of this paper is to investigate the effect of dendrosomal curcumin (DNC) on the expression of p53 in both p53 mutant cell lines SKBR3/SW480 and p53 wild-type MCF7/HCT116 in both RNA and protein levels. Curcumin, derived from Curcumin longa, is recently considered in cancer related researches for its cell growth inhibition properties. p53 is a common tumor-suppressor gene involved in cancers and its mutation not only inhibits tumor suppressor activity but also promotes oncogenic activity. Here, p53 mutant/Wild-type cells were employed to study the toxicity of DNC using MTT assay, Flow cytometry and Annexin-V, Real-time PCR and Western blot were used to analyze p53, BAX, Bcl-2, p21 and Noxa changes after treatment. During the time, DNC increased the SubG1 cells and decreased G1, S and G2/M cells, early apoptosis also indicated the inhibition of cell growth in early phase. Real-Time PCR assay showed an increased mRNA of BAX, Noxa and p21 during the time with decreased Bcl-2. The expression of p53 mutant decreased in SKBR3/SW480, and the expression of p53 wild-type increased in MCF7/HCT116. Consequently, p53 plays an important role in mediating the survival by DNC, which can prevent tumor cell growth by modulating the expression of genes involved in apoptosis and proliferation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

    Directory of Open Access Journals (Sweden)

    Yiran Feng

    2017-10-01

    Full Text Available Data in this article are associated with the research article “Highthroughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins” (Yang et al., 2017 [1]. This article provided data on how to develop an immunoturbidimetric assay (ITA of enzyme/mutants as proteins in cell lysates in high-throughput (HTP mode together with HTP assay of their activities to derive their specific activities in cell lysates for comparison, with Pseudomonas aeruginosa arylsulfatase (PAAS and Bacillus fastidious uricase (BFU plus their mutants as models. Data were made publicly available for further analyses.

  4. B-cell memory and primary immune deficiencies: interleukin-21 related defects.

    Science.gov (United States)

    Desjardins, Marylin; Mazer, Bruce D

    2013-12-01

    The purpose of this study is to describe recent advances in our understanding of the role of interleukin-21 (IL-21) in B-cell maturation, and how defects in IL-21 receptor (IL-21R) signalling pathways (IL-21R/γc/JAK3/STAT3) are related to primary immune deficiencies. Abnormal signalling through IL-21R/γc/JAK3/STAT3 pathway has been related to decreased specific antibody responses following vaccination, and to increased susceptibility to encapsulated bacterial infections. This is manifested in the hyper-IgE syndrome, X-linked and JAK3-related severe combined immunodeficiency (SCID) and loss-of-function mutations in the IL-21R gene. Common variable immunodeficiency is associated with impaired in-vitro development of peripheral blood mononuclear cells or purified B-cells into memory or CD38 B-cells following addition of IL-21. IL-21 is a key cytokine in development of B-cells into immunoglobulin-secreting cells. Abnormal signalling through the IL-21R/γc/JAK3/STAT3 pathway leads to defective humoral immune responses to both T-dependent and T-independent antigens and impairs the establishment of long-lasting B-cell memory. Studies involving patients with hyper-IgE syndrome demonstrated the nonredundant role of STAT3 in B-cell production of high-affinity specific antibodies, while total serum immunoglobulins could be maintained through STAT3-independent activation of AID (activation-induced cytidine-deaminase). IL-21 related defects may also be associated with reduced natural killer (NK)-cell cytotoxicity and TH17 cytokine production, indicating that abnormalities in the IL-21-IL-21R pathway have profound effects on crucial immune responses.

  5. Adipose stem cells used to reconstruct 13 cases with cranio-maxillofacial hard-tissue defects.

    Science.gov (United States)

    Sándor, George K; Numminen, Jura; Wolff, Jan; Thesleff, Tuomo; Miettinen, Aimo; Tuovinen, Veikko J; Mannerström, Bettina; Patrikoski, Mimmi; Seppänen, Riitta; Miettinen, Susanna; Rautiainen, Markus; Öhman, Juha

    2014-04-01

    Although isolated reports of hard-tissue reconstruction in the cranio-maxillofacial skeleton exist, multipatient case series are lacking. This study aimed to review the experience with 13 consecutive cases of cranio-maxillofacial hard-tissue defects at four anatomically different sites, namely frontal sinus (3 cases), cranial bone (5 cases), mandible (3 cases), and nasal septum (2 cases). Autologous adipose tissue was harvested from the anterior abdominal wall, and adipose-derived stem cells were cultured, expanded, and then seeded onto resorbable scaffold materials for subsequent reimplantation into hard-tissue defects. The defects were reconstructed with either bioactive glass or β-tricalcium phosphate scaffolds seeded with adipose-derived stem cells (ASCs), and in some cases with the addition of recombinant human bone morphogenetic protein-2. Production and use of ASCs were done according to good manufacturing practice guidelines. Follow-up time ranged from 12 to 52 months. Successful integration of the construct to the surrounding skeleton was noted in 10 of the 13 cases. Two cranial defect cases in which nonrigid resorbable containment meshes were used sustained bone resorption to the point that they required the procedure to be redone. One septal perforation case failed outright at 1 year because of the postsurgical resumption of the patient's uncontrolled nasal picking habit.

  6. MALDI-TOF MS and CE-LIF Fingerprinting of Plant Cell Wall Polysaccharide Digests as a Screening Tool for Arabidopsis Cell Wall Mutants

    NARCIS (Netherlands)

    Westphal, Y.; Schols, H.A.; Voragen, A.G.J.; Gruppen, H.

    2010-01-01

    Cell wall materials derived from leaves and hypocotyls of Arabidopsis mutant and wild type plants have been incubated with a mixture of pure and well-defined pectinases, hemicellulases, and cellulases. The resulting oligosaccharides have been subjected to MALDI-TOF MS and CE-LIF analysis. MALDI-TOF

  7. Nanodiamonds with silicon vacancy defects for nontoxic photostable fluorescent labeling of neural precursor cells.

    Science.gov (United States)

    Merson, Tobias D; Castelletto, Stefania; Aharonovich, Igor; Turbic, Alisa; Kilpatrick, Trevor J; Turnley, Ann M

    2013-10-15

    Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiV-containing NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

  8. Intrinsic Plasma Cell Differentiation Defects in B Cell Expansion with NF-κB and T Cell Anergy Patient B Cells

    Directory of Open Access Journals (Sweden)

    Swadhinya Arjunaraja

    2017-08-01

    Full Text Available B cell Expansion with NF-κB and T cell Anergy (BENTA disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound in vitro survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.

  9. Clinical light exposure, photoreceptor degeneration, and AP-1 activation: a cell death or cell survival signal in the rhodopsin mutant retina?

    Science.gov (United States)

    Gu, Danian; Beltran, William A; Li, Zexiao; Acland, Gregory M; Aguirre, Gustavo D

    2007-11-01

    The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.

  10. Treatment of periodontal intrabony defects using autologous periodontal ligament stem cells: a randomized clinical trial.

    Science.gov (United States)

    Chen, Fa-Ming; Gao, Li-Na; Tian, Bei-Min; Zhang, Xi-Yu; Zhang, Yong-Jie; Dong, Guang-Ying; Lu, Hong; Chu, Qing; Xu, Jie; Yu, Yang; Wu, Rui-Xin; Yin, Yuan; Shi, Songtao; Jin, Yan

    2016-02-19

    Periodontitis, which progressively destroys tooth-supporting structures, is one of the most widespread infectious diseases and the leading cause of tooth loss in adults. Evidence from preclinical trials and small-scale pilot clinical studies indicates that stem cells derived from periodontal ligament tissues are a promising therapy for the regeneration of lost/damaged periodontal tissue. This study assessed the safety and feasibility of using autologous periodontal ligament stem cells (PDLSCs) as an adjuvant to grafting materials in guided tissue regeneration (GTR) to treat periodontal intrabony defects. Our data provide primary clinical evidence for the efficacy of cell transplantation in regenerative dentistry. We conducted a single-center, randomized trial that used autologous PDLSCs in combination with bovine-derived bone mineral materials to treat periodontal intrabony defects. Enrolled patients were randomly assigned to either the Cell group (treatment with GTR and PDLSC sheets in combination with Bio-oss(®)) or the Control group (treatment with GTR and Bio-oss(®) without stem cells). During a 12-month follow-up study, we evaluated the frequency and extent of adverse events. For the assessment of treatment efficacy, the primary outcome was based on the magnitude of alveolar bone regeneration following the surgical procedure. A total of 30 periodontitis patients aged 18 to 65 years (48 testing teeth with periodontal intrabony defects) who satisfied our inclusion and exclusion criteria were enrolled in the study and randomly assigned to the Cell group or the Control group. A total of 21 teeth were treated in the Control group and 20 teeth were treated in the Cell group. All patients received surgery and a clinical evaluation. No clinical safety problems that could be attributed to the investigational PDLSCs were identified. Each group showed a significant increase in the alveolar bone height (decrease in the bone-defect depth) over time (p 0.05). This study

  11. Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ayres, Flavio Monteiro; Glickman, Barry W.; Steele, Patricia [University of Victoria, BC (Canada). Dept. of Biology. Centre for Environmental; Cruz, Aparecido Divino da [Universidade Estadual de Goias, Anapolis, GO (Brazil). Dept. de Biologia. Nucleo de Pesquisas Replicon]. E-mail: acruz@ucg.br

    2006-07-01

    The TK6 lymphoblastoid cell line is known to be mismatch repair (MMR) and p53 proficient. Deficiency in MMR results in a mutator phenotype characterized by microsatellite instability (MSI) and increased hprt mutant frequency (MF). Increased hprt MF is also a biomarker of effect for exposure to ionizing radiation. In order to test if a mutator phenotype could be induced by low doses of gamma ionizing radiation, an hprt cloning assay and a MSI investigation were performed after radiation exposure. The spontaneous MF was 1.6 x 10-6. The groups exposed to 0.2, 0.5 and 1.0 Gy had hprt MFs of 2.3, 3.3 and 2.2 x 10-6, respectively. The spontaneous MSI frequency per allele in non-selected cells was 5.4 x 10-3, as evidenced at the loci D11S35, nm23-H1, D8S135 and p53. MSI frequencies in the groups exposed to 0.2, 0.5 and 1.0 Gy were found to be < 4.7, < 7.7 and < 12 x 10-3, respectively. The frequencies of hprt mutants and MSI found in this study suggest that low doses of ionizing radiation increase hprt mutant frequency without triggering the mutator phenotype pathway. (author)

  12. Latent membrane protein 1 deletion mutants accumulate in reed-sternberg cells of human immunodeficiency virus-related Hodgkin's lymphoma.

    Science.gov (United States)

    Guidoboni, Massimo; Ponzoni, Maurilio; Caggiari, Laura; Lettini, Antonia A; Vago, Luca; De Re, Valli; Gloghini, Annunziata; Zancai, Paola; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-02-01

    The origin and biological significance of deletions at the 3' end of the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) gene are still controversial. We herein demonstrate that LMP-1 deletion mutants are highly associated with human immunodeficiency virus-related Hodgkin's lymphoma (HIV-HL) of Italian patients (29 of 31 cases; 93.5%), a phenomenon that is not due to a peculiar distribution of EBV strains in this area. In fact, although HIV-HL patients are infected by multiple EBV variants, we demonstrate that LMP-1 deletion mutants preferentially accumulate within neoplastic tissues. Subcloning and sequencing of the 3' LMP-1 ends of two HIV-HL genes in which both variants were present showed the presence of molecular signatures suggestive of a likely derivation of the LMP-1 deletion mutant from a nondeletion ancestor. This phenomenon likely occurs within tumor cells in vivo, as shown by the detection of both LMP-1 variants in single microdissected Reed-Sternberg cells, and may at least in part explain the high prevalence of LMP-1 deletions associated with HIV-HL.

  13. Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

    Science.gov (United States)

    Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

    2017-12-12

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo , erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity.

  14. Regenerative potential of tonsil mesenchymal stem cells on surgical cutaneous defect.

    Science.gov (United States)

    Shin, Sung-Chan; Seo, Yoojin; Park, Hee Young; Jung, Da-Woon; Shin, Tae-Hoon; Son, Haejin; Kim, Young Keum; Lee, Jin-Choon; Sung, Eui-Suk; Jang, Jeon Yeob; Kim, Hyung-Sik; Lee, Byung-Joo

    2018-02-07

    As tissue engineering and regenerative medicine have evolved recently, stem cell therapy has been investigated in the field of impaired wound healing. Several studies have reported that mesenchymal stem cells derived from various tissues including bone marrow and adipose tissue can exert the regenerative efficacy in the wound healing. Previously, we have demonstrated the isolation and characterization of tonsil-derived mesenchymal stem cells (TMSCs) with excellent proliferative property. In the present study, we aimed to evaluate the regenerative efficacy of TMSCs in the wound healing process. Two distinct cutaneous surgical defects were generated in the dorsum of mice. Each wound was treated with TMSCs or phosphate-buffered saline (PBS), respectively. After sacrifice, the skin and subcutaneous tissues around the surgical defect were harvested and assessed for inflammation, re-epithelialization, dermal regeneration, and granulation tissue formation. The administration of TMSCs into wound beds significantly promoted the repair of surgical defects in mice. Especially, TMSCs efficiently contributed to the attenuation of excessive inflammation in the surgical lesion, as well as the augmentation of epidermal and dermal regeneration. To elucidate the underlying mechanisms, TMSCs were analyzed for their potency in immunomodulatory ability on immune cells, stimulatory effect on the proliferation of keratinocytes, and fibroblasts, as well as the regulation of fibroblast differentiation. TMSCs inhibited the non-specific or T-cell-specific proliferation of peripheral blood mononuclear cells, as well as the M1 polarization of macrophage-like cells. Moreover, TMSCs augmented the proliferation of skin-constituting fibroblasts and keratinocytes while they suppressed the differentiation of fibroblasts into myofibroblasts. Taken together, our findings demonstrate the regenerative potential of TMSCs in wound healing process through the regulation on inflammation, proliferation

  15. Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects

    Directory of Open Access Journals (Sweden)

    Sucov Henry M

    2006-11-01

    Full Text Available Abstract Background Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood. Results We deleted the TGF-β type I receptor Alk5 specifically in the mouse neural crest cell lineage. Failure in signaling via ALK5 leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. While ALK5 is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells. Conclusion Our results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development.

  16. Modulation of mutant huntingtin N-terminal cleavage and its effect on aggregation and cell death

    NARCIS (Netherlands)

    Juenemann, Katrin; Weisse, Christina; Reichmann, Denise; Kaether, Christoph; Calkhoven, Cornelis F.; Schilling, Gabriele

    2011-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem

  17. Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific

    NARCIS (Netherlands)

    Jansen, Anne H. P.; van Hal, Maurik; Op den Kelder, Ilse C.; Meier, Romy T.; de Ruiter, Anna-Aster; Schut, Menno H.; Smith, Donna L.; Grit, Corien; Brouwer, Nieske; Kamphuis, Willem; Boddeke, H. W. G. M.; den Dunnen, Wilfred F. A.; van Roon, Willeke M. C.; Bates, Gillian P.; Hol, Elly M.; Reits, Eric A.

    2017-01-01

    Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions

  18. Frequency of Nuclear Mutant Huntingtin Inclusion Formation in Neurons and Glia is Cell-Type-Specific

    NARCIS (Netherlands)

    Jansen, Anne H P; van Hal, Maurik; op den Kelder, Ilse C.; Meier, Romy T.; de Ruiter, Anna-Aster; Schut, Menno H.; Smith, Donna L.; Grit, Corien; Brouwer, Nieske; Kamphuis, Willem; Boddeke, H. W. G. M.; den Dunnen, Wilfred F. A.; van Roon, Willeke M. C.; Bates, Gillian P.; Hol, Elly M.; Reits, Eric A.

    Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions

  19. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    International Nuclear Information System (INIS)

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-01-01

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3 H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture

  20. The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila.

    Science.gov (United States)

    Vogel, E W; Dusenbery, R L; Smith, P D

    1985-04-01

    Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in

  1. An ion-current mutant of Paramecium tetraurelia with defects in the primary structure and post-translational N-methylation of calmodulin

    International Nuclear Information System (INIS)

    Wallen-Friedman, M.A.

    1988-01-01

    My work on pantophobiac A 2 (pntA 2 ), a behavioral mutant of Paramecium tetraurelia, suggest that the Ca ++ -binding protein calmodulin (CaM), and post-translation N-methylation of CaM, are important for Ca ++ -related ion-current function. Calmodulin from wild-type Paramecium has two sites of lysine-N-methylation. Both of these sites are almost fully methylated in vivo; thus wild-type calmodulin is a poor substrate for N-methylation in vitro. In contrast, pntA/ 2 CaM can be heavily N-methylated in vitro, suggesting that the mutant calmodulin is under-methylated in vivo. Amino-acid composition analysis showed that CaM lysine 115 is undermethylated in pntA 2 . Once pntA 2 CaM is N-methylated, the [methyl- 3 H] group does not turn over in either wild-type or pntA 2 cytoplasmic fractions. The methylating enzymes in pntA 2 high-speed supernatant fractions are active, but may be less robust than those of the wild type, suggesting a possible control of these enzymes by CaM

  2. An ion-current mutant of Paramecium tetraurelia with defects in the primary structure and post-translational N-methylation of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Wallen-Friedman, M.A.

    1988-01-01

    My work on pantophobiac A{sup 2} (pntA{sup 2}), a behavioral mutant of Paramecium tetraurelia, suggest that the Ca{sup ++}-binding protein calmodulin (CaM), and post-translation N-methylation of CaM, are important for Ca{sup ++}-related ion-current function. Calmodulin from wild-type Paramecium has two sites of lysine-N-methylation. Both of these sites are almost fully methylated in vivo; thus wild-type calmodulin is a poor substrate for N-methylation in vitro. In contrast, pntA/{sup 2} CaM can be heavily N-methylated in vitro, suggesting that the mutant calmodulin is under-methylated in vivo. Amino-acid composition analysis showed that CaM lysine 115 is undermethylated in pntA{sup 2}. Once pntA{sup 2} CaM is N-methylated, the (methyl-{sup 3}H) group does not turn over in either wild-type or pntA{sup 2} cytoplasmic fractions. The methylating enzymes in pntA{sup 2} high-speed supernatant fractions are active, but may be less robust than those of the wild type, suggesting a possible control of these enzymes by CaM.

  3. A genetic screen for mutants defective in IAA1-LUC degradation in Arabidopsis thaliana reveals an important requirement for TOPOISOMERASE6B in auxin physiology.

    Science.gov (United States)

    Gilkerson, Jonathan; Callis, Judy

    2014-01-01

    Many plant growth and developmental processes are modulated by the hormone auxin. Auxin-modulated proteolysis of Aux/IAAs, a family of transcriptional repressors, represents a major mode of auxin action. Auxin facilitates the interaction of Aux/IAAs with TIR1/AFB F-box proteins, promoting their ubiquitination by the SCF(TIR1/AFB) ubiquitin E3 ligase leading to subsequent degradation by the 26S proteasome. To identify new genes regulating Aux/IAA proteolysis in Arabidopsis thaliana, we took a genetic approach, identifying individuals with altered degradation of an IAA1-luciferase fusion protein (IAA1-LUC). A mutant with 2-fold slower IAA1-LUC degradation rate compared with wild-type was isolated. Positional cloning identified the mutant as an allele of TOPOISOMERASE6B, named top6b-7. TOP6B encodes a subunit of a plant and archea-specific enzyme regulating endoreduplication, DNA damage repair and transcription in plants. T-DNA insertion alleles (top6b-8 and top6b-9) were also analyzed. top6b-7 seedlings are less sensitive to exogenous auxin than wild-type siblings in primary root growth assays, and experiments with DR5:GUS. Additionally, top6b-7 seedlings have a 40% reduction in the amount of endogenous IAA. These data suggest that increased IAA1-LUC half-life in top6b-7 probably results from a combination of both lower endogenous IAA levels and reduced sensitivity to auxin.

  4. Prevalence of Zygomatic Air Cell Defect in adults—A retrospective panoramic radiographic analysis

    International Nuclear Information System (INIS)

    Patil, Karthikeya; Mahima, V.G.; Malleshi, Suchetha N.; Srikanth, H.S.

    2012-01-01

    Objectives: This research involved retrospectively evaluating panoramic radiographs of patients from India with the intention of assessing the prevalence of Zygomatic Air Cell Defect (ZACD) and establishing its dominant location and type. Methods: Seven thousand seven hundred and fifty-five panoramic radiographs of routine outpatients aged between 19 and 91 years were concomitantly evaluated by four investigators for estimating the prevalence and characteristics of the Zygomatic Air Cell Defect. Results: The prevalence of ZACD was noted to be 1.82%, with male preponderance. Unilateralality and multilocular appearance of ZACD were the dominant patterns observed. Conclusion: The frequency of ZACD amongst Indian population is in harmony with most of the similar studies conducted on various geographic populations.

  5. Method of detecting defects in ion exchange membranes of electrochemical cells by chemochromic sensors

    Science.gov (United States)

    Brooker, Robert Paul; Mohajeri, Nahid

    2016-01-05

    A method of detecting defects in membranes such as ion exchange membranes of electrochemical cells. The electrochemical cell includes an assembly having an anode side and a cathode side with the ion exchange membrane in between. In a configuration step a chemochromic sensor is placed above the cathode and flow isolation hardware lateral to the ion exchange membrane which prevents a flow of hydrogen (H.sub.2) between the cathode and anode side. The anode side is exposed to a first reactant fluid including hydrogen. The chemochromic sensor is examined after the exposing for a color change. A color change evidences the ion exchange membrane has at least one defect that permits H.sub.2 transmission therethrough.

  6. Auditory hair cell defects as potential cause for sensorineural deafness in Wolf-Hirschhorn syndrome

    Directory of Open Access Journals (Sweden)

    Mohi Ahmed

    2015-09-01

    Full Text Available WHSC1 is a histone methyltransferase (HMT that catalyses the addition of methyl groups to lysine 36 on histone 3. In humans, WHSC1 haploinsufficiency is associated with all known cases of Wolf-Hirschhorn syndrome (WHS. The cardinal feature of WHS is a craniofacial dysmorphism, which is accompanied by sensorineural hearing loss in 15% of individuals with WHS. Here, we show that WHSC1-deficient mice display craniofacial defects that overlap with WHS, including cochlea anomalies. Although auditory hair cells are specified normally, their stereocilia hair bundles required for sound perception fail to develop the appropriate morphology. Furthermore, the orientation and cellular organisation of cochlear hair cells and their innervation are defective. These findings identify, for the first time, the likely cause of sensorineural hearing loss in individuals with WHS.

  7. Auditory hair cell defects as potential cause for sensorineural deafness in Wolf-Hirschhorn syndrome.

    Science.gov (United States)

    Ahmed, Mohi; Ura, Kiyoe; Streit, Andrea

    2015-09-01

    WHSC1 is a histone methyltransferase (HMT) that catalyses the addition of methyl groups to lysine 36 on histone 3. In humans, WHSC1 haploinsufficiency is associated with all known cases of Wolf-Hirschhorn syndrome (WHS). The cardinal feature of WHS is a craniofacial dysmorphism, which is accompanied by sensorineural hearing loss in 15% of individuals with WHS. Here, we show that WHSC1-deficient mice display craniofacial defects that overlap with WHS, including cochlea anomalies. Although auditory hair cells are specified normally, their stereocilia hair bundles required for sound perception fail to develop the appropriate morphology. Furthermore, the orientation and cellular organisation of cochlear hair cells and their innervation are defective. These findings identify, for the first time, the likely cause of sensorineural hearing loss in individuals with WHS. © 2015. Published by The Company of Biologists Ltd.

  8. Nondestructive method for detecting defects in photodetector and solar cell devices

    Science.gov (United States)

    Not Available

    The invention described herein is a method for locating semiconductor device defects and for measuring the internal resistance of such devices by making use of the intrinsic distributed resistance nature of the devices. The method provides for forward-biasing a solar cell or other device while it is scanning with an optical spot. The forward-biasing is achieved with either an illuminator light source or an external current source.

  9. Defects and Interfaces on PtPb Nanoplates Boost Fuel Cell Electrocatalysis.

    Science.gov (United States)

    Sun, Yingjun; Liang, Yanxia; Luo, Mingchuan; Lv, Fan; Qin, Yingnan; Wang, Lei; Xu, Chuan; Fu, Engang; Guo, Shaojun

    2018-01-01

    Nanostructured Pt is the most efficient single-metal catalyst for fuel cell technology. Great efforts have been devoted to optimizing the Pt-based alloy nanocrystals with desired structure, composition, and shape for boosting the electrocatalytic activity. However, these well-known controls still show the limited ability in maximizing the Pt utilization efficiency for achieving more efficient fuel cell catalysis. Herein, a new strategy for maximizing the fuel cell catalysis by controlling/tuning the defects and interfaces of PtPb nanoplates using ion irradiation technique is reported. The defects and interfaces on PtPb nanoplates, controlled by the fluence of incident C + ions, make them exhibit the volcano-like electrocatalytic activity for methanol oxidation reaction (MOR), ethanol oxidation reaction (EOR), and oxygen reduction reaction (ORR) as a function of ion irradiation fluence. The optimized PtPb nanoplates with the mixed structure of dislocations, subgrain boundaries, and small amorphous domains are the most active for MOR, EOR, and ORR. They can also maintain high catalytic stability in acid solution. This work highlights the impact and significance of inducing/controlling the defects and interfaces on Pt-based nanocrystals toward maximizing the catalytic performance by advanced ion irradiation strategy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

    Science.gov (United States)

    George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

  11. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

    Science.gov (United States)

    Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

    2017-11-24

    To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  12. Recombination in Perovskite Solar Cells: Significance of Grain Boundaries, Interface Traps, and Defect Ions.

    Science.gov (United States)

    Sherkar, Tejas S; Momblona, Cristina; Gil-Escrig, Lidón; Ávila, Jorge; Sessolo, Michele; Bolink, Henk J; Koster, L Jan Anton

    2017-05-12

    Trap-assisted recombination, despite being lower as compared with traditional inorganic solar cells, is still the dominant recombination mechanism in perovskite solar cells (PSCs) and limits their efficiency. We investigate the attributes of the primary trap-assisted recombination channels (grain boundaries and interfaces) and their correlation to defect ions in PSCs. We achieve this by using a validated device model to fit the simulations to the experimental data of efficient vacuum-deposited p-i-n and n-i-p CH 3 NH 3 PbI 3 solar cells, including the light intensity dependence of the open-circuit voltage and fill factor. We find that, despite the presence of traps at interfaces and grain boundaries (GBs), their neutral (when filled with photogenerated charges) disposition along with the long-lived nature of holes leads to the high performance of PSCs. The sign of the traps (when filled) is of little importance in efficient solar cells with compact morphologies (fused GBs, low trap density). On the other hand, solar cells with noncompact morphologies (open GBs, high trap density) are sensitive to the sign of the traps and hence to the cell preparation methods. Even in the presence of traps at GBs, trap-assisted recombination at interfaces (between the transport layers and the perovskite) is the dominant loss mechanism. We find a direct correlation between the density of traps, the density of mobile ionic defects, and the degree of hysteresis observed in the current-voltage ( J - V ) characteristics. The presence of defect states or mobile ions not only limits the device performance but also plays a role in the J - V hysteresis.

  13. Radiofrequency electromagnetic radiation from cell phone causes defective testicular function in male Wistar rats.

    Science.gov (United States)

    Oyewopo, A O; Olaniyi, S K; Oyewopo, C I; Jimoh, A T

    2017-12-01

    Cell phones have become an integral part of everyday life. As cell phone usage has become more widespread, concerns have increased regarding the harmful effects of radiofrequency electromagnetic radiation from these devices. The current study was undertaken to investigate the effects of the emitted radiation by cell phones on testicular histomorphometry and biochemical analyses. Adult male Wistar rats weighing 180-200 g were randomly allotted to control, group A (switched off mode exposure), group B (1-hr exposure), group C (2-hr exposure) and group D (3-hr exposure). The animals were exposed to radiofrequency electromagnetic radiation of cell phone for a period of 28 days. Histomorphometry, biochemical and histological investigations were carried out. The histomorphometric parameters showed no significant change (p electromagnetic radiation of cell phone leads to defective testicular function that is associated with increased oxidative stress and decreased gonadotropic hormonal profile. © 2017 Blackwell Verlag GmbH.

  14. Oncometabolite D-2-Hydroxyglutarate Inhibits ALKBH DNA Repair Enzymes and Sensitizes IDH Mutant Cells to Alkylating Agents.

    Science.gov (United States)

    Wang, Pu; Wu, Jing; Ma, Shenghong; Zhang, Lei; Yao, Jun; Hoadley, Katherine A; Wilkerson, Matthew D; Perou, Charles M; Guan, Kun-Liang; Ye, Dan; Xiong, Yue

    2015-12-22

    Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

    Science.gov (United States)

    Piekarowicz, Katarzyna; Machowska, Magdalena; Dratkiewicz, Ewelina; Lorek, Daria; Madej-Pilarczyk, Agnieszka; Rzepecki, Ryszard

    2017-08-01

    LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

  16. Renal Operational Tolerance Is Associated With a Defect of Blood Tfh Cells That Exhibit Impaired B Cell Help.

    Science.gov (United States)

    Chenouard, A; Chesneau, M; Bui Nguyen, L; Le Bot, S; Cadoux, M; Dugast, E; Paul, C; Malard-Castagnet, S; Ville, S; Guérif, P; Soulillou, J-P; Degauque, N; Danger, R; Giral, M; Brouard, S

    2017-06-01

    Renal operationally tolerant patients (TOL) display a defect in B cell differentiation, with a deficiency in plasma cells. Recently described, T follicular helper (Tfh) cells play a critical role in B cell differentiation. We analyzed blood Tfh subsets in TOL and transplanted patients with stable graft function under immunosuppression (STA). We observed a reduced proportion of blood activated and highly functional Tfh subsets in TOL, without affecting Tfh absolute numbers. Functionally, Tfh cells from TOL displayed a modified gene expression profile, failed to produce interleukin-21, and were unable to induce IgG production by naive B cells. This Tfh defect is linked to a low incidence of postgraft de novo donor-specific antibody (dnDSA) immunization, suggesting that the lack of Tfh cells in TOL may induce a protolerogenic environment with reduced risk of developing dnDSA. Finally, we showed that elevated Tfh in STA precedes the occurrence of dnDSA during an alloresponse. These data provide new insights into the mechanisms of antibody response in operational tolerance. Disrupted homeostasis and impaired Tfh function in TOL could lead to a reduced risk of developing dnDSA and suggest a predictive role of blood Tfh cells on the occurrence of dnDSA in transplant recipients. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  17. Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions

    Science.gov (United States)

    Waldren, C.; Vannais, D.; Drabek, R.; Gustafson, D.; Kraemer, S.; Lenarczyk, M.; Kronenberg, A.; Hei, T.; Ueno, A.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

  18. Improved defect analysis of Gallium Arsenide solar cells using image enhancement

    Science.gov (United States)

    Kilmer, Louis C.; Honsberg, Christiana; Barnett, Allen M.; Phillips, James E.

    1989-01-01

    A new technique has been developed to capture, digitize, and enhance the image of light emission from a forward biased direct bandgap solar cell. Since the forward biased light emission from a direct bandgap solar cell has been shown to display both qualitative and quantitative information about the solar cell's performance and its defects, signal processing techniques can be applied to the light emission images to identify and analyze shunt diodes. Shunt diodes are of particular importance because they have been found to be the type of defect which is likely to cause failure in a GaAs solar cell. The presence of a shunt diode can be detected from the light emission by using a photodetector to measure the quantity of light emitted at various current densities. However, to analyze how the shunt diodes affect the quality of the solar cell the pattern of the light emission must be studied. With the use of image enhancement routines, the light emission can be studied at low light emission levels where shunt diode effects are dominant.

  19. Specific TP53 Mutants Overrepresented in Ovarian Cancer Impact CNV, TP53 Activity, Responses to Nutlin-3a, and Cell Survival

    Directory of Open Access Journals (Sweden)

    Lisa K. Mullany

    2015-10-01

    Full Text Available Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC. As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17; copy number variation (R175H; chromosome 9; and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31% of HGSCs exhibit loss of heterozygosity, a significant number (24% maintain a wild-type (WT allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing “proof of principle” that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.

  20. Are Soft Short Tests Good Indicators of Internal Li-ion Cell Defects?

    Science.gov (United States)

    Jeevarajan, J.; Chung, J.-S.; Jung, K.; Park, J.

    2013-01-01

    The self discharge test at full state of charge, may not be a good one to detect subtle defects since the li-ion chemistry has the highest self discharge at full state of charge. One should characterize self discharge versus storage time for each cell manufacturer/design to differentiate between normal self discharge and that due to a subtle manufacturing defect. The various soft short test methods indicate that if this test is carried out at full discharge (0% SOC) with all capacity removed (by lowering the current load in a stepwise manner to the same end of discharge voltage), then the cells need to be placed in storage for more than 72 hours to get a good analysis on the presence of subtle defects since it takes more than 72 hours to achieve voltage stabilization. If the cells are to be charged up even to a small percentage (ex. 1%), 72 hours are sufficient to determine issues. However, the pass/fail criteria should be based on a valid OCV decline. Less than 10 mV voltage decline is not a good method to detect subtle defects. As mentioned in the first bullet, self discharge is a competing reaction when a charge is introduced and hence a characterization of the self discharge versus storage time is required to fully correlate voltage decline to a failure due to a subtle defect. Soft short test method cannot be relied on for defect detection because cells with and without voltage decline seemed to have similar defects and characteristics. Screening methods such as internal resistance and capacity as well as a 3-sigma range for OCV, mass and dimensions should be used to screen out outliers. A very critical aspect in the understanding of subtle defects is to carry out destructive analysis of cells from every lot to confirm the quality of production and screen all cells and batteries in a stringent manner to have a high quality set of flight cells. Self Discharge Test: Fully charged cells shall be placed in Open circuit stand for 72 hours (OCV measurement twice a

  1. Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency.

    Science.gov (United States)

    Vliet, Lydia K; Wilkinson, Terry G; Duval, Nathan; Vacano, Guido; Graham, Christine; Zikánová, Marie; Skopova, Vaclava; Baresova, Veronika; Hnízda, Aleš; Kmoch, Stanislav; Patterson, David

    2011-01-01

    Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Analysis of electronic models for solar cells including energy resolved defect densities

    Energy Technology Data Exchange (ETDEWEB)

    Glitzky, Annegret

    2010-07-01

    We introduce an electronic model for solar cells including energy resolved defect densities. The resulting drift-diffusion model corresponds to a generalized van Roosbroeck system with additional source terms coupled with ODEs containing space and energy as parameters for all defect densities. The system has to be considered in heterostructures and with mixed boundary conditions from device simulation. We give a weak formulation of the problem. If the boundary data and the sources are compatible with thermodynamic equilibrium the free energy along solutions decays monotonously. In other cases it may be increasing, but we estimate its growth. We establish boundedness and uniqueness results and prove the existence of a weak solution. This is done by considering a regularized problem, showing its solvability and the boundedness of its solutions independent of the regularization level. (orig.)

  3. Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

    International Nuclear Information System (INIS)

    Takubo, Keiyo; Hirao, Atsushi; Ohmura, Masako; Azuma, Masaki; Arai, Fumio; Nagamatsu, Go; Suda, Toshio

    2006-01-01

    Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16 Ink4a activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16 Ink4a -mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16 Ink4a -mediated senescence

  4. Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

    Science.gov (United States)

    De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

    2017-09-01

    Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics. Copyright © 2017 American Society for Microbiology.

  5. Tissue- and cell-specific mitochondrial defect in Parkin-deficient mice.

    Directory of Open Access Journals (Sweden)

    Maria Damiano

    Full Text Available Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be

  6. Cyr61/CCN1 and CTGF/CCN2 mediate the proangiogenic activity of VHL-mutant renal carcinoma cells.

    Science.gov (United States)

    Chintalapudi, Mastan R; Markiewicz, Margaret; Kose, Nurgun; Dammai, Vincent; Champion, Kristen J; Hoda, Rana S; Trojanowska, Maria; Hsu, Tien

    2008-04-01

    The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)-alpha subunits. Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic therapy specifically targeting VEGF signaling remains modest. In this study, we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2alpha function, the dominant HIF-alpha isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.

  7. Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

    Directory of Open Access Journals (Sweden)

    Schulz Burkhard

    2010-12-01

    Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

  8. Contribution of Implanted, Genetically Modified Muscle Progenitor Cells Expressing BMP-2 to New Bone Formation in a Rat Osseous Defect.

    Science.gov (United States)

    De La Vega, Rodolfo E; De Padilla, Consuelo Lopez; Trujillo, Miguel; Quirk, Nicholas; Porter, Ryan M; Evans, Christopher H; Ferreira, Elisabeth

    2018-01-03

    Because muscle contains osteoprogenitor cells and has a propensity to form bone, we have explored its utility in healing large osseous defects. Healing is achieved by the insertion of muscle fragments transduced with adenovirus encoding BMP-2 (Ad.BMP-2). However, it is not known whether the genetically modified muscle contributes osteoprogenitor cells to healing defects or merely serves as a local source of BMP-2. This question is part of the larger debate on the fate of progenitor cells introduced into sites of tissue damage to promote regeneration. To address this issue, we harvested fragments of muscle from rats constitutively expressing GFP, transduced them with Ad.BMP-2, and implanted them into femoral defects in wild-type rats under various conditions. GFP + cells persisted within defects for the entire 8 weeks of the experiments. In the absence of bone formation, these cells presented as fibroblasts. When bone was formed, GFP + cells were present as osteoblasts and osteocytes and also among the lining cells of new blood vessels. The genetically modified muscle thus contributed progenitor cells as well as BMP-2 to the healing defect, a property of great significance in light of the extensive damage to soft tissue and consequent loss of endogenous progenitors in problematic fractures. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  9. A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

    Science.gov (United States)

    Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

    1996-01-01

    We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

  10. Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

    Science.gov (United States)

    Saqcena, M; Mukhopadhyay, S; Hosny, C; Alhamed, A; Chatterjee, A; Foster, D A

    2015-05-14

    Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

  11. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    we have developed models of mutant ER driven cancer in which to characterize gene expression. Specifically, tetracycline inducible MCF7 cells that...expression profiling. Figure 1: Degradation of WT and mutant ER with ARN810. MCF7 cells stably transfected with tet-inducible WT and mutant...mutant ER. MCF7 cells stably transfected with tet- inducible WT and mutant ER are treated for 24 hours with estradiol and gene expression profiling

  12. Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette Tina Marie [Los Alamos National Laboratory

    2008-01-01

    The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

  13. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike

    2006-01-01

    derived from cHL are rare and therefore not notably involved in the pathogenesis of the malignant H&RS cells. Re-evaluating the expression in cHL-derived cell lines, we found that in 3/6 of these cell lines, TP53 transcripts are characterized by deletions within exon 4 (L428 cells) and nearly a complete...

  14. In vitro chondrogenesis and in vivo repair of osteochondral defect with human induced pluripotent stem cells.

    Science.gov (United States)

    Ko, Ji-Yun; Kim, Kyung-Il; Park, Siyeon; Im, Gun-Il

    2014-04-01

    The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells, chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG, SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture, greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9, type II collagen, and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen, type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state, suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects, and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Zezhang T Wen

    Full Text Available Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans.

  16. Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

    Directory of Open Access Journals (Sweden)

    Péter Nagy

    2016-05-01

    Full Text Available UV radiation resistance-associated gene (UVRAG is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent or late (Rab7-dependent steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans.

  17. Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects.

    Science.gov (United States)

    Nuñez, Javier; Sanchez, Nerea; Vignoletti, Fabio; Sanz-Martin, Ignacio; Caffesse, Raul; Santamaria, Silvia; Garcia-Sanz, Jose A; Sanz, Mariano

    2017-12-29

    The objective of this in vivo experimental study to evaluate the regenerative potential of a cell therapy combining allogenic periodontal ligament-derived cells within a xenogeneic bone substitute in a similar experimental model. In nine beagle dogs, critical size 6-mm supra-alveolar periodontal defects were created around the PIII and PIV. The resulting supra-alveolar defects were randomly treated with either 1.4 × 10 6 allogenic canine periodontal ligament-derived cells seeded on de-proteinized bovine bone mineral with 10% collagen (DBBM-C) (test group) or DBBM-C without cells (control group). Specimens were obtained at 3 months, and histological outcomes were studied. The histological analysis showed that total furcation closure occurred very seldom in both groups, being the extent of periodontal regeneration located in the apical third of the defect. The calculated amount of periodontal regeneration at the furcation area was comparable in both the test and control groups (1.93 ± 1.14 mm (17%) versus 2.35 ± 1.74 mm (22%), respectively (p = .37). Similarly, there were no significant differences in the amount of new cementum formation 4.49 ± 1.56 mm (41%) versus 4.97 ± 1.05 mm (47%), respectively (p = .45). This experimental study was unable to demonstrate the added value of allogenic cell therapy in supra-crestal periodontal regeneration. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Rattner Jerome B

    2009-12-01

    Full Text Available Abstract Background Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines. Methods Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis. Results We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis. Conclusions The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first

  19. Zygomatic air cell defect: A panoramic radiographic study of a south Indian population

    International Nuclear Information System (INIS)

    Hs, Srikanth; Patil, Karthikeya; Vg, Mahima

    2010-01-01

    To determine the prevalence, patterns of occurrence and variations of zygomatic air cell defects (ZACDs) using panoramic radiographs. Dental panoramic radiographs of 600 outpatients were examined to evaluate the variations and characteristics of ZACDs. ZACDs were identified in 15 subjects out of 600, giving an overall prevalence of 2.5%. Seven ZACDs were seen in males and eight in females. Among the 15 ZACDs, nine were unilateral and six were bilateral. The overall prevalence of ZACD is relatively low in south Indian population and careful radiographic evaluation is needed to detect these entities

  20. A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

    Directory of Open Access Journals (Sweden)

    S.C. Silva

    2010-02-01

    Full Text Available Bovine herpesvirus 5 (BoHV-5, the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99. The recombinants are defective in glycoprotein E (BoHV-5gEΔ, thymidine kinase (BoHV-5TKΔ and both proteins (BoHV-5gEΔTKΔ. Rabbits inoculated with the parental virus (N = 8 developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi. Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8 or BoHV-5gEΔTKΔ (N = 8 remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

  1. Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

    OpenAIRE

    Zhu, Z; DeLuca, N A; Schaffer, P A

    1996-01-01

    ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it h...

  2. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

    Directory of Open Access Journals (Sweden)

    Luyuan Li

    Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

  3. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira

    2007-01-01

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53ΔC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53ΔC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain

  4. Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

    Science.gov (United States)

    2010-01-01

    by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. The opinions, interpretations, conclusions, and... detergent -resistant microdomains on internal host cell mem- branes. Mol. Microbiol. 49:685–704. 30. Leiman, P. G., M. Basler, U. A. Ramagopal, J. B

  5. Irradiated fetal thymus transplantation in a patient with combined immunodeficiency with predominant T cell defect

    International Nuclear Information System (INIS)

    Higuchi, Shigenori; Yanabe, Yasuhide; Tsuchiya, Hiroyuki; Akahoshi, Izumi; Migita, Masahiro; Matsuda, Ichiro; Udaka, Keiji.

    1993-01-01

    A 6 month old boy was diagnosed as a case of combined immunodeficiency (with predominant T cell defect by previous classification). His T cell count was decreased, his B cell count in peripheral blood was increased, his serum IgG level was decreased, his serum IgM level was normal and the thymus was not evident on CT scans and magnetic resonance imaging. Administration of the thymus hormone, thymosin, led to a partial recovery of T cell function without normalization of the T cell count. At age 26 months the patient received an irradiated thymus transplantation from a 16 week old female fetus. After the transplantation, the T cell count (mainly CD4 + cells) increased by 50-70%. A mild graft-versus-host reaction (GVHR) occurred and several immunosuppressants were prescribed. Chromosome analysis showed that the T cells have both 46 XY and 46 XX karyotypes while the B cells have the 46 XY karyotype alone. His cellular immunity (skin tests, DNA synthesis, mixed lymphocyte reaction, cytotoxic activity and natural killer cell function) and his serum IgG level remained low. However, being on regular γ-globulin therapy and oral anti-fungal drugs, he is now living normally with almost no trouble at age 6 years and 3 months. This case showed that irradiated thymus transplantation might be a useful method when an adequate donor for bone marrow transplantation is not available. The unexpected observation that the increased T cells were mainly CD4 may be related to the mild GVHR and the clinical improvement. (author)

  6. Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

    Science.gov (United States)

    Barbaro, Vanessa; Nasti, Annamaria Assunta; Raffa, Paolo; Migliorati, Angelo; Nespeca, Patrizia; Ferrari, Stefano; Palumbo, Elisa; Bertolin, Marina; Breda, Claudia; Miceli, Francesco; Russo, Antonella; Caenazzo, Luciana; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-08-01

    : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative

  7. Macular Ganglion Cell Inner Plexiform Layer Thickness in Glaucomatous Eyes with Localized Retinal Nerve Fiber Layer Defects.

    Directory of Open Access Journals (Sweden)

    Chunwei Zhang

    Full Text Available To investigate macular ganglion cell-inner plexiform layer (mGCIPL thickness in glaucomatous eyes with visible localized retinal nerve fiber layer (RNFL defects on stereophotographs.112 healthy and 149 glaucomatous eyes from the Diagnostic Innovations in Glaucoma Study (DIGS and the African Descent and Glaucoma Evaluation Study (ADAGES subjects had standard automated perimetry (SAP, optical coherence tomography (OCT imaging of the macula and optic nerve head, and stereoscopic optic disc photography. Masked observers identified localized RNFL defects by grading of stereophotographs.47 eyes had visible localized RNFL defects on stereophotographs. Eyes with visible localized RNFL defects had significantly thinner mGCIPL thickness compared to healthy eyes (68.3 ± 11.4 μm versus 79.2 ± 6.6 μm respectively, P<0.001 and similar mGCIPL thickness to glaucomatous eyes without localized RNFL defects (68.6 ± 11.2 μm, P = 1.000. The average mGCIPL thickness in eyes with RNFL defects was 14% less than similarly aged healthy controls. For 29 eyes with a visible RNFL defect in just one hemiretina (superior or inferior mGCIPL was thinnest in the same hemiretina in 26 eyes (90%. Eyes with inferior-temporal RNFL defects also had significantly thinner inferior-temporal mGCIPL (P<0.001 and inferior mGCIPL (P = 0.030 compared to glaucomatous eyes without a visible RNFL defect.The current study indicates that presence of a localized RNFL defect is likely to indicate significant macular damage, particularly in the region of the macular that topographically corresponds to the location of the RNFL defect.

  8. External fixation of femoral defects in athymic rats: Applications for human stem cell implantation and bone regeneration

    Directory of Open Access Journals (Sweden)

    Terasa Foo

    2013-01-01

    Full Text Available An appropriate animal model is critical for the research of stem/progenitor cell therapy and tissue engineering for bone regeneration in vivo. This study reports the design of an external fixator and its application to critical-sized femoral defects in athymic rats. The external fixator consists of clamps and screws that are readily available from hardware stores as well as Kirschner wires. A total of 35 rats underwent application of the external fixator with creation of a 6-mm bone defect in one femur of each animal. This model had been used in several separate studies, including implantation of collagen gel, umbilical cord blood mesenchymal stem cells, endothelial progenitor cells, or bone morphogenetic protein-2. One rat developed fracture at the proximal pin site and two rats developed deep tissue infection. Pin loosening was found in nine rats, but it only led to the failure of external fixation in two animals. In 8 to 10 weeks, various degrees of bone growth in the femoral defects were observed in different study groups, from full repair of the bone defect with bone morphogenetic protein-2 implantation to fibrous nonunion with collagen gel implantation. The external fixator used in these studies provided sufficient mechanical stability to the bone defects and had a comparable complication rate in athymic rats as in immunocompetent rats. The external fixator does not interfere with the natural environment of a bone defect. This model is particularly valuable for investigation of osteogenesis of human stem/progenitor cells in vivo.

  9. Cell-Based Gene Therapy for Repair of Critical Size Defects in the Rat Fibula

    Science.gov (United States)

    Lazard, ZaWaunyka W.; Heggeness, Michael H.; Hipp, John A.; Sonnet, Corinne; Fuentes, Angie S.; Nistal, Rita P.; Davis, Alan R.; Olabisi, Ronke M.; West, Jennifer L.; Olmsted-Davis, Elizabeth A.

    2012-01-01

    More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies. PMID:21344484

  10. A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus.

    Science.gov (United States)

    Barateau, Alice; Vadrot, Nathalie; Vicart, Patrick; Ferreiro, Ana; Mayer, Michèle; Héron, Delphine; Vigouroux, Corinne; Buendia, Brigitte

    2017-01-01

    A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.

  11. Association of mutator activity with UV sensitivity in an aphidicolin-resistant mutant of Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Liu, P.K.; Chang, C.; Trosko, J.E.

    1982-01-01

    The spontaneous mutation rates of an ultraviolet light (UV)-sensitive aphidicolin-resistant mutant (aphsup(r)-4-2) and its revertants have been determined by 2 techniques. By using the fluctuation analysis, the mutant and its thymidine (TdR)-prototrophic 'revertant' were found to exhibit elevated spontaneous mutation rates at the 6-thioguanine- and diphtheria-toxin-resistant loci. In contrast, the TdR-auxotrophic 'revertant' did not show this property. Similar results were obtained by the multiple replating technique. From these comparative studies and other previous characterizations, it appears that a single gene mutation is responsible for the following pleiotropic phenotype: slow growth, UV sensitivity, high UV-induced mutability, high frequency of site-specific bromodeoxyuridine (BrdU)-dependent chromosome breaks and enhanced spontaneous mutation rate. Recent studies indicate that the mutation may be on the gene for DNA polymerase α. The results further indicate that thymidine auxotrophy or imbalance in nucleotide pools is not necessarily associated with the mutator activity in mammalian cells. (orig.)

  12. Aumento de linfocitos T regulatorios en los ganglios linfoides de ratones mutantes para catepsina L Increase of regulatory T cells in the lymph node of cathepsin L mutant mice

    Directory of Open Access Journals (Sweden)

    Gabriela Camicia

    2011-08-01

    Full Text Available Las células T regulatorias CD4+CD25+Foxp3+ (Treg han sido implicadas en el rechazo al trasplante de órganos alogeneicos, en cáncer, infecciones y enfermedades autoinmunes. Así, su modulación posee un enorme potencial para el tratamiento de estas enfermedades. En este trabajo se evaluó la influencia de la catepsina L (CTSL sobre la homeostasis de las células Treg. Los ratones CTSLnkt/nkt -mutantes para CTSLpresentaron una reducción en el número de células Treg en el timo. Contrariamente, en los ganglios linfáticos el número de células Treg y su frecuencia dentro de la población CD4+ se incrementaron. La ausencia de actividad de CTSL en las células CD4+ de los ganglios linfoides -y no en su entorno- incrementó su tasa de proliferación. Las células Treg y las T CD4+ convencionales (CD4+CD25-Foxp3- de los ratones mutantes mostraron aumentos similares en su tasa de proliferación, sugiriendo que la proliferación contribuye al incremento en el número de células Treg, pero el aumento en su frecuencia no derivaría exclusivamente del incremento en su proliferación. Por otra parte, no se observó una disminución en los niveles de apoptosis de células Treg. Teniendo en cuenta que la producción diaria de timocitos CD4+ está disminuida en los ratones CTSLnkt/nkt, estos resultados sugieren que el incremento de células Treg en los ganglios linfoides de estos mutantes no derivaría de una mayor exportación tímica, y permiten hipotetizar que la ausencia de actividad de CTSL favorecería la adquisición del fenotipo CD4+CD25+Foxp3+ a partir de células CD4+CD25-Foxp3- en la periferia.Regulatory CD4+CD25+Foxp3+ T cells (Treg have been implicated in different pathologies including cancer, infections and autoimmune diseases and in the rejection of allogeneic organ transplantation. Thus, modulation of Treg activity has a great potential in the treatment of these pathologies. Herein, we evaluated the influence of cathepsin L (CTSL on Treg

  13. Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Tadafumi Yokoyama

    Full Text Available The Wiskott-Aldrich syndrome (WAS is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg obtained from Was gene knockout (WKO mice and found that their numbers were significantly lower in these mice compared to wild type (WT controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

  14. Rapid detection of defects in fuel-cell electrodes using infrared reactive-flow-through technique

    Science.gov (United States)

    Das, Prodip K.; Weber, Adam Z.; Bender, Guido; Manak, Austin; Bittinat, Daniel; Herring, Andrew M.; Ulsh, Michael

    2014-09-01

    As fuel cells become more prominent, new manufacturing and production methods will need to be developed to deal efficiently and effectively with increased demand. One necessary component of this industrial growth is the accurate measurement of the variability in the manufacturing process. In this study, we present a diagnostic system that combines infrared thermography with a reactive-flow-through technique to detect catalyst-loading defects in fuel-cell gas-diffusion electrodes accurately with high spatial and temporal resolutions. Experimental results are compared with model predictions of thermal response with good agreement. Data analysis, operating-condition impacts, and detection limits are explored using both experiments and simulation. Overall, the results demonstrate the potential of this technique to measure defects on the millimeter length scale with temporal resolutions appropriate for use on a web-line. Thus we present the first development stage of a next-generation non-destructive diagnostic tool, which may be amenable to eventual use on roll-to-roll manufacturing lines.

  15. A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

    Directory of Open Access Journals (Sweden)

    Ioannis Gavvovidis

    Full Text Available Biallelic mutations in MCPH1 cause primary microcephaly (MCPH with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL and a second transcript lacking the six 3' exons (MCPH1Δe9-14. Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.

  16. Quantitative Analysis of Defects in Silicon. [to predict energy conversion efficiency of silicon samples for solar cells

    Science.gov (United States)

    Natesh, R.; Smith, J. M.; Qidwai, H. A.; Bruce, T.

    1979-01-01

    The evaluation and prediction of the conversion efficiency for a variety of silicon samples with differences in structural defects, such as grain boundaries, twin boundaries, precipitate particles, dislocations, etc. are discussed. Quantitative characterization of these structural defects, which were revealed by etching the surface of silicon samples, is performed by using an image analyzer. Due to different crystal growth and fabrication techniques the various types of silicon contain a variety of trace impurity elements and structural defects. The two most important criteria in evaluating the various silicon types for solar cell applications are cost and conversion efficiency.

  17. T-cell dysfunction in HIV infection: anergy due to defective antigen-presenting cell function?

    NARCIS (Netherlands)

    Meyaard, L.; Schuitemaker, H.; Miedema, F.

    1993-01-01

    Before CD4+ T cells are depleted, T cells in asymptomatic HIV-infected individuals are functionally abnormal. These T cells are programmed for death, are non-responsive and fail to produce interleukin-2 after antigenic stimulation. Our view is that these different T-cell abnormalities are explained

  18. Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

    Science.gov (United States)

    Beggah, Siham; Vogne, Christelle; Zenaro, Elena; Van Der Meer, Jan Roelof

    2008-01-01

    Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-dependent transcription activators, with the purpose of changing its effector recognition specificity from 2-hydroxybiphenyl (2-HBP, the cognate effector) to 2-chlorobiphenyl (2-CBP). Mutations were introduced in the hbpR gene part for the A-domain via error-prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR-dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2-CBP were enriched and separated in a flow cytometry-assisted cell-sorting procedure. Some 70 mutants were isolated and the A-domain mutations mapped. One of these had acquired true 2-CBP recognition but reacted hypersensitively to 2-HBP (20-fold more than the wild type), whereas others had reduced sensitivity to 2-HBP but a gain of 2-CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A-domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2-CBP or reduced 2-HBP recognition. Our data thus demonstrate that a one-step in vitro 'evolutionary' adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition.

  19. Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

    International Nuclear Information System (INIS)

    Hu, Bo; He, Xiusheng; Li, Ao; Qiu, Qingchao; Li, Cunxi; Liang, Dan; Zhao, Ping; Ma, Jie; Coffey, Robert J.; Zhan, Qimin; Wu, Guanqing

    2011-01-01

    Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1 -/- renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1 -/- cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1 -/- mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1 -/- kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

  20. BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer.

    Science.gov (United States)

    Karachaliou, Niki; Codony-Servat, Jordi; Teixidó, Cristina; Pilotto, Sara; Drozdowskyj, Ana; Codony-Servat, Carles; Giménez-Capitán, Ana; Molina-Vila, Miguel Angel; Bertrán-Alamillo, Jordi; Gervais, Radj; Massuti, Bartomeu; Morán, Teresa; Majem, Margarita; Felip, Enriqueta; Carcereny, Enric; García-Campelo, Rosario; Viteri, Santiago; González-Cao, María; Morales-Espinosa, Daniela; Verlicchi, Alberto; Crisetti, Elisabetta; Chaib, Imane; Santarpia, Mariacarmela; Luis Ramírez, José; Bosch-Barrera, Joaquim; Felipe Cardona, Andrés; de Marinis, Filippo; López-Vivanco, Guillermo; Miguel Sánchez, José; Vergnenegre, Alain; Sánchez Hernández, José Javier; Sperduti, Isabella; Bria, Emilio; Rosell, Rafael

    2015-12-07

    BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.

  1. BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer

    Science.gov (United States)

    Karachaliou, Niki; Codony-Servat, Jordi; Teixidó, Cristina; Pilotto, Sara; Drozdowskyj, Ana; Codony-Servat, Carles; Giménez-Capitán, Ana; Molina-Vila, Miguel Angel; Bertrán-Alamillo, Jordi; Gervais, Radj; Massuti, Bartomeu; Morán, Teresa; Majem, Margarita; Felip, Enriqueta; Carcereny, Enric; García-Campelo, Rosario; Viteri, Santiago; González-Cao, María; Morales-Espinosa, Daniela; Verlicchi, Alberto; Crisetti, Elisabetta; Chaib, Imane; Santarpia, Mariacarmela; Luis Ramírez, José; Bosch-Barrera, Joaquim; Felipe Cardona, Andrés; de Marinis, Filippo; López-Vivanco, Guillermo; Miguel Sánchez, José; Vergnenegre, Alain; Sánchez Hernández, José Javier; Sperduti, Isabella; Bria, Emilio; Rosell, Rafael

    2015-01-01

    BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR. PMID:26639561

  2. Isolation and characterization of mink lung epithelial cell mutants resistant to transforming growth factor β

    International Nuclear Information System (INIS)

    Chinkers, M.

    1987-01-01

    Mink lung epithelial cells resistant to growth inhibition by transforming growth factor β (TGF-β) have been isolated by chemical mutagenesis and growth in the presence of platelet extracts enriched in TGF-β. Several resistant clones were isolated, at least one of which stably retained its resistance to TGF-β when grown in the absence of the factor. The cells of this clone were similar to the parent cells in morphology and growth properties. However, unlike the parent cells, the resistant cells did not show any of the following responses to 125 I TGF-β: (1) inhibition of DNA synthesis and proliferation; (2) morphological changes involving increased cell spreading; or (3) stimulation of synthesis of a 48-kilodalton secreted 35 S-protein. The resistant cells do, however, retain a functional TGF-β receptor. The TGF-β resistant cell lines may be useful in genetic studies designed to identify the biochemical events required for inhibition of epithelial cell growth by this factor

  3. Pathogenic mitochondrial DNA-induced respiration defects in hematopoietic cells result in anemia by suppressing erythroid differentiation.

    Science.gov (United States)

    Inoue, Shin-Ichi; Yokota, Mutsumi; Nakada, Kazuto; Miyoshi, Hiroyuki; Hayashi, Jun-Ichi

    2007-05-01

    Anemia is a symptom in patients with Pearson syndrome caused by the accumulation of mutated mitochondrial DNA (mtDNA). Such mutated mtDNAs have been detected in patients with anemia. This suggested that respiration defects due to mutated mtDNA are responsible for the anemia. However, there has been no convincing experimental evidence to confirm the pathophysiological relation between respiration defects in hematopoietic cells and expression of anemia. We address this issue by transplanting bone marrow cells carrying pathogenic mtDNA with a large-scale deletion (DeltamtDNA) into normal mice. The bone marrow-transplanted mice carried high proportion of DeltamtDNA only in hematopoietic cells, and resultant the mice suffered from macrocytic anemia. They show abnormalities of erythroid differentiation and weak erythropoietic response to a stressful condition. These observations suggest that hematopoietic cell-specific respiration defects caused by mtDNAs with pathogenic mutations are responsible for anemia by inducing abnormalities in erythropoiesis.

  4. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    Directory of Open Access Journals (Sweden)

    Andreia Michelle Smith-Moritz

    2015-08-01

    Full Text Available The CELLULOSE SYNTHASE-LIKE F6 (CslF6 gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG, a cell wall polysaccharide that is hypothesized to be a tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of three day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell was of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  5. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    Directory of Open Access Journals (Sweden)

    Hongping Jin

    2016-07-01

    Full Text Available Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1 fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA and JQ1 had no effect, while suberanilohydroxamic acid (SAHA modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA, but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

  6. Sister chromatid cohesion defects are associated with chromosome instability in Hodgkin lymphoma cells

    International Nuclear Information System (INIS)

    Sajesh, Babu V; Lichtensztejn, Zelda; McManus, Kirk J

    2013-01-01

    Chromosome instability manifests as an abnormal chromosome complement and is a pathogenic event in cancer. Although a correlation between abnormal chromosome numbers and cancer exist, the underlying mechanisms that cause chromosome instability are poorly understood. Recent data suggests that aberrant sister chromatid cohesion causes chromosome instability and thus contributes to the development of cancer. Cohesion normally functions by tethering nascently synthesized chromatids together to prevent premature segregation and thus chromosome instability. Although the prevalence of aberrant cohesion has been reported for some solid tumors, its prevalence within liquid tumors is unknown. Consequently, the current study was undertaken to evaluate aberrant cohesion within Hodgkin lymphoma, a lymphoid malignancy that frequently exhibits chromosome instability. Using established cytogenetic techniques, the prevalence of chromosome instability and aberrant cohesion was examined within mitotic spreads generated from five commonly employed Hodgkin lymphoma cell lines (L-1236, KM-H2, L-428, L-540 and HDLM-2) and a lymphocyte control. Indirect immunofluorescence and Western blot analyses were performed to evaluate the localization and expression of six critical proteins involved in the regulation of sister chromatid cohesion. We first confirmed that all five Hodgkin lymphoma cell lines exhibited chromosome instability relative to the lymphocyte control. We then determined that each Hodgkin lymphoma cell line exhibited cohesion defects that were subsequently classified into mild, moderate or severe categories. Surprisingly, ~50% of the mitotic spreads generated from L-540 and HDLM-2 harbored cohesion defects. To gain mechanistic insight into the underlying cause of the aberrant cohesion we examined the localization and expression of six critical proteins involved in cohesion. Although all proteins produced the expected nuclear localization pattern, striking differences in RAD21

  7. Universal Approach toward Hysteresis-Free Perovskite Solar Cell via Defect Engineering.

    Science.gov (United States)

    Son, Dae-Yong; Kim, Seul-Gi; Seo, Ja-Young; Lee, Seon-Hee; Shin, Hyunjung; Lee, Donghwa; Park, Nam-Gyu

    2018-01-31

    Organic-inorganic halide perovskite is believed to be a potential candidate for high efficiency solar cells because power conversion efficiency (PCE) was certified to be more than 22%. Nevertheless, mismatch of PCE due to current density (J)-voltage (V) hysteresis in perovskite solar cells is an obstacle to overcome. There has been much lively debate on the origin of J-V hysteresis; however, effective methodology to solve the hysteric problem has not been developed. Here we report a universal approach for hysteresis-free perovskite solar cells via defect engineering. A severe hysteresis observed from the normal mesoscopic structure employing TiO 2 and spiro-MeOTAD is almost removed or does not exist upon doping the pure perovskites, CH 3 NH 3 PbI 3 and HC(NH 2 ) 2 PbI 3 , and the mixed cation/anion perovskites, FA 0.85 MA 0.15 PbI 2.55 Br 0.45 and FA 0.85 MA 0.1 Cs 0.05 PbI 2.7 Br 0.3 , with potassium iodide. Substantial reductions in low-frequency capacitance and bulk trap density are measured from the KI-doped perovskite, which is indicative of trap-hysteresis correlation. A series of experiments with alkali metal iodides of LiI, NaI, KI, RbI and CsI reveals that potassium ion is the right element for hysteresis-free perovskite. Theoretical studies suggest that the atomistic origin of the hysteresis of perovskite solar cells is not the migration of iodide vacancy but results from the formation of iodide Frenkel defect. Potassium ion is able to prevent the formation of Frenkel defect since K + energetically prefers the interstitial site. A complete removal of hysteresis is more pronounced at mixed perovskite system as compared to pure perovskites, which is explained by lower formation energy of K interstitial (-0.65 V for CH 3 NH 3 PbI 3 vs -1.17 V for mixed perovskite). The developed KI doping methodology is universally adapted for hysteresis-free perovskite regardless of perovskite composition and device structure.

  8. Natural Killer Cells from Patients with Recombinase-Activating Gene and Non-Homologous End Joining Gene Defects Comprise a Higher Frequency of CD56bright NKG2A+++ Cells, and Yet Display Increased Degranulation and Higher Perforin Content

    Directory of Open Access Journals (Sweden)

    Kerry Dobbs

    2017-07-01

    Full Text Available Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2 in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

  9. Theoretical analysis of the influence of defect parameters on photovoltaic performances of composition graded InGaN solar cells

    International Nuclear Information System (INIS)

    Gorge, V.; Migan-Dubois, A.; Djebbour, Z.; Pantzas, K.; Gautier, S.; Moudakir, T.; Suresh, S.; Ougazzaden, A.

    2013-01-01

    Highlights: ► We have modeled a p–i–n InGaN-based solar cell with gradual bandgap layers. ► InGaN defects have been modeled by two band tails and one localized energy level. ► Energetic position and band tail widths have a low effect on device efficiency. ► The localized defect FWHM has a significant impact on performance. ► The efficiency drops radically when the defect density is higher than the P-doping. - Abstract: In this paper, we have used simulations to evaluate the impact of the distribution of electrically active defects on the photovoltaic performances of InGaN-based solar cell. The simulations were carried out using Silvaco's ATLAS software. We have modeled a P-GaN/Grad-InGaN/i-In 0.53 Ga 0.47 N/Grad-InGaN/N-ZnO where Grad-InGaN corresponds to an InGaN layer with a graded composition. This layer is inserted to eliminate the band discontinuities at the interface between InGaN and the GaN and ZnO layers. The defects were modeled through the introduction of band tails and a Gaussian distribution of defects in i-InGaN material. We have evaluated the influence of band tail widths as well as the parameters of the Gaussian distribution (i.e. defect density, mean position and standard deviation) on the short-circuit current, the open-circuit voltage and the fill-factor (efficiency) of the solar cell. These results have allowed us to identify key structural parameters useful for the optimization of InGaN solar cells, as well as to give realistic estimates of the performances of such cells.

  10. Theoretical analysis of the influence of defect parameters on photovoltaic performances of composition graded InGaN solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorge, V.; Migan-Dubois, A. [LGEP, UMR 8507, CNRS, SUPELEC, UPMC, Universite Paris-Sud 11, 11 rue Joliot-Curie, Plateau de Moulon, 91192 Gif-sur-Yvette Cedex (France); Djebbour, Z., E-mail: zakaria.djebbour@uvsq.fr [LGEP, UMR 8507, CNRS, SUPELEC, UPMC, Universite Paris-Sud 11, 11 rue Joliot-Curie, Plateau de Moulon, 91192 Gif-sur-Yvette Cedex (France); Department of Physics and Engineering Science, University of Versailles UVSQ, 45 Av. Des Etats Unis, 78035 Versailles (France); Pantzas, K. [Georgia Institute of Technology, GT-Lorraine, 2 rue Marconi, 57 070 Metz (France); UMI 2958 Georgia Tech, CNRS, 2 rue Marconi, 57 070 Metz (France); Gautier, S. [UMI 2958 Georgia Tech, CNRS, 2 rue Marconi, 57 070 Metz (France); LMOPS, UMR 7132, CNRS, University of Metz, Supelec, 2 rue E. Belin, 57 070 Metz (France); Moudakir, T.; Suresh, S. [UMI 2958 Georgia Tech, CNRS, 2 rue Marconi, 57 070 Metz (France); Ougazzaden, A. [Georgia Institute of Technology, GT-Lorraine, 2 rue Marconi, 57 070 Metz (France); UMI 2958 Georgia Tech, CNRS, 2 rue Marconi, 57 070 Metz (France)

    2013-02-01

    Highlights: Black-Right-Pointing-Pointer We have modeled a p-i-n InGaN-based solar cell with gradual bandgap layers. Black-Right-Pointing-Pointer InGaN defects have been modeled by two band tails and one localized energy level. Black-Right-Pointing-Pointer Energetic position and band tail widths have a low effect on device efficiency. Black-Right-Pointing-Pointer The localized defect FWHM has a significant impact on performance. Black-Right-Pointing-Pointer The efficiency drops radically when the defect density is higher than the P-doping. - Abstract: In this paper, we have used simulations to evaluate the impact of the distribution of electrically active defects on the photovoltaic performances of InGaN-based solar cell. The simulations were carried out using Silvaco's ATLAS software. We have modeled a P-GaN/Grad-InGaN/i-In{sub 0.53}Ga{sub 0.47}N/Grad-InGaN/N-ZnO where Grad-InGaN corresponds to an InGaN layer with a graded composition. This layer is inserted to eliminate the band discontinuities at the interface between InGaN and the GaN and ZnO layers. The defects were modeled through the introduction of band tails and a Gaussian distribution of defects in i-InGaN material. We have evaluated the influence of band tail widths as well as the parameters of the Gaussian distribution (i.e. defect density, mean position and standard deviation) on the short-circuit current, the open-circuit voltage and the fill-factor (efficiency) of the solar cell. These results have allowed us to identify key structural parameters useful for the optimization of InGaN solar cells, as well as to give realistic estimates of the performances of such cells.

  11. Human Umbilical Cord MSCs as New Cell Sources for Promoting Periodontal Regeneration in Inflammatory Periodontal Defect.

    Science.gov (United States)

    Shang, Fengqing; Liu, Shiyu; Ming, Leiguo; Tian, Rong; Jin, Fang; Ding, Yin; Zhang, Yongjie; Zhang, Hongmei; Deng, Zhihong; Jin, Yan

    2017-01-01

    Human periodontal ligament stem cells (hPDLSCs) transplantation represents a promising approach for periodontal regeneration; however, the cell source is limited due to the invasive procedure required for cell isolation. As human umbilical cord mesenchymal stem cells (hUCMSCs) can be harvested inexpensively and inexhaustibly, here we evaluated the regenerative potentials of hUCMSCs as compared with hPDLSCs to determine whether hUCMSCs could be used as new cell sources for periodontal regeneration. Methods The characteristics of hUCMSCs, including multi-differentiation ability and anti-inflammatory capability, were determined by comparison with hPDLSCs. We constructed cell aggregates (CA) using hUCMSCs and hPDLSCs respectively. Then hPDLSCs-CA and hUCMSCs-CA were combined with β-tricalcium phosphate bioceramic (β-TCP) respectively and their regenerative potentials were determined in a rat inflammatory periodontal defect model. Results hPDLSCs showed higher osteogenic differentiation potentials than hUCMSCs. Meanwhile, hUCMSCs showed higher extracellular matrix secretion and anti-inflammatory abilities than hPDLSCs. Similar to hPDLSCs, hUCMSCs were able to contribute to regeneration of both soft and hard periodontal tissues under inflammatory periodontitis condition. There were more newly formed bone and periodontal ligaments in hPDLSCs and hUCMSCs groups than in non-cell treated group. Moreover, no significant differences of regenerative promoting effects between hPDLSCs and hUCMSCs were found. Conclusion : hUCMSCs generated similar promoting effects on periodontal regeneration compared with hPDLSCs, and can be used as new cell sources for periodontal regeneration.

  12. HEK293T cell lines defective for O-linked glycosylation.

    Directory of Open Access Journals (Sweden)

    James M Termini

    Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

  13. Purine biosynthesis in Chinese Hamster cell mutants and human fibroblasts partially deficient in adenylosuccinate lyase

    International Nuclear Information System (INIS)

    Laikind, P.K.; Gruber, H.E.; Hoffer, M.; Jansen, I.; Miller, L.; Seegmiller, J.E.

    1986-01-01

    In a study of purine nucleotide metabolism of CHO-Ade I cells and fibroblasts from an adenylocsuccinate (AS) and SAICA riboside over-producing pattient the authors examined the effect of ASMP lyase deficiency on brachpoint enzyme activities and on de novo synthesis by comparison to the control cell lines, wild type parent cells (CHO-K1) and fibroblasts from age-matched normal individuals, respectively. The rate of newly-formed purine synthesis and excretion was measured by determining the amount of C 14-formate incorporatted into adenine, hypoxanthine, and guanasine moities. The nucleotide, nucleoside and base content of cells and media were analyzed by hhigh-pressure liquid chrromatography. Results are presented

  14. Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Mortelmans, K.; Friedberg, E.C.; Slor, H.; Thomas, G.; Cleaver, J.E.

    1976-01-01

    Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [ 3 H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations

  15. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

    OpenAIRE

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative ma...

  16. Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency

    International Nuclear Information System (INIS)

    Gervais, Alban; Levêque, Jean; Bouet-Toussaint, Françoise; Burtin, Florence; Lesimple, Thierry; Sulpice, Laurent; Patard, Jean-Jacques; Genetet, Noelle; Catros-Quemener, Véronique

    2005-01-01

    Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs. The IL-4/GM-CSF (granulocyte–macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells. Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR - Lin - cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-γ production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR - Lin - cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed

  17. X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    O' Neill, J.P.; Flint, K.B.

    The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G0/G1 phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50-800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10-12 h. Cells irradiated at 5 h after serum addition, i.e. in G1, show a similar dose response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G1, S or G2, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G0/G1 phase allow more time for such interactions in the absence of semiconservative DNA replication. (orig.).

  18. Study on defects and impurities in cast-grown polycrystalline silicon substrates for solar cells

    International Nuclear Information System (INIS)

    Arafune, K.; Sasaki, T.; Wakabayashi, F.; Terada, Y.; Ohshita, Y.; Yamaguchi, M.

    2006-01-01

    We focused on the defects and impurities in polycrystalline silicon substrates, which deteriorate solar cell efficiency. Comparison of the minority carrier lifetime with the grain size showed that the region with short minority carrier lifetimes did not correspond to the region with small grains. Conversely, the minority carrier lifetime decreased as the etch-pit density (EPD) increased, suggesting that the minority carrier lifetime is strongly affected by the EPD. Electron beam induced current measurements revealed that a combination of grain boundaries and point defects had high recombination activity. Regarding impurities, the interstitial oxygen concentration was relatively low compared with that in a Czochralski-grown silicon substrate, the total carbon concentration exceeded the solubility limit of silicon melt. X-ray microprobe fluorescence measurements revealed a large amount of iron in the regions where there were many etch-pits and grain boundaries with etch-pits. X-ray absorption near edge spectrum analysis revealed trapped iron in the form of oxidized iron

  19. Controlling Arteriogenesis and Mast Cells Are Central to Bioengineering Solutions for Critical Bone Defect Repair Using Allografts

    Directory of Open Access Journals (Sweden)

    Ben Antebi

    2016-01-01

    Full Text Available Although most fractures heal, critical defects in bone fail due to aberrant differentiation of mesenchymal stem cells towards fibrosis rather than osteogenesis. While conventional bioengineering solutions to this problem have focused on enhancing angiogenesis, which is required for bone formation, recent studies have shown that fibrotic non-unions are associated with arteriogenesis in the center of the defect and accumulation of mast cells around large blood vessels. Recently, recombinant parathyroid hormone (rPTH; teriparatide; Forteo therapy have shown to have anti-fibrotic effects on non-unions and critical bone defects due to inhibition of arteriogenesis and mast cell numbers within the healing bone. As this new direction holds great promise towards a solution for significant clinical hurdles in craniofacial reconstruction and limb salvage procedures, this work reviews the current state of the field, and provides insights as to how teriparatide therapy could be used as an adjuvant for healing critical defects in bone. Finally, as teriparatide therapy is contraindicated in the setting of cancer, which constitutes a large subset of these patients, we describe early findings of adjuvant therapies that may present future promise by directly inhibiting arteriogenesis and mast cell accumulation at the defect site.

  20. Defective ciliogenesis in thyroid hürthle cell tumors is associated with increased autophagy

    Science.gov (United States)

    Lee, Junguee; Yi, Shinae; Kang, Yea Eun; Chang, Joon Young; Kim, Jung Tae; Sul, Hae Joung; Kim, Jong Ok; Kim, Jin Man; Kim, Joon; Porcelli, Anna Maria; Kim, Koon Soon; Shong, Minho

    2016-01-01

    Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy. PMID:27816963

  1. Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5.

    OpenAIRE

    Greenberg, J T; Silverman, F P; Liang, H

    2000-01-01

    Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneou...

  2. Traffic jam in the primitive streak: the role of defective mesoderm migration in birth defects.

    Science.gov (United States)

    Herion, Nils J; Salbaum, J Michael; Kappen, Claudia

    2014-08-01

    Gastrulation is the process in which the three germ layers are formed that contribute to the formation of all major tissues in the developing embryo. We here review mouse genetic models in which defective gastrulation leads to mesoderm insufficiencies in the embryo. Depending on severity of the abnormalities, the outcomes range from incompatible with embryonic survival to structural birth defects, such as heart defects, spina bifida, or caudal dysgenesis. The combined evidence from the mutant models supports the notion that these congenital anomalies can originate from perturbations of mesoderm specification, epithelial-mesenchymal transition, and mesodermal cell migration. Knowledge about the molecular pathways involved may help to improve strategies for the prevention of major structural birth defects. © 2014 Wiley Periodicals, Inc.

  3. Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line.

    Science.gov (United States)

    Carpenter, Adam J; Porter, Andrew C G

    2004-12-01

    DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

  4. REPAIR OF LARGE SKULL BASE DEFECT FOLLOWING EXCISION OF BASALOID SQUAMOUS CELL CARCINOMA OF MAXILLO - ETHMOID REGION : A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Monoj Mukherjee

    2015-02-01

    Full Text Available AIM: To present a case of basaloid squamous cell carcinoma of maxillo - ethmoid region with intracranial extradural extention and its surgical management including repair of the skull base defect. MATERIAL : A 30 year female presented with progressive bilateral nasal obstruction, facial deformity for 5 years duration. She developed blindness in last 6 months. Recent CT s can showed large heterogeneous enhancing soft tissue mass in right maxillary sinus, nasal cavity and right ethmoid sinus invading the skull base . INTERVENTION : She underwent excision of the mass by modified weber ferguson incision and repair of skull base defect with temporalis muscle flap. Skin defect over the face and nose was repaired by median forehead flap. RESULT : There was total tumor clearance and no CSF leakage following surgery. CONCLUSION : Sinonasal malignancy with intracranial extradural extenti on is not a contraindication for successful surgical management. Resultant skull base defect can be repaired by a temporalis muscle flap to prevent CSF leak and intracranial infection

  5. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    OpenAIRE

    Jin, Hongping; Li, Dongsheng; Sivakumaran, Haran; Lor, Mary; Rustanti, Lina; Cloonan, Nicole; Wani, Shivangi; Harrich, David

    2016-01-01

    ABSTRACT Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutive