Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO(2) atmosphere.

Science.gov (United States)

Pollock, Steve V; Colombo, Sergio L; Prout, Davey L; Godfrey, Ashley C; Moroney, James V

2003-12-01

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the BleR (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO2 atmosphere for optimal growth. The DNA flanking the BleR insert of one of the high-CO2-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO2-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO2-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.

Rubisco Activase Is Required for Optimal Photosynthesis in the Green Alga Chlamydomonas reinhardtii in a Low-CO2 Atmosphere1

Science.gov (United States)

Pollock, Steve V.; Colombo, Sergio L.; Prout, Davey L.; Godfrey, Ashley C.; Moroney, James V.

2003-01-01

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the BleR (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO2 atmosphere for optimal growth. The DNA flanking the BleR insert of one of the high-CO2-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO2-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO2-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii. PMID:14605215

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

Science.gov (United States)

Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

2016-10-07

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Optically Guided Photoactivity: Coordinating Tautomerization, Photoisomerization, Inhomogeneity, and Reactive Intermediates within the RcaE Cyanobacteriochrome.

Science.gov (United States)

Gottlieb, Sean M; Chang, Che-Wei; Martin, Shelley S; Rockwell, Nathan C; Lagarias, J Clark; Larsen, Delmar S

2014-05-01

The RcaE cyanobacteriochrome uses a linear tetrapyrrole chromophore to sense the ratio of green and red light to enable the Fremyella diplosiphon cyanobacterium to control the expression of the photosynthetic infrastructure for efficient utilization of incident light. The femtosecond photodynamics of the embedded phycocyanobilin chromophore within RcaE were characterized with dispersed femtosecond pump-dump-probe spectroscopy, which resolved a complex interplay of excited-state proton transfer, photoisomerization, multilayered inhomogeneity, and reactive intermediates. These reactions were integrated within a central model that incorporated a rapid (200 fs) excited-state Le Châtelier redistribution between parallel evolving populations ascribed to different tautomers. Three photoproducts were resolved and originates from four independent subpopulations, each with different dump-induced behavior: Lumi-Go was depleted, Lumi-Gr was unaffected, and Lumi-Gf was enhanced. This suggests that RcaE may be engineered to act either as an in vivo fluorescent probe (after single-pump excitation) or as an in vivo optogenetic sample (after pump and dump excitation).

Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

OpenAIRE

Zielke, Norman

2007-01-01

Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family ...

Tenth working group meeting of representatives of RCA Member States. Report

International Nuclear Information System (INIS)

1989-04-01

The Tenth RCA (Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology) Working Group meeting of representatives of RCA Member States was held at the Chinese Academy of Agricultural Sciences, Beijing, between 11-14 April 1988. The report on the meeting consists of a presentation of the seven technical sessions which dealt with topics such as nuclear techniques in industry, agriculture and medicine, nuclear power and of the project reports under RCA

RCA annual report 1988

International Nuclear Information System (INIS)

1989-09-01

The report covers the calendar year 1988. It contains an extended preface on Asian regional co-operation in the application of nuclear techniques, a summary of major RCA meetings, administrative questions, a summary of technical aspects of the programme and an analysis of the resources including a prediction of project activities for 1989-1991. 4 figs, 3 tabs

Sensitive magnetic biodetection using magnetic multi-core nanoparticles and RCA coils

Energy Technology Data Exchange (ETDEWEB)

Ahrentorp, Fredrik; Blomgren, Jakob; Jonasson, Christian; Sarwe, Anna [Acreo Swedish ICT AB, Arvid Hedvalls Backe 4, Göteborg (Sweden); Sepehri, Sobhan; Eriksson, Emil; Kalaboukhov, Alexei; Jesorka, Aldo; Winkler, Dag [Department of Microtechnology and Nanoscience – MC2, Chalmers University of Technology, Göteborg (Sweden); Schneiderman, Justin F. [Institute of Neuroscience and Physiology, University of Gothenburg and MedTech West, Göteborg (Sweden); Nilsson, Mats [Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm (Sweden); Albert, Jan [Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm (Sweden); Department of Clinical Microbiology, Karolinska University Hospital, Stockholm (Sweden); Zardán Gómez de la Torre, Teresa; Strømme, Maria [Uppsala University, Uppsala (Sweden); Johansson, Christer, E-mail: christer.johansson@acreo.se [Acreo Swedish ICT AB, Arvid Hedvalls Backe 4, Göteborg (Sweden)

2017-04-01

We use functionalized iron oxide magnetic multi-core particles of 100 nm in size (hydrodynamic particle diameter) and AC susceptometry (ACS) methods to measure the binding reactions between the magnetic nanoparticles (MNPs) and bio-analyte products produced from DNA segments using the rolling circle amplification (RCA) method. We use sensitive induction detection techniques in order to measure the ACS response. The DNA is amplified via RCA to generate RCA coils with a specific size that is dependent on the amplification time. After about 75 min of amplification we obtain an average RCA coil diameter of about 1 µm. We determine a theoretical limit of detection (LOD) in the range of 11 attomole (corresponding to an analyte concentration of 55 fM for a sample volume of 200 µL) from the ACS dynamic response after the MNPs have bound to the RCA coils and the measured ACS readout noise. We also discuss further possible improvements of the LOD. - Highlights: • Biosensing using Brownian relaxation of functionalized magnetic nanoparticles. • Rolling circle amplification and magnetic nanoparticles enables biosensing. • Theoretical limit of detection estimated from the signal noise gives about 55 fM.

LFI Radiometric Chain Assembly (RCA) data handling ``Rachel''

Science.gov (United States)

Malaspina, M.; Franceschi, E.; Battaglia, P.; Binko, P.; Butler, R. C.; D'Arcangelo, O.; Fogliani, S.; Frailis, M.; Franceschet, C.; Galeotta, S.; Gasparo, F.; Gregorio, A.; Lapolla, M.; Leonardi, R.; Maggio, G.; Mandolesi, N.; Manzato, P.; Maris, M.; Meharga, M.; Meinhold, P.; Morisset, N.; Pasian, F.; Perrotta, F.; Rohlfs, R.; Sandri, M.; Tomasi, M.; Türler, M.; Zacchei, A.; Zonca, A.

2009-12-01

Planck's Low Frequency Instrument is an array of 22 pseudo-correlation radiometers at 30, 44, and 70 GHz. Before integrating the overall array assembly, a first set of tests has been performed for each radiometer chain assembly (RCA), consisting of two radiometers. In this paper, we describe Rachel, a software application which has been purposely developed and used during the RCA test campaign to carry out both near-realtime on-line data analysis and data storage (in FITS format) of the raw output from the radiometric chains.

LFI Radiometric Chain Assembly (RCA) data handling 'Rachel'

International Nuclear Information System (INIS)

Malaspina, M; Franceschi, E; Butler, R C; Mandolesi, N; Battaglia, P; Franceschet, C; Lapolla, M; Binko, P; Meharga, M; D'Arcangelo, O; Fogliani, S; Frailis, M; Galeotta, S; Gasparo, F; Maggio, G; Manzato, P; Maris, M; Gregorio, A; Leonardi, R; Meinhold, P

2009-01-01

Planck's Low Frequency Instrument is an array of 22 pseudo-correlation radiometers at 30, 44, and 70 GHz. Before integrating the overall array assembly, a first set of tests has been performed for each radiometer chain assembly (RCA), consisting of two radiometers. In this paper, we describe Rachel, a software application which has been purposely developed and used during the RCA test campaign to carry out both near-realtime on-line data analysis and data storage (in FITS format) of the raw output from the radiometric chains.

19. working group meeting of representatives of RCA member States. Report

International Nuclear Information System (INIS)

1997-03-01

The 19. RCA Working Group Meeting was hosted by the Myanmar Atomic Energy Committee in Yangon from 10-14 March 1997. The meeting marked the 25. anniversary of the RCA which commenced in 1972. It was attended by 29 delegates from 15 visiting RCA Member States, 15 participants from Myanmar and 4 from the IAEA. The report contains the statements of the following countries: Australia, Bangladesh, China, India, Indonesia, Japan, Republic of Korea, Malaysia, Mongolia, Myanmar, New Zealand, Pakistan, Philippines, Singapore, Sri Lanka, Thailand, and Viet Nam. Tabs

19. working group meeting of representatives of RCA member States. Report

Energy Technology Data Exchange (ETDEWEB)

NONE

1997-03-01

The 19. RCA Working Group Meeting was hosted by the Myanmar Atomic Energy Committee in Yangon from 10-14 March 1997. The meeting marked the 25. anniversary of the RCA which commenced in 1972. It was attended by 29 delegates from 15 visiting RCA Member States, 15 participants from Myanmar and 4 from the IAEA. The report contains the statements of the following countries: Australia, Bangladesh, China, India, Indonesia, Japan, Republic of Korea, Malaysia, Mongolia, Myanmar, New Zealand, Pakistan, Philippines, Singapore, Sri Lanka, Thailand, and Viet Nam. Tabs.

The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP.

Science.gov (United States)

Wilson, R B; Renault, G; Jacquet, M; Tatchell, K

1993-07-05

The high affinity cAMP phosphodiesterase, encoded by PDE2, is an important component of the cAMP-dependent protein kinase signaling system in Saccharomyces cerevisiae. An unexpected phenotype of pde2 mutants is sensitivity to external cAMP. This trait has been found independently for rca1 mutants and has been used to monitor the effects of cAMP on several biological processes. We demonstrate here that RCA1 is identical to PDE2. Further analysis of the phenotype of pde2 deletions reveal that exogenously added cAMP results in an increase in the internal level of cAMP. This increase slows down the rate of cell division by increasing the length of the G1 phase of the cell cycle and leads to increased cell volume. Also, cells with a disrupted PDE2 gene previously arrested by nutrient starvation rapidly lose thermotolerance when incubated with exogenous cAMP. From these observations we propose that a role of the PDE2-encoded phosphodiesterase may be to help insulate the internal cAMP pools from the external environment. This protective role might also be important in other eukaryotic organisms where cAMP is a key second messenger.

RCA/IAEA third external dosimetry intercomparisons in East Asia region

International Nuclear Information System (INIS)

Yamamoto, H.; Yoshizawa, M.; Murakami, H.; Momose, T.; Tsujimura, N.; Kanai, K.; Cruz-Suarez, R.

2007-01-01

Several intercomparison exercises were organised by the International Atomic Energy Agency (IAEA) on the determination of operational quantities at the regional or inter-regional basis. In East Asia region, a third phase of the intercomparison finished in mid 2004. It was organised within the frame of the Regional Cooperation Agreement (RCA) as a follow-up to previous exercises carried out during 1990-1992 and 1995-1996. The results of this intercomparison for the determination of operational quantities were satisfactory for all Member States. The laboratories demonstrated a good performance in quantities tested. The purpose of this paper is to present the results of the RCA/IAEA intercomparison and the future of RCA activities in support of assessment of occupational exposure by organising intercomparison runs. (authors)

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function.

Science.gov (United States)

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-12-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp-Cullin-F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C-Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1-S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1-S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry.

RCA activities in the Asian and Pacific Region

International Nuclear Information System (INIS)

Kobayashi, M.

1984-01-01

So-called ''RCA'' activities - practical work undertaken within the framework of a Regional Co-operative Agreement for Research, Development and Training related to Nuclear Science and Technology for the Asian and Pacific region - are widely regarded as an example of successful co-operative effort in the application of nuclear techniques at a regional level. Activities undertaken to promote the transfer of nuclear technology within the framework of the RCA cover a large spectrum of nuclear applications in agriculture and food production, medicine, study of the environment, industry, and physics. Fourteen projects are operational this year (1984)

Three genes for mitochondrial proteins suppress null-mutations in both Afg3 and Rca1 when over-expressed.

Science.gov (United States)

Rep, M; Nooy, J; Guélin, E; Grivell, L A

1996-08-01

The AFG3 gene of Saccharomyces cerevisiae encodes a mitochondrial inner membrane protein with ATP-dependent protease activity. To gain more insight into the function of this protein, multi-copy suppressors of an afg3-null mutation were isolated. Three genes were found that restored partial growth on non-fermentable carbon sources, all of which affect the biogenesis of respiratory competent mitochondria: PIM1(LON) encodes a matrix-localized ATP-dependent protease involved in the turnover of matrix proteins; OXA1(PET1402) encodes a putative mitochondrial inner membrane protein involved in the biogenesis of the respiratory chain; and MBA1 encodes a mitochondrial protein required for optimal respiratory growth. All three genes also suppressed a null mutation in a related gene, RCA1, as well as in the combination of afg3- and rca1-null.

VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

Science.gov (United States)

Chen, H T; Alexander, C B; Young-Cooper, G O; Mage, R G

1993-04-01

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

OpenAIRE

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-01-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the ...

Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

Science.gov (United States)

Bissinger, P H; Wieser, R; Hamilton, B; Ruis, H

1989-03-01

In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

International Nuclear Information System (INIS)

Wu, Hsu-Pin; Hsu, Shu-Yuan; Wu, Wen-Ai; Hu, Ji-Wei; Ouyang, Pin

2014-01-01

Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB +/− mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species. The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes

Energy Technology Data Exchange (ETDEWEB)

Wu, Hsu-Pin; Hsu, Shu-Yuan [Department of Anatomy, Chang Gung University Medical College, Taiwan (China); Wu, Wen-Ai; Hu, Ji-Wei [Transgenic Mouse Core Laboratory, Chang Gung University, Taiwan (China); Ouyang, Pin, E-mail: ouyang@mail.cgu.edu.tw [Department of Anatomy, Chang Gung University Medical College, Taiwan (China); Transgenic Mouse Core Laboratory, Chang Gung University, Taiwan (China); Molecular Medicine Research Center, Chang Gung University, Taiwan (China)

2014-01-03

Highlights: •Pnn CCD domain functions as a dominant negative mutant regulating Pnn expression and function. •Pnn CCD mutant Tg mice have a muscle wasting phenotype during development and show dystrophic histological features. •Pnn mutant muscles are susceptible to slow fiber type gene transition and NEB reduction. •The Tg mouse generated by overexpression of the Pnn CCD domain displays many characteristics resembling NEB{sup +/−} mice. -- Abstract: Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species. The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.

Characterization of D-myo-inositol 3-phosphate synthase gene expression in two soybean low phytate mutants

International Nuclear Information System (INIS)

Yuan Fengjie; Dong Dekun; Li Baiquan; Yu Xiaomin; Fu Xujun; Zhu Danhua; Zhu Shenlong; Yang Qinghua

2013-01-01

1D-myo-inositol 3-phosphate synthase (MIPS) gene plays a significant role in phytic acid biosynthesis. In this study, we used two low phytic acid mutants Gm-lpa-TW-1, Gm-lpa-ZC-2 and their respective wild type parents Taiwan75 and Zhechun No.3 to analyze the expression pattern and characterization of MIPS1 gene. The results showed that there was a common expression pattern of MIPS1 in soybean developing seeds. Expression was weak as detected by RT-PCR in initial stage, increased in the following stages, and the peak expression was appeared in 22 day after flowering (DAF). The expression of MIPS1 gene of non-seed tissues in mutant Gm-lpa-TW-1 and its wildtype Taiwan75 was very weak. In the developing seeds, the MIPS1 expression by qRT-PCR revealed a significant reduction in 22 DAF in mutant Gm-lpa-TW-1 as compared with the wildtype. Similarly, the expression of MIPS1 gene in non-seed tissue of Zhenchun No.3 and Gm-lpa-ZC-2 was very weak. However, stronger expression in developing seeds of the mutant Gm-lpa-ZC-2 than Zhechun No.3 was found. We concluded that the MIPS1 gene expression in the developing seed exhibited an up-regulation pattern in mutant Gm-lpa-ZC-2, but a down-regulation pattern in the mutant Gm-lpa-TW-1. (authors)

The 2015 Resuscitation Council of Asia (RCA) guidelines on adult basic life support for lay rescuers.

Science.gov (United States)

Chung, Sung Phil; Sakamoto, Tetsuya; Lim, Swee Han; Ma, Mathew Huei-Ming; Wang, Tzong-Luen; Lavapie, Francis; Krisanarungson, Sopon; Nonogi, Hiroshi; Hwang, Sung Oh

2016-08-01

This paper introduces adult basic life support (BLS) guidelines for lay rescuers of the resuscitation council of Asia (RCA) developed for the first time. The RCA BLS guidelines for lay rescuers have been established by expert consensus among BLS Guidelines Taskforce of the RCA on the basis of the 2015 International Consensus on Cardiopulmonary Resuscitation (CPR) and Emergency Cardiovascular Care Science with Treatment Recommendations. The RCA recommends compression-only CPR for lay rescuers and emphasizes high-quality CPR with chest compression depth of approximately 5cm and chest compression rate of 100-120min(-1). Role of emergency medical dispatchers in helping lay rescuers recognize cardiac arrest and perform CPR is also emphasized. The RCA guidelines will contribute to help Asian countries establish and implement their own CPR guidelines in the context of their domestic circumstances. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Predicting of the compressive strength of RCA concrete

Directory of Open Access Journals (Sweden)

Jaskulski Roman

2017-01-01

Full Text Available The paper presents the results of predicting the strength of 61 concretes made with the use of recycled concrete aggregate (RCA. Five models in the form of first-order polynomials containing two to six variables characterizing the composition of the mixture were formulated for this purpose. Factors for unknowns were selected using linear regression in two variants: with and without additional coefficient. For each model, the average absolute error of the concrete strength estimation was determined. Because of the various consequences of underestimation and overestimation of the results, the analysis of models quality was carried out with the distinction of the two cases. The results indicate that the key to improving the quality of models is to take into account the quality of the aggregate expressed by the ACV parameter. Better match results were also obtained for models with more variables and the additional coefficient.

The effect of recycled concrete aggregate properties on the bond strength between RCA concrete and steel reinforcement

International Nuclear Information System (INIS)

Butler, L.; West, J.S.; Tighe, S.L.

2011-01-01

The purpose of this study was to investigate the influence that replacing natural coarse aggregate with recycled concrete aggregate (RCA) has on concrete bond strength with reinforcing steel. Two sources of RCA were used along with one natural aggregate source. Numerous aggregate properties were measured for all aggregate sources. Two types of concrete mixture proportions were developed replacing 100% of the natural aggregate with RCA. The first type maintained the same water-cement ratios while the second type was designed to achieve the same compressive strengths. Beam-end specimens were tested to determine the relative bond strength of RCA and natural aggregate concrete. On average, natural aggregate concrete specimens had bond strengths that were 9 to 19% higher than the equivalent RCA specimens. Bond strength and the aggregate crushing value seemed to correlate well for all concrete types.

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.

RCA : resource consumptions accounting

OpenAIRE

Valeska Rodriguez Lucas de Freitas

2013-01-01

Devido à grande concorrência do mercado o controle de custos passa a ser peça fundamental no auxílio à tomada de decisão quanto à redução dos custos, visando uma lucratividade maior por parte das empresas. Essa dissertação trata sobre um novo modelo de custos, o RCA – Resource Consumption Accounting, que ainda não foi adotado por nenhuma empresa. Esse novo método de custeio foi aplicado como modelo piloto em uma empresa Americana onde os resultados foram favoráveis a ele em relação aos modelo...

P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences.

OpenAIRE

Milla, M. E.; Brown, B. M.; Sauer, R. T.

1993-01-01

Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10- to 20-fold and, ...

The fourth UNDP/RCA/IAEA/meeting of national co-ordinators for radiation technology. Report

International Nuclear Information System (INIS)

1996-01-01

The objectives of the Meeting were to provide information for the Terminal Report of the joint UNDP/RCA/IAEA project RAS/92/073 and to look into future activities under the Radiation Technology project. The main achievements of this Meeting are: The Meeting reviewed the implementation of all radiation technology sub-projects and agreed that all of them were successful but not yet equally developed among RCA Member States. The Meeting recommended to have three projects carried out in the form of Co-ordinated Research Programs and requested the IAEA to find new ways to implement the organized in RCA Member States to carry these CRPs out. Figs, tabs

The economic and social significance of RCA regional co-operative projects

International Nuclear Information System (INIS)

Fowler, E.E.

1979-01-01

The regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology (RCA) is an established and valuable instrument between the International Atomic Energy Agency (IAEA) and Member States in South Asia, Southeast Asia and the Pacific or Far East for assisting in the transfer of modern technology to areas having economic and social importance to the region. The purpose of this review is to identify on-going or planned work which is designed to help achieve this goal. Currently approved RCA Regional Co-operative Research Projects are listed

Immunomodulatory response of mice splenocytes induced by RcaL, a lectin isolated from cobia fish (Rachycentron canadum) serum.

Science.gov (United States)

Coriolano, Marília Cavalcanti; Silva, Cynarha Daysy Cardoso da; Melo, Cristiane Moutinho Lagos de; Bezerra, Ranilson de Souza; Santos, Athiê Jorge Guerra; Pereira, Valéria Rêgo Alves; Coelho, Luana Cassandra Breitenbach Barroso

2012-11-01

This work reports the isolation of a serum lectin from cobia fish (Rachycentron canadum) named RcaL. Immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production were also performed. RcaL was obtained through precipitation with ammonium sulphate and affinity chromatography on a Concanavalin A-Sepharose 4B column. The ammonium sulphate fraction F3 showed the highest specific hemagglutinating activity and was applied to affinity chromatography. The lectin was eluted with methyl-α-D-mannopyranoside. RcaL showed highest affinity for methyl-α-D-mannopyranoside and D-mannose; eluted fractions of RcaL agglutinated rabbit erythrocytes (titre, 128(-1)) retained 66 % of chromatographed lectin activity, and the obtained purification factor was 1.14. Under reducing conditions, a polypeptide band of 19.2 kDa was revealed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (PAGE). PAGE confirmed RcaL as an acidic protein revealed in a single band. Cytotoxic and immunomodulatory assays with RcaL in mice splenocyte cultures showed that the lectin was not cytotoxic and induced higher interferon gamma and nitric oxide production in splenocyte cultures. Purified RcaL induced preferential Th1 response, suggesting that it acts as an immunomodulatory compound.

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.

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

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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.

Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

Science.gov (United States)

Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

2011-09-01

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Functional analysis of the rice rubisco activase promoter in transgenic Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Yang, Zhipan; Lu, Qingtao; Wen, Xiaogang [Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China); Chen, Fan [Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Lu, Congming, E-mail: lucm@ibcas.ac.cn [Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 (China)

2012-02-17

Highlights: Black-Right-Pointing-Pointer Rice rubisco activase promoter was analyzed in transgenic Arabidopsis system. Black-Right-Pointing-Pointer Region conferring tissue specific and light inducible expression of Rca was identified. Black-Right-Pointing-Pointer -58 to +43 bp region mediates tissue-specific expression of rice Rca. Black-Right-Pointing-Pointer Light inducible expression of rice Rca is mediated by -297 to -58 bp region. Black-Right-Pointing-Pointer Rice nuclear proteins bind specifically with the light inducible region. -- Abstract: To gain a better understanding of the regulatory mechanism of the rice rubisco activase (Rca) gene, variants of the Rca gene promoter (one full-length and four deletion mutants) fused to the coding region of the bacterial reporter gene {beta}-glucuronidase (GUS) were introduced into Arabidopsis via Agrobacterium-mediated transformation. Our results show that a 340 bp fragment spanning from -297 to +43 bp relative to the transcription initiation site is enough to promote tissue-specific and light-inducible expression of the rice Rca gene as done by the full-length promoter (-1428 to +43 bp). Further deletion analysis indicated that the region conferring tissue-specificity of Rca expression is localized within a 105 bp fragment from -58 to +43 bp, while light-inducible expression of Rca is mediated by the region from -297 to -58 bp. Gel shift assays and competition experiments demonstrated that rice nuclear proteins bind specifically with the fragment conferring light responsiveness at more than one binding site. This implies that multiple cis-elements may be involved in light-induced expression of the rice Rca gene. These works provide a useful reference for understanding transcriptional regulation mechanism of the rice Rca gene, and lay a strong foundation for further detection of related cis-elements and trans-factors.

Functional analysis of the rice rubisco activase promoter in transgenic Arabidopsis

International Nuclear Information System (INIS)

Yang, Zhipan; Lu, Qingtao; Wen, Xiaogang; Chen, Fan; Lu, Congming

2012-01-01

Highlights: ► Rice rubisco activase promoter was analyzed in transgenic Arabidopsis system. ► Region conferring tissue specific and light inducible expression of Rca was identified. ► −58 to +43 bp region mediates tissue-specific expression of rice Rca. ► Light inducible expression of rice Rca is mediated by −297 to −58 bp region. ► Rice nuclear proteins bind specifically with the light inducible region. -- Abstract: To gain a better understanding of the regulatory mechanism of the rice rubisco activase (Rca) gene, variants of the Rca gene promoter (one full-length and four deletion mutants) fused to the coding region of the bacterial reporter gene β-glucuronidase (GUS) were introduced into Arabidopsis via Agrobacterium-mediated transformation. Our results show that a 340 bp fragment spanning from −297 to +43 bp relative to the transcription initiation site is enough to promote tissue-specific and light-inducible expression of the rice Rca gene as done by the full-length promoter (−1428 to +43 bp). Further deletion analysis indicated that the region conferring tissue-specificity of Rca expression is localized within a 105 bp fragment from −58 to +43 bp, while light-inducible expression of Rca is mediated by the region from −297 to −58 bp. Gel shift assays and competition experiments demonstrated that rice nuclear proteins bind specifically with the fragment conferring light responsiveness at more than one binding site. This implies that multiple cis-elements may be involved in light-induced expression of the rice Rca gene. These works provide a useful reference for understanding transcriptional regulation mechanism of the rice Rca gene, and lay a strong foundation for further detection of related cis-elements and trans-factors.

Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

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Wright Anthony PH

2010-01-01

Full Text Available Abstract Background Histone acetyltransferase enzymes (HATs are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.

RCA and its regional office for cooperative activities in the applications of nuclear techniques in Asia and the Pacific

International Nuclear Information System (INIS)

Lee, Jae-Sol

2008-01-01

The Regional Cooperative Agreement for Research, Development and Training Related to Nuclear Science and Technology (RCA) is an intergovernmental agreement among 17 Member States of the International Atomic Energy Agency (IAEA), in South Asia, South East Asia, Far East and the Pacific, established for promoting regional cooperation in the peaceful uses of nuclear science and technology. Established in 1972, the RCA is a unique development within the technical cooperation framework coordinated by IAEA is support of the regional cooperation in the applications of nuclear technology to the benefit of its Member States. It has a well established network of scientists trained in the use of nuclear technologies in the agricultural, medical, industrial, and environmental sectors, with a good potentiality to use its regional technical and scientific resources to contribute to the solution of some of the significant problems in the region. The achievements recorded in the several decades of RCA history cover a wide spectrum of areas and these achievements have been published a Success Stories. Having recognized a need to enhance ownership of the region, the RCA Member States had wished to establish its Regional Office (RCARO). The Regional Office which is hosted by the Government of Republic of Korea was established in 2002. Located in Daejeon, the RCARO has been active in its endeavors toward increasing visibility of the RCA and establishing cooperative partnership between RCA and other international organizations. The RCARO has also played a key role in successfully implementing an RCA Project on Post-Tsunami Environment Impact Assessment in partnership with UNDP (Republic of Korea) with the participation of 14 RCA Member States. A number of projects for capacity building in the region are also being implemented by RCARO, mostly by providing training opportunities for young professionals in the region. The RCA community welcomes any contacts from potential end-users or

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.

Analysis on expression of gene for flower shape in Dendrobium sonia mutants using differential display technique

International Nuclear Information System (INIS)

Affrida Abu Hassan; Ahmad Syazni Kamarudin; Nurul Nadia Aminuddin; Mohd Nazir Basiran

2004-01-01

In vitro mutagenesis on Dendrobium Sonia in MINT has produced mutants with wide range of flower form and colour variations. Among the mutants are plants with different flower size and shape. These changes could be caused by alterations to the expression level of the genes responsible for the characteristics. In this studies, Differential Display technique was used to identify and analyse altered gene expression at the mRNA level. Total RNA of the control and mutants were reversed transcribed using three anchored oligo-d T primers. Subsequently, these cDNAs were Pcr amplified in combination with 16 arbitrary primers. The amplified products were electrophoresed side by side on agarose gel. Differentially expressed bands are isolated for further analysis. (Author)

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

Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53.

Science.gov (United States)

Loging, W T; Reisman, D

1999-11-01

The wild-type p53 protein is a DNA-binding transcription factor that activates genes such as p21, MDM2, GADD45, and Bax that are required for the regulation of cell cycle progression or apoptosis in response to DNA damage. Mutant forms of p53, which are transforming oncogenes and are expressed at high levels in tumor cells, generally have a reduced binding affinity for the consensus DNA sequence. Interestingly, some p53 mutants that are no longer effective at binding to the consensus DNA sequence and transactivating promoters containing this target site have acquired the ability to transform cells in culture, in part through their ability to transactivate promoters of a number of genes that are not targets of the wild-type protein. Certain p53 mutants are therefore considered to be gain-of-function mutants and appear to be promoting proliferation or transforming cells through their ability to alter the expression of novel sets of genes. Our goal is to identify genes that have altered expression in the presence of a specific mutant p53 (Arg to Trp mutation at codon 248) protein. Through examining differential gene expression in cells devoid of p53 expression and in cells that express high levels of mutant p53 protein, we have identified three ribosomal protein genes that have elevated expression in response to mutant p53. Consistent with these findings, the overexpression of a number of ribosomal protein genes in human tumors and evidence for their contribution to oncogenic transformation have been reported previously, although the mechanism leading to this overexpression has remained elusive. We show results that indicate that expression of these specific ribosomal protein genes is increased in the presence of the R248W p53 mutant, which provides a mechanism for their overexpression in human tumors.

RCA/IAEA third external dosimetry intercomparison in East Asia

International Nuclear Information System (INIS)

Momose, T.; Yamamoto, H.; Cruz Suarez, R.

2005-01-01

Full text: Several intercomparison exercises were organized by the International Atomic Energy Agency (IAEA) on the determination of operational quantities at the regional or interregional basis. These exercises revealed significant differences in the approach, methods and assumptions, and consequently in the measurement results obtained by participating laboratories. In the East Asia region, the third phase of the Hp(10) intercomparison, organized within the frame of the Regional Cooperation Agreement (RCA) as a follow-up to previous exercises during 1990-92 and 1995-96, was completed mid-2004. The first phase grouped 25 laboratories from 16 member states, and 4 Secondary Standards Dosimetry Laboratories irradiated dosimeters in 6 different qualities for photon and beta radiations. In the second phase, 23 laboratories from 16 member states participated, and 3 Secondary Standards Dosimetry Laboratories provided irradiation in 5 different radiation qualities simulating workplace fields. The results of the second phase for the determination of operational quantities Hp(d) were satisfactory for all participating Member States, with marked improvement from the first phase; the laboratories demonstrated good performance in both quantities tested. These results underline the importance of such an intercomparison programme as a key element towards the harmonization of quantities and units on an international level. This paper presents the results of this RCA/IAEA intercomparison, and also the forthcoming RCA activities supporting intercomparison runs for the assessment of occupational exposure. Member states strongly recommend that the IAEA continue acting as a focal point for, inter alia, training in all forms, particularly in measurements and dosimetry techniques. This exercise also stressed the importance for the IAEA to take an active role in establishing a network of monitoring laboratories for radiation protection purposes, as it would provide for better information

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response.

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Céline Schaeffer

Full Text Available Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle's loop (TAL and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD, characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER, is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR, and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and

Dynamic range studies of the RCA streak tube in the LLL streak camera

International Nuclear Information System (INIS)

Thomas, S.W.; Phillips, G.E.

1979-01-01

As indicated by tests on several cameras, the dynamic range of the Lawrence Livermore Laboratory streak-camera system appears to be about two orders of magnitude greater than those reported for other systems for 10- to 200-ps pulses. The lack of a fine mesh grid in the RCA streak tube used in these cameras probably contributes to a lower system dynamic noise and therefore raises the dynamic range. A developmental tube with a mesh grid was tested and supports this conjecture. Order-of-magnitude variations in input slit width do not affect the spot size on the phosphor or the dynamic range of the RCA tube. (author)

Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

Directory of Open Access Journals (Sweden)

Andrea L Frump

Full Text Available More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH. More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations. These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2 in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+ mice. The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+ mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12.

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Jarukit Edward Long

Full Text Available Repairing DNA damage begins with its detection and is often followed by elicitation of a cellular response. In E. coli, RecA polymerizes on ssDNA produced after DNA damage and induces the SOS Response. The RecA-DNA filament is an allosteric effector of LexA auto-proteolysis. LexA is the repressor of the SOS Response. Not all RecA-DNA filaments, however, lead to an SOS Response. Certain recA mutants express the SOS Response (recA(C in the absence of external DNA damage in log phase cells.Genetic analysis of two recA(C mutants was used to determine the mechanism of constitutive SOS (SOS(C expression in a population of log phase cells using fluorescence of single cells carrying an SOS reporter system (sulAp-gfp. SOS(C expression in recA4142 mutants was dependent on its initial level of transcription, recBCD, recFOR, recX, dinI, xthA and the type of medium in which the cells were grown. SOS(C expression in recA730 mutants was affected by none of the mutations or conditions tested above.It is concluded that not all recA(C alleles cause SOS(C expression by the same mechanism. It is hypothesized that RecA4142 is loaded on to a double-strand end of DNA and that the RecA filament is stabilized by the presence of DinI and destabilized by RecX. RecFOR regulate the activity of RecX to destabilize the RecA filament. RecA730 causes SOS(C expression by binding to ssDNA in a mechanism yet to be determined.

Report of the IAEA/RCA-NDT education project

International Nuclear Information System (INIS)

Ooka, Kiichi; Terada, Kunio; Ohtani, Kiyoshi; Niwa, Noboru.

1988-01-01

The Japan Non-Destructive Test (NDT) Association has been carried out an NDT technical education project with support from IAEA. Phase I of the project was successfully completed last year. The present report briefly outlines the second five-year plan (Phase II) to be carried out under the project. The project was first established in 1981 after receiving a request from IAEA to provide NDT education for participants from the RCA countries (13 countries in Asian and Pacific area). Phase I was mainly designed to provide lectures on NDT techniques. Prior to the start of Phase II, the International Education Subcommittee was established in December 1986 to take the leadership in carrying out Phase II of the project. In addition, the NDT Expert Working Group (formally called Advisory Group) has been installed which consists of representative from Japan. Australia India and Singapore. The Group held nine meetings during Phase I. Three lecture meetings at Singapore and another three at Tokyo were held to provide education according to a plan developed by the Group. It has been decided that Japan be support the lecture meetings to be held during Phase II. To conduct such meetings, a group comprising all of the National Coordinators from the RCA countries was set up to replace the Working Group. The National Coordinator Conference was held four times since its establishment. (N.K.)

Report of the IAEA/RCA-NDT education project

Energy Technology Data Exchange (ETDEWEB)

Ooka, Kiichi; Terada, Kunio; Ohtani, Kiyoshi; Niwa, Noboru

1988-11-01

The Japan Non-Destructive Test (NDT) Association has been carried out an NDT technical education project with support from IAEA. Phase I of the project was successfully completed last year. The present report briefly outlines the second five-year plan (Phase II) to be carried out under the project. The project was first established in 1981 after receiving a request from IAEA to provide NDT education for participants from the RCA countries (13 countries in Asian and Pacific area). Phase I was mainly designed to provide lectures on NDT techniques. Prior to the start of Phase II, the International Education Subcommittee was established in December 1986 to take the leadership in carrying out Phase II of the project. In addition, the NDT Expert Working Group (formally called Advisory Group) has been installed which consists of representative from Japan. Australia India and Singapore. The Group held nine meetings during Phase I. Three lecture meetings at Singapore and another three at Tokyo were held to provide education according to a plan developed by the Group. It has been decided that Japan be support the lecture meetings to be held during Phase II. To conduct such meetings, a group comprising all of the National Coordinators from the RCA countries was set up to replace the Working Group. The National Coordinator Conference was held four times since its establishment. (N.K.).

A new member of a family of ATPases is essential for assembly of mitochondrial respiratory chain and ATP synthetase complexes in Saccharomyces cerevisiae.

Science.gov (United States)

Tzagoloff, A; Yue, J; Jang, J; Paul, M F

1994-10-21

Respiration-defective pet mutants of Saccharomyces cerevisiae, assigned to complementation group G25, are grossly deficient in mitochondrial respiratory and ATPase complexes. This phenotype is usually found in strains impaired in mitochondrial protein synthesis. The G25 mutants, however, synthesize all of the proteins encoded by mitochondrial DNA. The mutants are also able to import and process cytoplasmically derived subunits of these enzymes. These results are most compatible with the idea that the gene defined by G25 mutants (RCA1) codes for a protein essential for the assembly of functional respiratory and ATPase complexes. The RCA1 gene has been cloned by complementation of an rca1 mutant with a yeast genomic library. The sequence of the encoded product shows Rca1 protein to be a new member of a recently described family of ATPases. The Rca1 protein is a mitochondrial membrane protein and is the third known member of this family implicated to function in the biogenesis of mitochondria. The primary structure of Rca1 protein indicates several distinct domains in addition to the common purine nucleotide binding region shared by all members of this protein family. One, located in the amino-terminal half, contains two hydrophobic stretches of sufficient length to span a membrane lipid bilayer.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo

International Nuclear Information System (INIS)

Perryman, L.A.; Blair, J.M.; Kingsley, E.A.; Szymanska, B.; Ow, K.T.; Wen, V.W.; MacKenzie, K.L.; Vermeulen, P.B.; Jackson, P.; Russell, P.J.

2006-01-01

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous 'take rate' in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation

Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse

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Maryam Rahimi Balaei

2016-01-01

Full Text Available Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2 mouse (nax—naked-ataxia mutant mouse correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5. In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

Science.gov (United States)

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Aplicación de la tribología y el análisis de la causa raíz (RCA en motores de combustión interna. // Application of tribology and root cause analysis (RCA on diesel engine.

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F. Martínez Pérez

2008-09-01

Full Text Available En el trabajo, se hace empleo de la herramienta de trabajo Análisis de la Causa Raíz (RCA para llegar a unas primerasconclusiones acerca de fallos producidos en el nudo tribológico válvula – guía de válvula en un motor de combustióninterna, y así pasar a hallar la causa final y dar solución al problema. El método permite arribar a conclusiones en cuanto ala causa del problema.Palabras claves: Tribología, Análisis causa raíz, RCA, desgaste, motor de combustión interna.______________________________________________________________________________Abstract:It is used the process root cause in the analysis of a problem in the tribological pair valve- valve guide of an internalcombustion engine. Using this process was possible to have the first conclusions in the problem and finally to arriveto a final conclusion about the cause of the problem.Key words: Tribology, RCA, wear, engine.

Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

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Tali Gidalevitz

2009-03-01

Full Text Available Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.

Results of the IAEA/RCA personal dosemeter intercomparison

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Tsujimura, Norio; Momose, Takumaro; Hayashi, Naomi [Environment and Safety Division, Tokai Works, Japan Nuclear Cycle Development Inst., Tokai, Ibaraki (Japan)

2000-06-01

The intercomparison of personal dosemeters for photon was carried out between 1990 and 1996 as part of the IAEA/RCA 'strengthening of radiation protection infrastructure' project. Japan Nuclear Cycle Development Institute participated in the intercomparison as one of the in-house personal dosimetry service organizations in Japan and also served the host irradiating laboratory. This report summarizes the dose evaluation results obtained from the JNC-TLD badges in the past four intercomparison programs. In the latest intercomparison the evaluated doses agreed to the reference doses with an accuracy of 10%. (author)

Contributions of Pakistan in the IAEA/RCA/UNDP regional project on management of marine coastal environment and its pollution

International Nuclear Information System (INIS)

Qureshi, R.M.; Mashiatullah, A.; Fazil, M.; Ahmad, E.; Tasneem, M.A.; Khan, H.A.; Sajjad, M.I.

2002-01-01

The International Atomic Energy Agency (IAEA), Vienna, launched a five years (duration: 1998 - 2002) Joint Project on 'Better Management of the Environment and Industrial Growth Through Isotope and Radiation Technology (RAS/97/030)' in co-operation with the RCA (Regional Co-operative Agreement) office, Vienna, and United Nations Development Programme (UNDP). The Marine Sub-project entitled 'Management of Marine Coastal Environment and its Pollution (RAS/8/083)' is 'Output 1.2' of this joint project. Pakistan is very actively participating in activities of the IAEA/RCA/UNDP Marine Sub-Project that were planned in two Project Formulation Meetings (PFMs) held at Manila, Philippines, during 1998. In Pakistan, various activities of the national marine pollution project are being administered by the nuclear institute namely, Pakistan Institute of Nuclear Science and Technology (PINSTECH), in collaboration with national end user institutions. To-date, Pakistan has significantly contributed in this project, both at national level and at RCA regional level. This paper highlights the progress and some accomplishments of Pakistan, up to the year 2001, for marine pollution studies related to the IAEA/RCA regional marine sub project. (author)

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

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Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack

1998-01-01

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions. PMID:9813103

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

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Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

2017-06-01

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

Recombination reduction at the c-Si/RCA oxide interface through Ar-H2 plasma treatment

Science.gov (United States)

Landheer, Kees; Bronsveld, Paula C. P.; Poulios, Ioannis; Tichelaar, Frans D.; Kaiser, Monja; Schropp, Ruud E. I.; Rath, Jatin K.

2017-02-01

An Ar-H2 plasma treatment was applied on an ultrathin RCA oxide to create well-passivated silicon wafers with symmetric c-Si/SiOx:H/a-Si:H passivation layer stacks. The effective lifetime of these samples increased from 10 μs to 4 ms after annealing at 200 °C through Ar-H2 plasma treatment of the oxide. The results indicate that the plasma treatment can modify the RCA oxide and this enables atomic hydrogen diffusion at low annealing temperature, leading to a well passivated c-Si/SiOx:H interface. This might provide new possibilities to use wet chemical oxides in c-Si solar cells, for example as tunnel contacts.

Design rules for RCA self-aligned silicon-gate CMOS/SOS process

Science.gov (United States)

1977-01-01

The CMOS/SOS design rules prepared by the RCA Solid State Technology Center (SSTC) are described. These rules specify the spacing and width requirements for each of the six design levels, the seventh level being used to define openings in the passivation level. An associated report, entitled Silicon-Gate CMOS/SOS Processing, provides further insight into the usage of these rules.

The IAEA regional cooperative agreement programme (RCA) on enhancement and harmonization of radiation protection in the Asia-Pacific Region (1998-2002)

International Nuclear Information System (INIS)

Cameron, R.F.; Oresegun, M.O.

2000-01-01

The IAEA Regional Cooperative Agreement (RCA) was initiated in 1972, and currently consists of 17 Member States from East Asia and Oceania. These countries are Australia, Bangladesh, China, India, Indonesia, Japan, Korea, Malaysia, Myanmar, Mongolia, New Zealand, Pakistan, Philippines, Singapore, Sri Lanka, Thailand and Vietnam. The RCA is one of three cooperative agreements under the aegis of the IAEA, the other two being for Africa (AFRA) and Latin America (ARCAL). The RCA agreement has been extended five times, as it meets particular needs of the participating countries. The region covered by the RCA comprises nearly half of the world's population and is undergoing a rapid expansion in nuclear power development and in the uses of radiation sources (radioactive materials and irradiating equipment). A number of activities in a wide range of areas of Agency interest are carried out under RCA. The Project to Strengthen Radiation Protection Infrastructures was initiated in 1988, as a five year programme, to offer a regional response to some of the radiation protection issues that were raised following the Chernobyl accident. It was extended into Phase 2 from 1993 to 1998 and has now reached a new phase. The IAEA has a dual role to establish standards in radiation protection and safety and to provide for their application. Since the status of the implementation of radiation safety laws and regulations is variable within the RCA countries, the primary focus has been on providing assistance to member states in the drafting of laws and regulations to ensure that sources are authorized and are properly controlled. A number of accidents worldwide have resulted from inappropriate or a complete lack of regulations on the control of sources. However as countries develop, their needs change and it is important that the RCA project in radiation protection changes with them. Hence the new Phase 3 programme in radiation protection has a mixture of events to strengthen radiation

Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

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David A. Dunn

2012-01-01

Full Text Available Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M or wildtype (A6W mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P0.05. This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

Co-expression and characterization of enterocin CRL35 and its mutant in Escherichia coli Rosetta

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Masías Emilse

2014-01-01

Full Text Available Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream of PelB sequence in the pET22b (+ expression vector. E. coli Rosetta (DE3 pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF and HPLCelectrospray (ESI-Q-TOF tandem mass spectrometry (MS/ MS sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system.

Purification, gene cloning, gene expression, and mutants of Dps from the obligate anaerobe Porphyromonas gingivalis.

Science.gov (United States)

Ueshima, Junichi; Shoji, Mikio; Ratnayake, Dinath B; Abe, Kihachiro; Yoshida, Shinichi; Yamamoto, Kenji; Nakayama, Koji

2003-03-01

The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR. The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9

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Susann Pohlers

2017-01-01

Full Text Available Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases—metalloenzymes that have been found in all domains of life—enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2. Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata. Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi.

Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

Science.gov (United States)

Ding, Mingquan; Jiang, Yurong; Cao, Yuefen; Lin, Lifeng; He, Shae; Zhou, Wei; Rong, Junkang

2014-02-10

Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

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Postina Rolf

2009-02-01

Full Text Available Abstract Background In a transgenic mouse model of Alzheimer disease (AD, cleavage of the amyloid precursor protein (APP by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant. Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice. Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. Conclusion In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of

In retrospect. 7th workshop RCA; Nachlese. 7. Workshop RCA. Conference report

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Knappik, Reinhard [Verein fuer Kernverfahrenstechnik und Analytik Rossendorf e.V. (VKTA), Dresden-Rossendorf (Germany). Programmausschuss Workshop RCA

2013-08-15

The 7th Workshop RCA with 27 lectures and 12 poster presentation took place on 10 and 11 June 2013 in Dresden-Rossendorf. The traditional German event brought over 90 participants together from Germany, Austria, France, Switzerland and the Netherlands. This time the workshop put forward a special subject for the first time ('Obstacles and pitfalls when characterising waste packages'). A remarkable range of contributions was presented, which demonstrated the diversity and the problems of this subject matter. Medium to low radioactive waste and their final disposal was discussed. Other topics were the upcoming approval of recycling materials according to paragraph 29 StrlSchV as well as those from NORM (Naturally Occurring Radioactive Materials) regarding radionuclide analytics, the range of matrices to be processed and the practical problems this causes. Although the radiological feature was the central focus, other material aspects were dealt with, above all the approval of recycling materials when nuclear power plants are dismantled, but also in research institutes, which is demanding more and more attention. The lectures by Jessica Riemer (GSI Darmstadt) on the topic 'Waste material management of a research centre with heavy ion accelerators' and Elvira Mesalic (TU Munich) on 'Determining the activity inventory of bituminized old wastes' as well as the poster by Sina Brockmann on the 'Locally resolved analysis or uranium species using a couple systems of CLSM and LIFS' were awarded prizes for being outstanding presentations. (orig.)

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

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Phanchaisri, Boonrak [Science and Technology Research Institute, Chiang Mai University, Chiang Mai 50200 (Thailand); Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Samsang, Nuananong [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, Liang Deng; Singkarat, Somsorn [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Anuntalabhochai, Somboon, E-mail: soanu.1@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

2012-06-01

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants

International Nuclear Information System (INIS)

Phanchaisri, Boonrak; Samsang, Nuananong; Yu, Liang Deng; Singkarat, Somsorn; Anuntalabhochai, Somboon

2012-01-01

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50–60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

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Mirian Perez Maluf

2009-01-01

Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient

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Alice Barateau

2017-04-01

Full Text Available Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A.

Current status of food irradiation in overseas (2013). From the meeting report of RCA food irradiation project

International Nuclear Information System (INIS)

Todoriki, Setsuko

2013-01-01

The report introduces the activity of RCA (Regional Cooperative Agreement for Research Development and Training Related to Nuclear Science and Technology for Asia and Pacific) food irradiation project (RAS/5/057) and information obtained at the two workshops on current status of Asia and Pacific areas together with EU, USA and Japan. Also current trends of RAS/5/057 Implementing Best Practices for food irradiation for plant sanitary and phytosanitary purposes are described. Amount of food irradiation products of RCA member countries, minimum adopted doses for quarantine harmful insects (70 to 232 Gy) and import amount of fruits of USA (79 to 5500 tons) are tabulated. Finally current status of Japan is explained. (S. Ohno)

The differential gene expression of key enzyme in the gibberellin pathway in the potato (solanum tuberosum) mutant

International Nuclear Information System (INIS)

Shi, J.B.; Ye, G.J.; Yang, Y.Z.; Wang, F.; Zhou, Y; Wang, J.

2016-01-01

In the present study, the expression patterns of the key genes in the gibberellin synthesis pathway in the potato dwarf mutant M4P-9 were detected using quantitative real-time PCR. Using Actin as an internal control, CPS1, KS, KO, GA20ox1, and GA2ox1, genes for key gibberellin synthesis enzymes, were evaluated, along with a gibberellin receptor gene. The standard curves were obtained from dilutions of PCR product; the correlation coefficient for Actin was 0.995, and those for the target genes varied from 0.994 to 1.000. The expression patterns of gibberellin pathway genes in different growth stages and tissues were calculated according to the method of Pfaffl. These genes showed expression patterns that varied based on growth stage and tissue type. The higher expression levels of CPS1 and GA2ox1 in roots, the lower expression levels of GA20ox1 in roots during tuber formation stage; as well as the increased expression of GA20ox1 and GA2ox1 genes in stems during the tuber formation stage, likely play key roles in the plant height phenotype in M4P-9 mutant materials. This article provides a basis for researching the mechanism of gibberellin synthesis in potato. (author)

Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

Science.gov (United States)

Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

2014-01-01

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

Science.gov (United States)

Kia, Azadeh; McAvoy, Kevin; Krishnamurthy, Karthik; Trotti, Davide

2018-01-01

Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS. PMID:29380416

45 CFR 400.66 - Eligibility and payment levels in a publicly-administered RCA program.

Science.gov (United States)

2010-10-01

... 45 Public Welfare 2 2010-10-01 2010-10-01 false Eligibility and payment levels in a publicly... REFUGEE RESETTLEMENT PROGRAM Refugee Cash Assistance § 400.66 Eligibility and payment levels in a publicly-administered RCA program. (a) In administering a publicly-administered refugee cash assistance program, the...

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

International Nuclear Information System (INIS)

Wang Qishan; Bag, Jnanankur

2008-01-01

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO 4 , and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein

Mutants of Agrobacterium tumefaciens with elevated vir gene expression

International Nuclear Information System (INIS)

Pazour, G.J.; Ta, C.N.; Das, A.

1991-01-01

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG

[hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis].

Science.gov (United States)

Xia, Zhen Wei; Cui, Wen Jun; Zhou, Wen Pu; Zhang, Xue Hong; Shen, Qing Xiang; Li, Yun Zhu; Yu, Shan Chang

2004-10-01

Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.

Production of a Marfan cellular phenotype by expressing a mutant human fibrillin allele on a normal human or murine genetic background

Energy Technology Data Exchange (ETDEWEB)

Eldadah, Z.A.; Dietz, H.C. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States); Brenn, T. [Stanford Univ. Medical Center, CA (United States)] [and others

1994-09-01

The Marfan Syndrome (MFS) is a heritable disorder of connective tissue caused by defects in fibrillin (FBN1), a 350 kD glycoprotein and principal component of the extracellular microfibril. Previous correlations of mutant transcript level and disease severity suggested a dominant negative model of MFS pathogenesis. To address this hypothesis we assembled an expression construct containing the mutant allele from a patient with severe MFS. This mutation causes skipping of FBN1 exon 2 and a frame shift, leading to a premature termination codon in exon 4. The predicted peptide would thus consist of 55 wild type and 45 missense amino acids. The construct was stably transfected into cultured human and mouse fibroblasts, and several clonal cell populations were established. Human and mouse cells expressing the truncated peptide exhibited markedly diminished fibrillin deposition and disorganized microfibrillar architecture by immunofluorescence. Pulse-chase analysis of these cells demonstrated normal levels of fibrillin synthesis but substantially decreased fibrillin deposition into the extracellular matrix. These data illustrate that expression of a mutant FBN1 allele, on a background of two normal alleles, is sufficient to disrupt normal fibrillin aggregation and reproduce the MFS cellular phenotype. This provides confirmation of a dominant negative model of MFS pathogenesis and may offer mutant allele knockout as a strategy for gene therapy. In addition, these data underscore the importance of the FBN1 amino-terminus in normal multimer formation and suggest that expression of the human extreme 5{prime} FBN1 coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Indeed, transgenic mice harboring this mutant allele have been produced, and phenotype analysis is currently in progress.

Whole genome-wide transcript profiling to identify differentially expressed genes associated with seed field emergence in two soybean low phytate mutants.

Science.gov (United States)

Yuan, Fengjie; Yu, Xiaomin; Dong, Dekun; Yang, Qinghua; Fu, Xujun; Zhu, Shenlong; Zhu, Danhua

2017-01-18

Seed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 . Numerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways. Seed germination in the soybean low phytate mutants is a very complex process

Mechanical properties of concrete containing recycled concrete aggregate (RCA) and ceramic waste as coarse aggregate replacement

Science.gov (United States)

Khalid, Faisal Sheikh; Azmi, Nurul Bazilah; Sumandi, Khairul Azwa Syafiq Mohd; Mazenan, Puteri Natasya

2017-10-01

Many construction and development activities today consume large amounts of concrete. The amount of construction waste is also increasing because of the demolition process. Much of this waste can be recycled to produce new products and increase the sustainability of construction projects. As recyclable construction wastes, concrete and ceramic can replace the natural aggregate in concrete because of their hard and strong physical properties. This research used 25%, 35%, and 45% recycled concrete aggregate (RCA) and ceramic waste as coarse aggregate in producing concrete. Several tests, such as concrete cube compression and splitting tensile tests, were also performed to determine and compare the mechanical properties of the recycled concrete with those of the normal concrete that contains 100% natural aggregate. The concrete containing 35% RCA and 35% ceramic waste showed the best properties compared with the normal concrete.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

KAUST Repository

Lissanu Deribe, Yonathan

2016-03-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

Science.gov (United States)

Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B; Wu, Chia-Chin; Akdemir, Kadir C; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T; Welch, Heidi C E; Garraway, Levi A; Chin, Lynda

2016-03-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

KAUST Repository

Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

2016-01-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Operation EUFOR TCHAD/RCA and the European Union’s Common Security and Defense Policy

Science.gov (United States)

2010-10-01

Lessons from EUFOR Tchad/RCA, March 2010. 152. For a more detailed description of the EU Battlegroups, see Gustav Lindstrom , “Enter the EU Battlegroups...Major General Gregg F. Martin Commandant ***** STRATEGIC STUDIES INSTITUTE Director Professor Douglas C. Lovelace, Jr. Director of Research Dr. Antulio J

Problem-Solving Test: Tryptophan Operon Mutants

Science.gov (United States)

Szeberenyi, Jozsef

2010-01-01

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

DEFF Research Database (Denmark)

Hoshino, Tatsuhiko; Schramm, Andreas

2010-01-01

target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal-to-noise ratio but low detection frequency (up to 15% for single-copy genes and up to 43% for the multi-copy 16S rRNA gene...... as Candidatus Accumulibacter phosphatis by combining in situ RCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells.......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

IAEA-RCA Co-ordinated Research Program on Reference Asian Man

International Nuclear Information System (INIS)

Koyanagi, Taku

1990-01-01

The Research Coordination Meeting was held in Mito City, Japan on October 17-21, 1988, inviting the chief investigating scientists from 11 RCA member countries to discuss practical plans for the Coordinated Research Program (CRP) on 'Compilation of Anatomical, Physiological and Metabolic Characteristics for a Reference Asian Man' based on the decision taken at the Project Formulation Meeting for the RCA Project 'Strengthening Radiation Protection' Tokyo, November 1987. Significance of the setting 'Reference Man' for Asian peoples to estimate more realistic radiation doses by applying the real typical physical, physiological and metabolic parameters for them instead of those recommended by ICRP based on the data for 'Caucasian Man' has been indicated by whole member countries and recognized again at the Meeting. The present status of 'Reference Man-oriented Studies' in each countries was presented by the participants and certain difference or difficulties were pointed out among the countries depending on the geographical, social, or economical conditions as well as the ethnic circumstances. After the mutual discussions and exchange of up-to-date information, the general conclusions were drawn as follows: acknowledging the importance of the CRP, research works should be carried out in each country with the expected supports from IAEA and other member countries. The first priority is given on the measurements of human physique (and internal organs) followed by the food consumption survey. Trace element analysis would be done by the countries where possible. The standard manual for data collection might be necessary. The establishments of Co-ordination Center or central body with data base and also subgroup systems are desirable to promote the CRP. (author)

P01.29 Mutant (R132H) IDH1-driven cellular transformation makes cells dependent on continued wild type IDH1 expression in a model of in vitro gliomagenesis

Science.gov (United States)

Johannessen, T.; Mukherjee, J.; Wood, M.; Viswanath, P.; Ohba, S.; Ronen, S.; Berkvig, R.; Pieper, R.

2017-01-01

Abstract Introduction: Missense R132H mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. We recently showed (Mol Cancer Res 14: 976–983, 2016) that although mutant IDH1 expression in hTERT-immortalized, p53/pRb-deficient astrocytes can drive cellular transformation and gliomagenesis, selective pharmacologic inhibition and elimination of 2-HG by the mutant IDH1 inhibitor AGI-5198 has little effect on the growth or clonagenicity of these transformed cells. To address the possible role of WT IDH1 in the growth of mutant IDH-driven tumor cells, we used a slightly different gliomagenesis model in which the transformation of TERT-deficient, p53/pRb-deficient astrocytes (pre-crisis cells) occurs only after prolonged expression of mutant IDH and passage through cellular crisis (post-crisis cells, Cancer Res 76:6680–6689, 2016). METHODS AND MATERIALS: Using this system we introduced AGI-5198, or siRNA targeting both WT and mutant forms of IDH1 into p53/pRb-deficient, mutant IDH1-expressing human astrocytes prior to or following their transformation, and compared the effects on cell growth and clonagenicity. Results: AGI-5198 exposure decreased levels of 2HG by greater than 90%, and as previously reported had no effect on the growth of either the pre-or post-crisis cell populations. A one-day exposure to a pan IDH1 siRNA resulted in a similar, prolonged (greater than 6 day), 80% inhibition of both WT and mutant IDH1 protein levels and 2HG in both cell groups. While the growth of the mutant IDH-expressing, non-transformed cells was similar to that of scramble siRNA controls, the growth

Requirements of cyclin a for mitosis are independent of its subcellular localization.

Science.gov (United States)

Dienemann, Axel; Sprenger, Frank

2004-06-22

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. We show that interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Here, we tested if the unique mitotic function of CycA requires nuclear accumulation. We fused subcellular localization signals to CycA and tested their mitotic capability. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA was able to induce mitosis. We noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16(th) embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein.

Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants

Science.gov (United States)

Mustapha, Mirna; Fang, Qing; Gong, Tzy-Wen; Dolan, David F.; Raphael, Yehoash; Camper, Sally A.; Duncan, R. Keith

2012-01-01

The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been utilized in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1dw), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated β-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1dw mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1dw mutant outer hair cells and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1dw mice. PMID:19176829

Differentially expressed genes in the head of the 2nd instar pre-molting larvae of the nm2 mutant of the silkworm, Bombyx mori.

Directory of Open Access Journals (Sweden)

Pingyang Wang

Full Text Available Molting is an important physiological process in the larval stage of Bombyx mori and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2nd instar (nm2 is incapable of molting in the 2nd instar and dies after seven or more days. The ecdysone titer in the nm2 mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of BmCPG10 is responsible for the phenotype of the nm2 mutant. To explore the possible relationship between BmCPG10 and the ecdysone titer as well as the genes affected by BmCPG10, digital gene expression (DGE profile analysis was conducted in the nm2 mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in nm2. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the nm2 mutant.

Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

Science.gov (United States)

Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

2017-09-15

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

Nimotuzumab enhances temozolomide?induced growth suppression of glioma cells expressing mutant EGFR in vivo

OpenAIRE

Nitta, Yusuke; Shimizu, Saki; Shishido?Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

Abstract A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti?EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild?type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and pho...

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

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Karen L Posey

2010-04-01

Full Text Available Mutations in cartilage oligomeric matrix protein (COMP, a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

Science.gov (United States)

Regales, Lucia; Balak, Marissa N; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A; Solit, David B; Rosen, Neal; Zakowski, Maureen F; Pao, William

2007-08-29

The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

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Lucia Regales

2007-08-01

Full Text Available The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M-expressing animals develop tumors with longer latency than EGFR(L858R+T790M-bearing mice and in the absence of additional kinase domain mutations.These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis

NARCIS (Netherlands)

Bokma, Evert; Rozeboom, Henriëtte J.; Sibbald, Mark; Dijkstra, Bauke W.; Beintema, Jaap J.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as

Thylakoid redox signals are integrated into organellar-gene-expression-dependent retrograde signalling in the prors1-1 mutant

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Luca eTadini

2012-12-01

Full Text Available Perturbations in organellar gene expression (OGE and the thylakoid redox state (TRS activate retrograde signalling pathways that adaptively modify nuclear gene expression (NGE, according to developmental and metabolic needs. The prors1-1 mutation in Arabidopsis down-regulates the expression of the nuclear gene Prolyl-tRNA Synthetase1 (PRORS1 which acts in both plastids and mitochondria, thereby impairing protein synthesis in both organelles and triggering OGE-dependent retrograde signalling. Because the mutation also affects thylakoid electron transport, TRS-dependent signals may likewise have an impact on the changes in NGE observed in this genotype. In this study, we have investigated whether signals related to TRS are actually integrated into the OGE-dependent retrograde signalling pathway. To this end, the chaos mutation (for chlorophyll a/b binding protein harvesting-organelle specific, which shows a partial loss of PSII antennae proteins and thus a reduction in PSII light absorption capability, was introduced into the prors1-1 mutant background. The resulting double mutant displayed a prors1-1-like reduction in plastid translation rate and a chaos-like decrease in PSII antenna size, whereas the hyper-reduction of the thylakoid electron transport chain, caused by the prors1-1 mutation, was alleviated, as determined by monitoring chlorophyll (Chl fluorescence and thylakoid phosphorylation. Interestingly, a substantial fraction of the nucleus-encoded photosynthesis genes down-regulated in the prors1-1 mutant are expressed at nearly wild-type rates in prors1-1 chaos leaves, and this recovery is reflected in the steady-state levels of their protein products in the chloroplast. We therefore conclude that signals related to photosynthetic electron transport and TRS, and indirectly to carbohydrate metabolism and energy balance, are indeed fed into the OGE-dependent retrograde pathway to modulate NGE and adjust the abundance of chloroplast proteins.

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

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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.

Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster

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Noriko Wakabayashi-Ito

2015-07-01

Full Text Available Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1, the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG deletion that results in the loss of a glutamic acid residue (ΔE302/303 in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4 levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis.

Science.gov (United States)

Zhang, Yong Q; Friedman, David B; Wang, Zhe; Woodruff, Elvin; Pan, Luyuan; O'donnell, Janis; Broadie, Kendal

2005-03-01

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

Structure and expression of cytochrome f in an Oenothera plastome mutant.

Science.gov (United States)

Johnson, E M; Sears, B B

1990-06-01

The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.

Recombination reduction at the c-Si/RCA oxide interface through Ar-H2 plasma treatment

NARCIS (Netherlands)

Landheer, K.; Bronsveld, P.C.P.; Poulios, I.; Tichelaar, F.D.; Kaiser, M.; Schropp, R.E.I.; Rath, J.K.

2017-01-01

An Ar‑H2 plasma treatment was applied on an ultrathin RCA oxide to create well-passivated silicon wafers with symmetric c‑Si/SiOx:H/a‑Si:H passivation layer stacks. The effective lifetime of these samples increased from 10 μs to 4 ms after annealing at 200 °C through Ar‑H2 plasma treatment of the

Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

International Nuclear Information System (INIS)

Wang Qishan; Bag, Jnanankur

2006-01-01

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including α-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis

Role of nuclear energy in Bangladesh: experience with IAEA and RCA

International Nuclear Information System (INIS)

Quaiyum, M.A.

1998-01-01

Electricity is one of the most convenient forms of energy for sustaining and accelerating industrial growth and thus augmenting the poverty alleviation. All conventional modes of generation of electricity have adverse effects on environment. The nature, quantities and extents of such effects are, however, specific to the type of generations. Taking Bangladesh as a representative developing country the role of nuclear energy is presented. Demand projections for electricity, energy resources and energy development are discussed. It is highlighted that the lack of suitably qualified and experienced manpower has to be addressed properly. Hence training in selected areas is being arranged at home and abroad with own resources and also under different IAEA, RCA and bilateral assistance programmes. The paper also discusses the safety and environmental aspects of nuclear energy and highlights the contribution of IAEA and RCA in establishing global safety culture and pollution free environment. It is concluded that Bangladesh has to depend heavily on imported fuel. Among the important fuels, nuclear is a viable proposition and hence needs careful consideration. This is of course true for all other developing countries like Bangladesh. It is hoped that in order to dispel the apprehension of nuclear proliferation, the concept of regional reprocessing centre may be thought of. This centre may be planned and implemented by pulling resources and expertise of developing countries, specially the neighbouring ones, so as to recover the fissile materials from the spent fuel and use them in the fast breeder reactor which may be considered as the energy source of the future, and thus sustainable development can be ensured. (author)

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

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David Ramonet

2011-04-01

Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene cause late-onset, autosomal dominant familial Parkinson's disease (PD and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s through which familial mutations precipitate neuronal degeneration and PD.

The contribution of the UNDP/RCA/IAEA programmes to the development of nuclear technology in the Asia Pacific Region

International Nuclear Information System (INIS)

Easey, J.F.

1994-01-01

The UNDP/RCA/IAEA Programmes are involving Member States in the Asia Pacific Region in projects to develop and increase the use of nuclear technologies. These efforts have already increased the level of awareness amongst the Region's scientists, engineers, technologists, and decision makers and has provided significant training to the scientists and engineers to enable them to play a role in technical back stopping at a national level. A new project has been started in 1993 and this will seek to expand the contribution of the nuclear technologies into further industrial and environmental fields. Much of the success of the programmes is credited to the high level of Regional Co-operative Agreement (RCA) for research, development and training related to nuclear science and technology in Asia and the Pacific

Bulk magnetic characterization of RCaCrO4 (Rrl02=Y, Pr, Sm ) oxides

International Nuclear Information System (INIS)

Martinez, J.L.; Fernandez-Diaz, M.T.; Chen, Q.; Prieto, C.; Andres, A. de; Saez-Puche, R.; Romero, J.

1995-01-01

The system RCaCrO 4 (R=Y, Sm, Pr) presents an orthorhombic structure (space group Bmab) at room temperature (RT), similar to that observed in La 2 MO 4 (M=Cu, Ni, Co). The magnetic susceptibility for RCaCrO 4 shows a weak temperature dependence down to 250 K, probably due to the antiferromagnetic ordering of the Cr sublattice above RT. Below RT there is a strong upturn anomaly at 210, 190 and 130 K for Pr, Sm and Y, respectively. This anomaly is associated with the appearance of a weak ferromagnetic component, and could be related to a low temperature structural phase transition, similar to that observed in the related compounds R 2 NiO 4 or La 1.88 Ba 0.12 CuO 4 . In the case of YCaCrO 4 this ferromagnetic component produces a hysteresis curve at 4.5 K with a coercive field of 0.7 T. For PrCaCrO 4 the coercive field is very small ( 4 shows a more complicated behavior with a low temperature magnetic transition (T N2 ∼40 K), which could be associated with either the antiferromagnetic ordering of the Sm sublattice or a spin reorientation in the Cr sublattice. ((orig.))

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

International Nuclear Information System (INIS)

Van Rossom, Sofie; Op de Beeck, Ken; Franssens, Vanessa; Swinnen, Erwin; Schepers, Anne; Ghillebert, Ruben; Caldara, Marina; Van Camp, Guy; Winderickx, Joris

2012-01-01

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 2–6. 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 Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

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

Energy Technology Data Exchange (ETDEWEB)

Van Rossom, Sofie [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Op de Beeck, Ken [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Franssens, Vanessa; Swinnen, Erwin [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Schepers, Anne [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Ghillebert, Ruben; Caldara, Marina [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium); Van Camp, Guy [Department of Biomedical Sciences, Center of Medical Genetics, University of Antwerp, Wilrijk-Antwerp (Belgium); Winderickx, Joris, E-mail: guy.vancamp@ua.ac.be, E-mail: joris.winderickx@bio.kuleuven.be [Department of Biology, Functional Biology, KU Leuven, Leuven-Heverlee (Belgium)

2012-07-25

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 2–6. 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 Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

Retrospective Molecular Epidemiology Study of PD-L1 Expression in Patients with EGFR-Mutant Non-small Cell Lung Cancer.

Science.gov (United States)

Cho, Jong Ho; Zhou, Wei; Choi, Yoon-La; Sun, Jong-Mu; Choi, Hyejoo; Kim, Tae-Eun; Dolled-Filhart, Marisa; Emancipator, Kenneth; Rutkowski, Mary Anne; Kim, Jhingook

2018-01-01

Data are limited on programmed death ligand 1 (PD-L1) expression in epidermal growth factor receptor ( EGFR )-mutant non-small cell lung cancer (NSCLC). We retrospectively evaluated the relationship between PD-L1 expression and recurrence-free survival (RFS) and overall survival in 319 patients with EGFR -mutant NSCLC who were treated at Samsung Medical Center from 2006 to 2014. Membranous PD-L1 expression on tumor cells was measured using the PD-L1 IHC 22C3 pharmDx antibody and reported as tumor proportion score (TPS). Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis. All patients had ≥1 EGFR mutation-54% in exon 19 and 39% in exon 21. Overall, 51% of patients had PD-L1-positive tumors. The prevalence of PD-L1 positivity was higher among patients with stages II-IV versus stage I disease (64% vs. 44%) and among patients with other EGFR mutations (75%) than with L858R mutation (39%) or exon 19 deletion (52%). PD-L1 positivity was associated with shorter RFS, with an adjusted hazard ratio of 1.52 (95% confidence interval [CI], 0.81 to 2.84; median, 18 months) for the PD-L1 TPS ≥ 50% group, 1.51 (95% CI, 1.02 to 2.21; median, 31 months) for the PD-L1 TPS 1%-49% group, and 1.51 (95% CI, 1.05 to 2.18) for the combined PD-L1-positive groups (TPS ≥ 1%) compared with the PD-L1-negative group (median, 35 months). PD-L1 expression is associated with disease stage and type of EGFR mutation. PD-L1 positivity might be associated with worse RFS among patients with surgically treated EGFR -mutant NSCLC.

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

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Nannenga Brent L

2011-12-01

Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

Analysis of the N-glycosylation of the serum glycoproteins defined by Con A, PHA-E, RCA1 and WGA in Chinese patients with gastrointestinal diseases.

Science.gov (United States)

Zhang, J; Chen, Y; Dai, X; Wang, S

1996-03-01

Glycoproteins from tumor cells isolated by Con A, PHA-E and RCA1, were coupled to horse radish peroxidase and then used as reporters to evaluate the oligosaccharides change on serum glycoproteins in digestive tract diseases and individual healthy persons. A value 2 standard deviation above the mean of the healthy person group was considered positive. The results indicated that the positive rates of the oligosaccharides bound with Con A, PHA-E and RCA1 were 51% (61/119), 70.6% (84/119), and 76.4% (91/119) in patients with gastric cancer; 53.3% (16/30), 46.7% (14/30) and 66.7% (20/30) in esophageal cancers; 28.5% (15/49), 49.0% (24/49) and 46.9% (23/49) in colorectal cancer; 78.1% (25/32), 81.3% (26/32) and 31.3% (10/32) in gallbladder tumors; 60.4% (29/48), 54.1% (26/48) and 39.5% (19/48) in hepatocellular cancer. The positive rates among the patients with benign diseases were also found to be 12.7% (16/126), 15.5% (20/126) and 26.2% (33/126). However, the expression of the oligosaccharide moieties recognized by WGA on the glycoprotein bound with Con A, was significantly lower in the serum of patients with tumors than in those with benign disease and in healthy individuals (P < 0.01, P < 0.01).

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

DEFF Research Database (Denmark)

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

1992-01-01

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

Mutant number distribution in an exponentially growing population

Science.gov (United States)

Keller, Peter; Antal, Tibor

2015-01-01

We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491-511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process.

The use of mutant stock for semi-dwarf plant type and early maturity as cross-breeding materials in rice (coordinated programme on semi-dwarf mutants for rice improvement in Asia and the Pacific Region - RCA)

International Nuclear Information System (INIS)

Pookamana, P.

1985-11-01

Development of semi-dwarf type varieties has led to a substantial yield increase in Thailand, where rice is cultivated on areas of about 8 million hectars. Of 19 recommended Thai rice varieties, 10 possess the DGWG gene which determines semi-dwarf character. This situation can create some problems in terms of genetic vulnerability towards pests, diseases and other adverse stresses. The authors found that susceptibility to disease and insect pests as well as poor grain quality are major drawbacks associated with the DGWG gene. With this background, the major project goal was to obtain, by mutation techniques, a new genetic source of semi-dwarfness. For this purpose 3 tall local Thai varieties (Leaung Awn 29, Niaw Sanpah Tawng and Khao Dawk Mali) with very good grain quality, were chosen as parent material. Using radiation techniques 2 glutinous and 7 non-glutinous semi-dwarf mutants were obtained. Results of agronomic trials indicated that the mutants from local varieties were more promising and higher yielding in comparison to introduced varieties from IRRI, Taiwan, China, France and the USA. The preliminary genetics analysis of these mutants indicated an allelic relationship to the DGWG gene. This result is very important for the rice breeding programme because it made available the semi-dwarf gene in a new genetic background - with desirable characteristics for cultivation in Thailand. These positive results stimulated efforts to obtain new semi-dwarf mutants from other local, lowland rice varieties such as Leaung Tawang and Leb Mue Nahng 111. 49 and 35 semi-dwarf mutants were already selected after irradiation and are now under agronomic evaluation

Der Zellzyklusregulator Rca1 - Inhibitor und Substrat des Anaphase-Promoting-Komplexes in Drosophila melanogaster

OpenAIRE

Morgenthaler, Christoph

2014-01-01

Ein wichtiger Kontrollmechanismus des Zellzyklus ist die irreversible Proteolyse von Zellzyklus-Regulatoren. Dabei markieren E3-Ligasen Zielproteine durch Ubiquitinmoleküle. Für den Abbau in der Mitose und der G1-Phase reguliert der APC/C-Komplex (Anaphase-Promoting-Complex/Cyclosome) als E3-Ligase den zeitlichen Verlauf des Zellzyklus. Die Aktivität des APC/C wiederum wird in den übrigen Zellzyklusstadien durch Phosphorylierung und durch Proteine der Rca1/Emi1-Proteinfamilie inaktiv gehalten...

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)

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.

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

International Nuclear Information System (INIS)

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

2013-01-01

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

Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

Science.gov (United States)

García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

2015-01-01

Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

X-ray-induced mutants resistant to 8-azaguanine

International Nuclear Information System (INIS)

Carver, J.H.; Dewey, W.C.; Hopwood, L.E.

1976-01-01

Asynchronous Chinese hamster ovary cells were irradiated and colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 μg/ml 8-Azaguanine (AG). Data indicated that a reproducible assay for the system was dependent upon controlling cell density at least two days prior to induction as well as throughout the expression period. Generally, spontaneous and radiation-induced mutant frequencies decreased when cell densities exceeded a critical density of 3-6 x 10 4 cells/cm 2 . Infrequently, the critical density was exceeded by a factor of two with no observed decrease, possibly correlated with a longer cell doubling time. Drug depletion artifacts can occur because of drug degradation, or because wild-type cells utilize the drug or produce conditions which reduce uptake of the drug. Thus, as the effective drug concentration is lowered, the observed mutant frequency increases because a spectrum of mutants resistant to only low concentrations can now survive. In fact, refeeding with AG at intervals during the incubation period lowered spontaneous and radiation-induced frequencies approx. 5-fold. Therefore, to standardize conditions, cells were trypsinized at the end of the expression time and replated at a constant cell number for mutant selection by AG. Over two generations of growth during the expression period were required for optimal manifestation of induced mutants, and when densities were kept below 4 x 10 4 cells/cm 2 at all times, observed mutant frequencies did not change significantly over a period between 80 and 140 h post-induction (over 4 generations for irradiated cells and over 6 generations for controls). Previous reports of observed mutant frequencies decreasing beyond three generations may be due to cell interaction prior to mutant selection

Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics

DEFF Research Database (Denmark)

Li, Haiying; Rasmussen, Morten I; Larsen, Martin R

2015-01-01

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heteroge...

Mutant number distribution in an exponentially growing population

International Nuclear Information System (INIS)

Keller, Peter; Antal, Tibor

2015-01-01

We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491–511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process. (paper)

[The Expression of Pokemon in Endometrial Carcinoma Tissue and the Correlation with Mutant p53].

Science.gov (United States)

Yi, Tian-jin; Wang, Ping

2016-05-01

To detect the expression of Pokemon in endometrial carcinoma (EC), to provide preliminary theoretical basis for clarifying pathogenesis and searching for effective targets. Ninety-eight cases of endometrial tissue paraffin specimens form July 2012 to July 2014 in West China Second University Hospital, Sichuan University, were collected, including: EC group, consisting of adenocarcinoma 23 cases, adenosquamous 12 cases, serous 3 cases, mucinous 11 cases and clear cell 9 cases, and control group, consisting of atypical hyperplasia endometrium 20 cases and normal endometrium 20 cases (secretory 10 cases, hyperplasia 10 cases). Immunohistochemistry was used to detect the expression of Pokemonin each section, analyzing the correlation of Pokemon expression with clinicopathologic characteristics and p53 expression. The positive rate of Pokemon in normal endometrium was 25% (5/20), significantly lower than that in atypical hyperplasia endometrium (60.0%, 12/20) and EC (93.1%, 54/58) (P Pokemon in III-IV stage, type II and Ki-67 ≥ 50 EC tissue was much higher (P = 0.012, 0.023, 0.029). In type II EC tissue, the correlation index between Pokemon and p53 is 0.669 (P = 0.000). The over expression of Pokemon upregulates the expression of mutant p53, which may be one of the carcinogenesis modes in type II EC.

In retrospect. 7th workshop RCA

International Nuclear Information System (INIS)

Knappik, Reinhard

2013-01-01

The 7th Workshop RCA with 27 lectures and 12 poster presentation took place on 10 and 11 June 2013 in Dresden-Rossendorf. The traditional German event brought over 90 participants together from Germany, Austria, France, Switzerland and the Netherlands. This time the workshop put forward a special subject for the first time ('Obstacles and pitfalls when characterising waste packages'). A remarkable range of contributions was presented, which demonstrated the diversity and the problems of this subject matter. Medium to low radioactive waste and their final disposal was discussed. Other topics were the upcoming approval of recycling materials according to paragraph 29 StrlSchV as well as those from NORM (Naturally Occurring Radioactive Materials) regarding radionuclide analytics, the range of matrices to be processed and the practical problems this causes. Although the radiological feature was the central focus, other material aspects were dealt with, above all the approval of recycling materials when nuclear power plants are dismantled, but also in research institutes, which is demanding more and more attention. The lectures by Jessica Riemer (GSI Darmstadt) on the topic 'Waste material management of a research centre with heavy ion accelerators' and Elvira Mesalic (TU Munich) on 'Determining the activity inventory of bituminized old wastes' as well as the poster by Sina Brockmann on the 'Locally resolved analysis or uranium species using a couple systems of CLSM and LIFS' were awarded prizes for being outstanding presentations. (orig.)

Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

Science.gov (United States)

Gaballo, Antonio; Signorile, Anna; Tanzarella, Paola; Pacelli, Consiglia; Di Paola, Marco

2017-01-01

In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts. PMID:29138676

Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

Directory of Open Access Journals (Sweden)

Daniele Vergara

2017-01-01

Full Text Available In this study, we investigated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson’s disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson’s disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

Andrographolide induces degradation of mutant p53 via activation of Hsp70.

Science.gov (United States)

Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

2018-05-22

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

A Report on IAEA/RCA C7-RAS 6/061-004 Training Course in Chiba, Japan in 2014

International Nuclear Information System (INIS)

Kosuda, Shigeru; Saga, Tsuneo; Paez, Diana

2015-01-01

The C7-RAS 6/061-004 training course by the International Atomic Energy Agency/Regional Cooperative Agreement (IAEA/RCA) was held in Chiba in 2014. The syllabus, pre- and post-course evaluations, and survey questionnaire results were assembled in this course. The post-course evaluation, including 32 questions similar to the pre-course evaluation, was performed right after the end of the final educational lecture. The mean score showed an improvement, with the score rising from 57.0 points at the beginning to 66.5 points at the end. Among 22 trainees, the greatest score was in a higher range, with an improvement from 82 points at the beginning to 88 points at the end. The grading distribution, with regard to the training course, was as follows: excellent (68.2%), good (31.8%), average (0%), fair (0%), and poor (0%). This report on the training course, held in Chiba in 2014, will contribute to the future global plans of IAEA/RCA. Continuous training courses in member states are required to decrease the present disparities in the knowledge level, instrumentation, and human resources

Transgenic mice display hair loss and regrowth overexpressing mutant Hr gene.

Science.gov (United States)

Zhu, Kuicheng; Xu, Cunshuan; Zhang, Jintao; Chen, Yingying; Liu, Mengduan

2017-10-30

Mutations in the hairless (Hr) gene in both mice and humans have been implicated in the development of congenital atrichia, but the role of Hr in skin and hair follicle (HF) biology remains unknown. Here, we established transgenic mice (TG) overexpressing mutant Hr to investigate its specific role in the development of HF. Three transgenic lines were successfully constructed, and two of them (TG3 and TG8) displayed a pattern of hair loss and regrowth with alternation in the expression of HR protein. The mutant Hr gene inhibited the expression of the endogenous gene in transgenic individuals, which led to the development of alopecia. Interestingly, the hair regrew with the increase in the endogenous expression levels resulting from decreased mutant Hr expression. The findings of our study indicate that the changes in the expression of Hr result in hair loss or regrowth.

Methods of producing protoporphyrin IX and bacterial mutants therefor

Science.gov (United States)

Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

2016-03-01

The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

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)

Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

Science.gov (United States)

Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

2000-01-01

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9.

Science.gov (United States)

Pohlers, Susann; Martin, Ronny; Krüger, Thomas; Hellwig, Daniela; Hänel, Frank; Kniemeyer, Olaf; Saluz, Hans Peter; Van Dijck, Patrick; Ernst, Joachim F; Brakhage, Axel; Mühlschlegel, Fritz A; Kurzai, Oliver

2017-01-31

Adaptation to alternating CO 2 concentrations is crucial for all organisms. Carbonic anhydrases-metalloenzymes that have been found in all domains of life-enable fixation of scarce CO 2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO 2 ). Expression of NCE103 is regulated in response to CO 2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO 2 -dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO 2 -dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata Deletion of SCH9 homologues of both species impaired CO 2 -dependent regulation of NCE103 expression, which indicates a conservation of the CO 2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO 2 adaptation to lipid signaling via Pkh1/2 in fungi. All living organisms have to cope with alternating CO 2 concentrations as CO 2 levels range from very low in the atmosphere (0.04%) to high (5% and more) in other niches, including the human body. In fungi, CO 2 is sensed via two pathways. The first regulates virulence in

Spectrum of induced floral mutants in Petunia

International Nuclear Information System (INIS)

Padmaja, V.; Sudhakar, P.

1987-01-01

A total of six floral mutants of garden Petunia isolated from the populations raised from the seed treatment with γ-rays, 2, 4-D and sodium azide are described. Five of the mutants viz. stellata, Campyloflora, Rubriflora mixed, Grandiflora and Albiflora mixed originated as segregants in M 2 generation while the chimeral floral phenotype was expressed in M 1 generation itself. Breeding behaviour of these horticulturally interesting altered floral phenotypes were studied in subsequent generations and appropriate conclusions were drawn regarding mode of inheritance of the mutant traits. 15 refs., 4 figures, 1 table. (author)

Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant

Energy Technology Data Exchange (ETDEWEB)

Shinkyo, Raku; Inouye, Kuniyo [Kyoto Univ. (Japan). Div. of Food Science and Biotechnology; Kamakura, Masaki; Ikushiro, Shin-ichi; Sakaki, Toshiyuki [Toyama Prefectural Univ. (Japan). Biotechnology Research Center

2006-09-15

Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s ({delta}1A1 and {delta}F240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that {delta}F240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both {delta}F240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E. coli cell culture at a final concentration of 10 {mu}M, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing {delta}F240A to the bioremediation of PCDD-contaminated soil. (orig.)

Two rubisco activase genes from ipomoea batatas have different roles in photosynthesis of arabidopsis

International Nuclear Information System (INIS)

Jiang, Y.

2014-01-01

Rubisco activase (RCA) that functions as a molecular chaperone regulates the activity of the Calvin-Benson cycle via regulation of the Rubisco activity. In plants such as Arabidopsis thaliana, Spinacia oleracea, and Oryza sativa, there are two RCA isoforms from two mRNAs that are produced from alternative splicing of the transcribed pre-mRNA of a single RCA gene. However, this research reported that the transcripts of the two IbRCA isoforms in sweet potato (Ipomoea batatas) were transcribed from two different genes. To study the roles of these two IbRCA isoforms in photosynthesis, we inserted these two IbRCA genes into the genome of Arabidopsis with deletion of RCA gene (RCA), resulting in IbRCAs- and IbRCAl-expressing plants, respectively. Analysis of these transgenic Arabidopsis indicated that the IbRCAs-expressing plants were similar to wild-type plants under ambient CO/sub 2/ concentration and 22 degree C conditions, suggesting that expression of IbRCAs gene was sufficient for functional complementation of RCA plants under normal conditions. However, IbRCAs-expressing plants were more susceptible to moderate heat stress (26 degree C) compared to wild-type plants. In contrast, although the IbRCAl-expressing plants had to grow normally in high CO/sub 2/ concentration conditions, there were almost no differences in growth and photosynthesis between normally grown and heat-treated plants, implying that IbRCAl-expressing plants had a better heat-resistance than IbRCAs-expressing plants. (author)

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib.

Science.gov (United States)

Booth, Laurence; Roberts, Jane L; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler, Richard E; Lalani, Alshad S; Dent, Paul

2018-02-01

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics

DEFF Research Database (Denmark)

Gravesen, Anne; Sorensen, K.; Aarestrup, Frank Møller

2001-01-01

-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology), The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wildtype strains, indicating...

Comparative Genomic Analysis of Transgenic Poplar Dwarf Mutant Reveals Numerous Differentially Expressed Genes Involved in Energy Flow

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Su Chen

2014-09-01

Full Text Available In our previous research, the Tamarix androssowii LEA gene (Tamarix androssowii late embryogenesis abundant protein Mrna, GenBank ID: DQ663481 was transferred into Populus simonii × Populus nigra. Among the eleven transgenic lines, one exhibited a dwarf phenotype compared to the wild type and other transgenic lines, named dwf1. To uncover the mechanisms underlying this phenotype, digital gene expression libraries were produced from dwf1, wild-type, and other normal transgenic lines, XL-5 and XL-6. Gene expression profile analysis indicated that dwf1 had a unique gene expression pattern in comparison to the other two transgenic lines. Finally, a total of 1246 dwf1-unique differentially expressed genes were identified. These genes were further subjected to gene ontology and pathway analysis. Results indicated that photosynthesis and carbohydrate metabolism related genes were significantly affected. In addition, many transcription factors genes were also differentially expressed in dwf1. These various differentially expressed genes may be critical for dwarf mutant formation; thus, the findings presented here might provide insight for our understanding of the mechanisms of tree growth and development.

An overview of the RCA/IAEA activities in the Australasian region using nuclear analysis techniques for monitoring air pollution

International Nuclear Information System (INIS)

Markwitz, Andreas

2005-01-01

The International Atomic Energy Agency (IAEA) via the Regional Co-operative Agreement (RCA) has identified air particulate matter pollution as a major transboundary environmental issue in the Australasian region. Sixteen countries in the region spanning from Pakistan to the Philippines and from China to New Zealand are participating in the regional programme RAS/7/013-Improved information of urban air quality management in the RCA region' that started in 1997. New Zealand is the lead-country for this programme in which nuclear analytical techniques, such as particle induced X-ray emission (PIXE), neutron activation analysis (NAA) and X-ray fluorescence spectrometry (XRF) are used to measure key elements in PM 2.5-0 and PM 10-2.5 filters from GENT stacked samplers collected twice weekly. Major sources of air particulate matter pollution are identified using statistical source apportionment techniques. To identify transboundary air particulate matter pollution events, the data is collated in a large database. Additionally, the data is used by end-users of the participating countries in the programme. An overview is presented. (author)

Results of the IAEA/RCA personal dosemeter intercomparison in the Asian and Pacific region

International Nuclear Information System (INIS)

Murakami, H.; Minami, K.; Griffith, R.V.

1994-01-01

In 1988, the International Atomic Energy Agency started the Regional Co-operative Agreement project for strengthening the radiation protection infrastructure in the Asian and Pacific region. One component of the initial programme was a regional personal dosimetry intercomparison, which was subsequently conducted in three phases over three years during the period 1990 to 1992. Seventeen organisations of all 14 member states participated in the programmes. Irradiations and data compilation were conducted by the Japan Atomic Energy Research Institute. This paper describes the results of the intercomparison programmes and discusses some features and personal dosimetry problems in the RCA member states. (author)

The Arabidopsis mutant iop1 exhibits induced over-expression of the plant defensin gene PDF1.2 and enhanced pathogen resistance

NARCIS (Netherlands)

Penninckx, I.A.M.A.; Eggermont, K.; Schenk, P.M.; Ackerveken, van den G.; Cammue, B.P.A.; Thomma, B.P.H.J.

2003-01-01

Jasmonate and ethylene are concomitantly involved in the induction of the Arabidopsis plant defensin gene PDF1.2. To define genes in the signal transduction pathway leading to the induction of PDF1.2, we screened for mutants with induced over-expression of a β-glucuronidase reporter, under the

Transcriptome changes associated wtih delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor

Science.gov (United States)

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr 1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr 1-1 gene was over-expressed under the control of a c...

Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo

International Nuclear Information System (INIS)

Nitta, Yusuke; Shimizu, Saki; Shishido-Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O 6 -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy

Mutant matrix metalloproteinase-9 reduces postoperative peritoneal adhesions in rats.

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Atta, Hussein; El-Rehany, Mahmoud; Roeb, Elke; Abdel-Ghany, Hend; Ramzy, Maggie; Gaber, Shereen

2016-02-01

Postoperative peritoneal adhesions continue to be a major source of morbidity and occasional mortality. Studies have shown that matrix metalloproteinase-9 (MMP-9) levels are decreased postoperatively which may limits matrix degradation and participate in the development of peritoneal adhesions. In this proof-of-principle study, we evaluated the effect of gene therapy with catalytically inactive mutant MMP-9 on postoperative peritoneal adhesions in rats. Adenovirus encoding mutant MMP-9 (Ad-mMMP-9) or saline was instilled in the peritoneal cavity after cecal and parietal peritoneal injury in rats. Expression of mutant MMP-9 transcript was verified by sequencing. Adenovirus E4 gene expression, adhesion scores, MMP-9, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-β1 (TGF-β1) expression were evaluated at sacrifice one week after treatment. Both mutant MMP-9 transcripts and adenovirus E4 gene were expressed in Ad-mMMP-9 treated adhesions. Adhesions severity decreased significantly (p = 0.036) in the Ad-mMMP-9-treated compared with saline-treated adhesions. Expression of MMP-9 mRNA and protein were elevated (p = 0.001 and p = 0.029, respectively) in the Ad-mMMP-9-treated adhesions compared with saline-treated adhesions. While tPA levels were increased (p = 0.02) in Ad-mMMP-9 treated adhesions compared with saline-treated adhesions, TGF-β1 and PAI-1 levels were decreased (p = 0.017 and p = 0.042, respectively). No difference in mortality were found between groups (p = 0.64). Mutant MMP-9 gene therapy effectively transduced peritoneal adhesions resulting in reduction of severity of primary peritoneal adhesions. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

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Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Anatomia foliar de microtomateiros fitocromo-mutantes e ultra-estrutura de cloroplastos Leaf anatomy of micro-tomato phytochrome-mutants and chloroplast ultra-structure

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Hyrandir Cabral de Melo

2011-02-01

Full Text Available Plantas fitocromo-mutantes têm sido utilizadas com o intuito de caracterizar isoladamente, dentre os demais fotorreceptores, a ação dos fitocromos sobre eventos ligados à fotomorfogênese. Raros são os estudos que relatam a ação dos fitocromos sobre aspectos estruturais, embora sejam fundamentais à compreensão do desenvolvimento das plantas. Neste trabalho, objetivou-se analisar características ultraestruturais de cloroplastos e aspectos anatômicos foliares dos microtomateiros (Solanum lycopersicum L. cv. Micro-Tom fitocromo-mutantes aurea (subexpressa fitocromos, hp1 e atroviolacea (ambos supra-responsivos a eventos mediados por fitocromo em plantas em estágio de floração. Observou-se que os fitocromos são responsáveis pela expressão de muitas características anatômicas da epiderme foliar, assim como do mesofilo e da ultraestrutura dos cloroplastos.Phytochrome-mutant plants have been used for phytochrome action characterization among all photoreceptors, in events of photomorphogenesis. Studies relating the phytochrome action on structural aspects, which are fundamental to the comprehension of plant development, are rare. The objective of this work was to analyze chloroplast ultra structure and leaf anatomical characteristics of micro-tomatos (Solanum lycopersicum L. cv. Micro-Tom phytochrome-mutants aurea (sub express phytochrome, hp1 and atroviolacea (both super express phytochrome events-mediated in plants in the flowering stage. The results show that phytochromes are responsible for the expression of many characteristics of leaf epidermis, mesophyll and chloroplast ultra-structure.

Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

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Reza Saberianfar

Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

Science.gov (United States)

Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

2013-01-23

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

Science.gov (United States)

Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

2016-12-01

During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3'-end.

Science.gov (United States)

Merkiene, Egle; Gaidamaviciute, Edita; Riauba, Laurynas; Janulaitis, Arvydas; Lagunavicius, Arunas

2010-08-01

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3' --> 5' single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3'-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3'-end and customize this technique for the inner RNA sequence analysis.

Comparison of expression of key sporulation, solventogenic and acetogenic genes in C. beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A.

Science.gov (United States)

Kolek, J; Diallo, M; Vasylkivska, M; Branska, B; Sedlar, K; López-Contreras, A M; Patakova, P

2017-11-01

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

Perturbation of formate pathway for hydrogen production by expressions of formate hydrogen lyase and its transcriptional activator in wild Enterobacter aerogenes and its mutants

Energy Technology Data Exchange (ETDEWEB)

Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084 (China)

2009-06-15

To examine perturbation effects of formate pathway on hydrogen productivity in Enterobacter aerogenes (Ea), formate dehydrogenase FDH-H gene (fdhF) and formate hydrogen lyase activator protein FHLA gene (fhlA) originated from Escherichia coli, were overexpressed in the wild strain Ea, its hycA-deleted mutant (A) by knockout the formate hydrogen lyase repressor and hybO-deleted mutant (O) by knockout of the uptake hydrogenase, respectively. Overexpression of fdhF and fhlA promoted cell growth and volumetric hydrogen production rates of all the strains, and the hydrogen production per gram cell dry weight (CDW) for Ea, A and O was increased by 38.5%, 21.8% and 5.25%, respectively. The fdhF and fhlA overexpression improved the hydrogen yield per mol glucose of strains Ea and A, but declined that of strain O. The increase of hydrogen yield of the strain Ea with fdhF and fhlA expression was mainly attributed to the increase of formate pathway, while for the mutant A, the improved hydrogen yield with fdhF and fhlA expression was mainly due to the increase of NADH pathway. Analysis of the metabolites and ratio of ethanol-to-acetate showed that the cellular redox state balance and energy level were also changed for these strains by fdhF and fhlA expression. These findings demonstrated that the hydrogen production was not only dependent on the hydrogenase genes, but was also affected by the regulation of the whole metabolism. Therefore, fdhF and fhlA expression in different strains of E. aerogenes could exhibit different perturbation effects on the metabolism and the hydrogen productivity. (author)

Plant mutation reports. Vol. 1, No. 3, December 2007

International Nuclear Information System (INIS)

2007-12-01

This issue of the Plant Mutation Reports emphasises the recently concluded IAEA-RCA project on Mutant Multi-location Trial and Mutational Enhancement of genetic Diversity (RAS/5/040) which has developed 33 mutant varieties and dozens of promising mutant breeding lines with high yield potantial and/or improved agronomic characters, resistance and end user quality traits

The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

OpenAIRE

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Kirkwood, John; Sander, Cindy; Avogadri-Connors, Francesca; Cutler Jr, Richard E.; Lalani, Alshad S.; Dent, Paul

2017-01-01

ABSTRACT The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFR...

Studies of Genetic Differences between KDML 105 and its Photo period-insensitive Mutants using DNA techniques

International Nuclear Information System (INIS)

Boonsirichai, Kanokporn; Klakhaeng, Kanchana; Phadvibulya, Valailak

2007-08-01

Full text: Photo period-insensitive mutants of KDML 105 could be planted for grains during and outside the regular cropping season. From genetic studies, the mutant characteristics appeared recessive. A DNA-fingerprinting technique was used to compare gene expression profiles in the leaves of mutants and KDML 105. Differences in the level of expression were found for several loci. Examination of the essential part of the gene for fragrance showed no differences between the mutants and the parental KDML 105

The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

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Sven Vilain

2012-01-01

Full Text Available Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC; however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

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ÁNGELA D ARMENDÁRIZ

2006-01-01

Full Text Available The role of metallothioneins (MT in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-. As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80% belong to two major categories: 1 metabolism; and 2 cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

Programmed cell death in the leaves of the Arabidopsis spontaneous necrotic spots (sns-D mutant correlates with increased expression of the eukaryotic translation initiation factor eIF4B2

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Gwenael M.D.J.-M. Gaussand

2011-04-01

Full Text Available From a pool of transgenic Arabidopsis (Arabidopsis thaliana plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD. The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD.

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

Science.gov (United States)

Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization and expression study of a histidine auxotrophic mutant (his1-) of Nicotiana plumbaginifolia.

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El Malki, F; Jacobs, M

2001-01-01

The histidine auxotroph mutant his 1(-) isolated from Nicotiana plumbaginifolia haploid protoplasts was first characterized to be deficient for the enzyme histidinol phosphate aminotransferase that is responsible for one of the last steps of histidine biosynthesis. Expression of the mutated gene at the RNA level was assessed by northern analysis of various tissues. Transcriptional activity was unimpaired by the mutation and, in contrast, a higher level of expression was obtained when compared to the wild-type. The cDNA sequence encoding the mutated gene was isolated by RT-PCR and compared to the wild-type gene. A single point mutation corresponding to the substitution of a G nucleotide by A was identified at position 1212 starting from the translation site. The alignment of the deduced amino acid sequences from the mutated and wild-type gene showed that this mutation resulted in the substitution of an Arg by a His residue at position 381. This Arg residue is a conserved amino acid for histidinol phosphate aminotransferase of many species. These results indicate that the identified mutation results in an altered histidinol phosphate aminotransferase enzyme that is unable to convert the substrate imidazole acetol phosphate to histidinol phosphate and thereby leads to the blockage of histidine biosynthesis. Possible consequences of this blockage on the expression of other amino acid biosynthesis genes were evaluated by analysing the expression of the dhdps gene encoding dihydrodipicolinate synthase, the first key enzyme of the lysine pathway.

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.

Science.gov (United States)

Park, Kyeong-Su; Kim, Ju Hee; Shin, Hee Won; Chung, Kyung-Sook; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2015-10-26

Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

Potent inhibition of HIV-1 replication by a Tat mutant.

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Luke W Meredith

Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

Science.gov (United States)

Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

2017-06-01

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43 Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43 Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

International Nuclear Information System (INIS)

Kane, W.H.; Devore-Carter, D.; Ortel, T.L.

1990-01-01

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 ± 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 ± 0.012 to 0.124 ± 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 ± 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1,800 ± 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1,700-2,000 units/mg. Immunoprecipitation of [ 35 S]methionine-labeled rHFV revealed a single high molecular mass component. Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. The authors have also expressed a mutant, rHFV-des-B 811-1441 , that lacks a large portion of the highly glycosylated connecting region that is present in factor V. This mutant constitutively expressed 38 ± 7% of the activity of the RVV-V-activated protein. These results suggest that one of the functions of the large connecting region in factor V is to inhibit constitutive procoagulant activity

Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.

Science.gov (United States)

Godon, C; Krapp, A; Leydecker, M T; Daniel-Vedele, F; Caboche, M

1996-02-25

This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

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...

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

Science.gov (United States)

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

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

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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

A wheat cold resistance mutant derived from space mutagenesis

International Nuclear Information System (INIS)

Li Peng; Sun Mingzhu; Zhang Fengyun; Gao Guoqiang; Qiu Denglin; Li Xinhua

2012-01-01

A cold resistance mutant, obtained by spaceflight mutagenesis on the seeds of wheat variety Han6172, and the DNA of cold resistance mutant and contrast Han6172 were compared by SRAP technique. 380 pairs of primers were screened, 6 pairs of them had polymorphisms between mutant and contrast, the rate was 1.58%, and this data indicated that there are no obvious DNA differences between mutant and contrast Six specific fragments were obtained, 3 fragments of them were amplified in mutant. Homology analysis in GenBank showed that Me3-Em7-Mt, Me4-Em11-CK, Me7-Em19-CK and Me6-Em9-Mt all had homologous sequences with wheat chromosome 3B-specific BAC library, and this result indicated that the gene and regulator sequences associated with mutant cold resistance might locate on 3B chromosome. It was speculated that space mutation induced the mutation of 3B chromosome primary structure, and influenced the expressions of cold resistance genes, which resulted in the mutation of cold resistance ability. (authors)

A wheat cold resistance mutant derived from space mutagenesis

International Nuclear Information System (INIS)

Li Peng; Sun Mingzhu; Zhang Fengyun; Gao Guoqiang; Qiu Denglin; Li Xinhua

2011-01-01

A cold resistance mutant, obtained by spaceflight mutagenesis on the seeds of wheat variety Han6172, and the DNA of cold resistance mutant and contrast Han6172 were compared by SRAP technique. 380 pairs of primers were screened, 6 pairs of them had polymorphisms between mutant and contrast, the rate was 1.58%, and this data indicated that there are no obvious DNA differences between mutant and contrast. Six specific fragments were obtained, 3 fragments of them were amplified in mutant. Homology analysis in GenBank showed that Me3-Em7-Mt, Me4-Em11-CK, Me7-Em19-CK and Me6-Em9-Mt all had homologous sequences with wheat chromosome 3B-specific BAC library, and this result indicated that the gene and regulator sequences associated with mutant cold resistance might locate on 3B chromosome. It was speculated that space mutation induced the mutation of 3B chromosome primary structure, and influenced the expressions of cold resistance genes, which resulted in the mutation of cold resistance ability. (authors)

The text of the third agreement to extend the 1987 Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology (RCA). Extension of agreement

International Nuclear Information System (INIS)

2002-01-01

The text of the Third Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology, 'the 1987 RCA', is reproduced herein for the information of all Members. Pursuant to Article 1 of the Third Agreement to Extend the 1987 Regional Co-operative Agreement, the 1987 RCA shall continue in force for a further period of five years with effect from 12 June 2002, i.e., through 11 June 2007. As of 15 May 2002, notifications of acceptance had been received by the Director General from the Governments of Bangladesh, China, India, Indonesia, Republic of Korea, Malaysia, Pakistan, Sri Lanka and Viet Nam. The latest status list is attached

Nanoformulated cell-penetrating survivin mutant and its dual actions

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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

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.

Analyzing the international competitiveness of the industry in Portugal, Ireland, Greece and Spain using revealed comparative advantages (RCA) indicators

OpenAIRE

Oelgemöller, Jens

2012-01-01

This paper sheds light on the export structure of the four European countries Portugal, Ireland, Greece and Spain, the so called PIGS countries. These countries were all hit by the economic downturn in the course of the financial crisis and have been struggling with the national debt crisis and recession. One way to identify sectoral international competitiveness is provided by the revealed comparative advantage index developed by Balassa (RCA 1). This indicator evolved through several studie...

Identification of a novel ga-related bush mutant in pumpkin (cucurbita moschata duchesne)

International Nuclear Information System (INIS)

Wu, T.; Cao, J.

2015-01-01

Pumpkin (Cucurbita moschata Duchesne) bush mutant plants were characterized by short stems. The sensitivity of pumpkin bush mutant plants to exogenous hormones was identified in this study. Results revealed that internode elongation of bush mutant plants could respond to gibberellins (GA4+7 and GA3), but not to indole acetic acid (IAA) and brassinosteroids (BR); by contrast, the mutant phenotype of bush mutant plants could not be fully rescued by GA4+7 and GA3. The internode of bush mutant plants yielded a lower KS expression level than that of vine plants. Therefore, pumpkin bush mutant plants were designated as GA-related mutant plants eliciting a partial response to GAs; the action of IAA and BR might not be involved in the internode growth of pumpkin bush mutant plants, specifically Cucurbita moschata Duch. (author)

Genome-wide identification and expression profiling reveal tissue-specific expression and differentially-regulated genes involved in gibberellin metabolism between Williams banana and its dwarf mutant.

Science.gov (United States)

Chen, Jingjing; Xie, Jianghui; Duan, Yajie; Hu, Huigang; Hu, Yulin; Li, Weiming

2016-05-27

Dwarfism is one of the most valuable traits in banana breeding because semi-dwarf cultivars show good resistance to damage by wind and rain. Moreover, these cultivars present advantages of convenient cultivation, management, and so on. We obtained a dwarf mutant '8818-1' through EMS (ethyl methane sulphonate) mutagenesis of Williams banana 8818 (Musa spp. AAA group). Our research have shown that gibberellins (GAs) content in 8818-1 false stems was significantly lower than that in its parent 8818 and the dwarf type of 8818-1 could be restored by application of exogenous GA3. Although GA exerts important impacts on the 8818-1 dwarf type, our understanding of the regulation of GA metabolism during banana dwarf mutant development remains limited. Genome-wide screening revealed 36 candidate GA metabolism genes were systematically identified for the first time; these genes included 3 MaCPS, 2 MaKS, 1 MaKO, 2 MaKAO, 10 MaGA20ox, 4 MaGA3ox, and 14 MaGA2ox genes. Phylogenetic tree and conserved protein domain analyses showed sequence conservation and divergence. GA metabolism genes exhibited tissue-specific expression patterns. Early GA biosynthesis genes were constitutively expressed but presented differential regulation in different tissues in Williams banana. GA oxidase family genes were mainly transcribed in young fruits, thus suggesting that young fruits were the most active tissue involved in GA metabolism, followed by leaves, bracts, and finally approximately mature fruits. Expression patterns between 8818 and 8818-1 revealed that MaGA20ox4, MaGA20ox5, and MaGA20ox7 of the MaGA20ox gene family and MaGA2ox7, MaGA2ox12, and MaGA2ox14 of the MaGA2ox gene family exhibited significant differential expression and high-expression levels in false stems. These genes are likely to be responsible for the regulation of GAs content in 8818-1 false stems. Overall, phylogenetic evolution, tissue specificity and differential expression analyses of GA metabolism genes can provide a

Regional Cooperation Agreement for Asia and the Pacific (RCA). A mechanism for nuclear technology transfer

International Nuclear Information System (INIS)

Bin Muslim, N.

1993-01-01

The paper presents the regional cooperation programs of the IAEA which have as purpose to promote the applications of peaceful uses of atomic energy and to transfer technology to the developing countries. The paper focusses on the (RCA) program for Asia and the Pacific, it is considered the most important mechanism for genuine technology transfer. The annex no 1 lists the full text of the Regional Cooperative Agreement for Research, Development and Training Related to Nuclear Science and Technology, 1987 (13 articles). The annex no.3 lists also the full text of the African Regional Cooperative Agreement for Research, Development and training Related to Nuclear Science and Technology (14 articles). 11 refs., 17 tabs

Exogenous Expressions of FTO Wild-Type and R316Q Mutant Proteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells as Demonstrated by DIGE Analysis

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Nil Guzel

2017-01-01

Full Text Available Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations.

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

Science.gov (United States)

Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

2017-06-01

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Is auxin involved in the induction of genetic instability in barley homeotic double mutants?

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Šiukšta, Raimondas; Vaitkūnienė, Virginija; Rančelis, Vytautas

2018-02-01

The triggers of genetic instability in barley homeotic double mutants are tweaky spike -type mutations associated with an auxin imbalance in separate spike phytomeres. Barley homeotic tweaky spike;Hooded (tw;Hd) double mutants are characterized by an inherited instability of spike and flower development, which is absent in the single parental constituents. The aim of the present study was to show that the trigger of genetic instability in the double mutants is the tw mutations, which are associated with an auxin imbalance in the developing spikes. Their pleiotropic effects on genes related to spike/flower development may cause the genetic instability of double mutants. The study of four double-mutant groups composed of different mutant alleles showed that the instability arose only if the mutant allele tw was a constituent of the double mutants. Application of auxin inhibitors and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated the relationship of the instability of the double mutants and the phenotype of the tw mutants to auxin imbalance. 2,4-D induced phenocopies of the tw mutation in wild-type plants and rescued the phenotypes of three allelic tw mutants. The differential display (dd-PCR) method allowed the identification of several putative candidate genes in tw that may be responsible for the initiation of instability in the double mutants by pleiotropic variations of their expression in the tw mutant associated with auxin imbalance in the developing spikes. The results of the present study linked the genetic instability of homeotic double mutants with an auxin imbalance caused by one of the constituents (tw). The genetic instability of the double mutants in relation to auxin imbalance was studied for the first time. A matrocliny on instability expression was also observed.

Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA.

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Zhao, Carolyn Y; Grinkevich, Vera V; Nikulenkov, Fedor; Bao, Wenjie; Selivanova, Galina

2010-05-01

Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.

Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni

International Nuclear Information System (INIS)

Tourigny, David S.; Elliott, Paul R.; Edgell, Louise J.; Hudson, Gregg M.; Moody, Peter C. E.

2010-01-01

The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported. The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP + or NAD + as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4 1 22, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme

Molecular Mechanisms Regulating Ocular Apoptosis in Zebrafish gdf6a Mutants

DEFF Research Database (Denmark)

Pant, Sameer D.; March, Lindsey D.; Famulski, Jakub K.

2013-01-01

intrinsic or extrinsic apoptotic mechanisms were involved, morpholino antisense oligonucleotides targeting baxa, baxb, and p53 were employed. Caspase-3 immunohistochemistry (IHC) was performed to assay apoptosis. Pharmacologic inhibition (using SB203580) and IHC were used to investigate the role of p38...... occurs 28 hours post fertilization (hpf) in gdf6a(-/-) mutants that is mediated independently of p53 by intrinsic mechanisms involving Bax proteins. Also, gdf6a(-/-) mutants exhibit markedly increased p38 MAP kinase activation that can be inhibited to significantly reduce retinal apoptosis. A reduction...... in retinal smad1 expression was also noted in gdf6a(-/-) mutants. CONCLUSIONS. gdf6a(-/-)-induced apoptosis is characterized by the involvement of intrinsic apoptotic pathways, p38 MAP kinases, and dysregulated smad expression. Modulation of key mediators can inhibit retinal apoptosis offering potential...

Indy mutants: live long and prosper

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Stewart eFrankel

2012-02-01

Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

Science.gov (United States)

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Fine Mapping of a Degenerated Abdominal Legs Mutant (Edl in Silkworm, Bombyx mori.

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Honglei Wang

Full Text Available In insects, abdominal appendages, also called prolegs, vary due to adaptive evolution. Mutations on prolegs within species provide insights to better understand the mechanisms underlying appendage development and diversity. In silkworm Bombyx mori, extra-crescents and degenerated abdominal legs (Edl mutant, belonging to the E pseudoallele group, is a spontaneous mutation that adds crescents and degenerates prolegs on the third abdominal segment (A3. This mutation may be a homeotic transformation of A3 to A2. In this study, the Edl locus was mapped within approximately a 211 Kb region that is 10 Kb upstream of Bmabdominal-A (Bmabd-A. RT-quantitative PCR (RT-qPCR and Western blot analysis of Bmabd-A expression showed a slight but significant decrease, while the expression of BmUltrabithorax (BmUbx was up-regulated in the Edl mutant compared to wildtype (Dazao. Moreover, we also found that BmDistal-less (BmDll, which regulated the development of distal proleg structures, was missing at the tips of the A3 prolegs in the Edl mutant compared to BmDll expression in normally developed prolegs in both the wildtype and mutant. Collectively, we identified approximately a 211 Kb region in the Edl locus that regulates BmUbx and Bmabd-A expression and found that changes in BmUbx and Bmabd-A expression may lead to the loss of distal proleg structures in B. mori.

The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses.

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Ellis, Christine; Karafyllidis, Ioannis; Wasternack, Claus; Turner, John G

2002-07-01

Biotic and abiotic stresses stimulate the synthesis of jasmonates and ethylene, which, in turn, induce the expression of genes involved in stress response and enhance defense responses. The cev1 mutant has constitutive expression of stress response genes and has enhanced resistance to fungal pathogens. Here, we show that cev1 plants have increased production of jasmonate and ethylene and that its phenotype is suppressed by mutations that interrupt jasmonate and ethylene signaling. Genetic mapping, complementation analysis, and sequence analysis revealed that CEV1 is the cellulose synthase CeSA3. CEV1 was expressed predominantly in root tissues, and cev1 roots contained less cellulose than wild-type roots. Significantly, the cev1 mutant phenotype could be reproduced by treating wild-type plants with cellulose biosynthesis inhibitors, and the cellulose synthase mutant rsw1 also had constitutive expression of VSP. We propose that the cell wall can signal stress responses in plants.

Inheritance and gene expression of a root-growth inhibiting mutant in rice (Oryza sativa L.)

International Nuclear Information System (INIS)

Kitano, H.; Futsuhara, Y.

1990-01-01

Full text: A root-growth inhibiting mutant was induced in the dwarf mutant line, 'Fukei 71', through ethylene-imine. The mutant is characterised by the excessive inhibition of both seminal and crown roots elongation just after germination, although its shoots grow nearly normal. To study the genetics, the mutant was crossed with its original line 'Fukei 71' and some other normal cultivars. Results show that the root-growth inhibition is controlled by a recessive gene (rt), independent of the dwarf gene, d-50(t) locus in Fukei 71. For elucidating the gene action on root morphogenesis, histological and cytological experiments were carried out using a longitudinal and transverse thin section of seminal and/or crown root tips. Observations suggest that the rt gene affects the normal formation of the epidermal system which is differentiated from the protoderm of the root apical meristem. (author)

Special report for UNDP on regional RCA project for Asia and the Pacific on industrial applications of isotopes and radiation technology. Technical, economic and commercial analyses

International Nuclear Information System (INIS)

1980-01-01

The report contains seven sub-projects for RCA countries on the following problems: tracer technology in industry; non-destructive testing practice; radiation processing; nucleonic control systems for paper manufacture and steel manufacture; mineral exploration, mining and process control; nuclear instruments maintenance practices

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

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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.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

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Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

2015-08-24

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Bacterio-opsin mutants of Halobacterium halobium

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Betlach, Mary; Pfeifer, Felicitas; Friedman, James; Boyer, Herbert W.

1983-01-01

The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the bop mRNA might be processed from a large mRNA transcript. Images PMID:16593291

Mutants induced in winter rye (Secale cereale L.): Short straw-mutant No. 2714 and late-senescence mutant

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Muszynski, S; Darlewska, M [Department of Plant Breeding and Seed Science, Warsaw Agricultural University, Warsaw (Poland)

1990-01-01

Full text: Mutants were induced by treating dormant seeds with ionizing radiation (fast neutrons) or chemicals (N-nitroso-N-ethyl urea or sodium azide). Among several mutants obtained, of special value is the short-straw mutant No. 2714 and a late senescent mutant. (author)

Dysregulation of gene expression in the striatum of BACHD rats expressing full-length mutant huntingtin and associated abnormalities on molecular and protein levels.

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Yu-Taeger, Libo; Bonin, Michael; Stricker-Shaver, Janice; Riess, Olaf; Nguyen, Hoa Huu Phuc

2017-05-01

Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for the huntingtin protein (HTT). Mutant HTT (mHTT) has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Utilizing the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, both of which carry full-length human mHTT but with different expression levels. By defining the threshold of significance at p < 0.01, we found 1608 genes and 871 genes differentially expressed in both TG5 and TG9 rats when compared to the wild type littermates, respectively. We only chose the highly up-/down-regulated genes for further analysis by setting an additional threshold of 1.5 fold change. Comparing gene expression profiles of human HD brains and BACHD rats revealed a high concordance in both functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant to HD. In addition, we investigated the causes leading to gene expression changes at molecular and protein levels in BACHD rats including the involvement of polyQ-containing transcription factors TATA box-binding protein (TBP), Sp1 and CBP as well as the chromatin structure. We demonstrate that the BACHD rat model recapitulates the gene expression changes of the human disease supporting its role as a preclinical research animal model. We also show for the first time that TFIID complex formation is reduced, while soluble TBP is increased in an HD model. This finding suggests that mHTT is a competitor instead of a recruiter of polyQ-containing transcription factors in the transcription process in HD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Estudo de Caso: Análise de um Relatório de Controle Ambiental (RCA Referente à Atividade de Levantamento Sísmico

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Igor Leonardo Morais Nascimento

2010-01-01

Full Text Available Este trabalho aborda a análise de um Relatório de Controle Ambiental (RCA, da atividade de sísmica, submetido à obtenção de licenciamento ambiental junto ao Instituto de Defesa do Meio Ambiente – IDEMA. Realiza uma comparação dos itens exigidos no Termo de Referência (TR do IDEMA com o Estudo ambiental citado, a fim de identificar se todos os itens exigidos legalmente foram contemplados no RCA e se os mesmos estão em conformidade com o Termo. Utiliza a técnica de estudo de impacto ambiental baseado em um contexto legal que estabelece requisitos a serem observados e procedimentos a serem cumpridos, tal técnica é a análise formal da qualidade de um estudo ambiental. Verifica, também, a repetição ou não das principais deficiências apontadas no elemento Mitigação e compensação de impactos com base no estudo do Ministério Público Federal - MPF, a partir da técnica de análise de conteúdo de estudo ambiental.

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

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Seok-Hyung Kim

2013-07-01

Tuberous sclerosis complex (TSC is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1 kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

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Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants.

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Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-06-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cbeta. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

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Farshad Farshidfar

2017-03-01

Full Text Available Cholangiocarcinoma (CCA is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region

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Neelam Taneja

2015-01-01

Full Text Available Background & objectives: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Methods: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 µg/ml, SFM2 (≥4 µg/ml and SFM3 (≥32 µg/ml were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 µg/ml and SDM2 (≥4 µg/ml were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Results: Mutations were observed in gyrA Ser [83]→Leu, Asp [87]→Asn/Gly, Val [196]→Ala and in parC Phe [93]→Val, Ser [80]→Ile, Asp [101]→Glu and Asp [110]→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance ( p0 <0.05; while tolC and acrR expression levels did not. Interpretation & conclusions: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val [196]→Ala in gyrA in clinical isolates and Phe [93]→Val, Asp [101]→Glu, Asp [110]→Glu and in parC in majority of laboratory-grown mutants.

Meiotic UV-sensitive mutant that causes deletion of duplications in neurospora

International Nuclear Information System (INIS)

Newmeyer, D.; Galeazzi, D.R.

1978-01-01

The meiotic-3 (mei-3) mutant of Neurospora crassa has several effects: (1) when homozygous, it almost completely blocks meiosis and ascospore formation, (2) it is sensitive to uv, (3) its growth is inhibited by histidine, and (4) it increases the instability of nontandem duplications. This was shown for duplications produced by five different rearrangements and was demonstrated by two different criteria. The effects on meiosis and duplication instability are expressed strongly at 25 0 ; the effects on sensitivity to uv and to histidine are expressed strongly at 38.5 0 but only slightly at 25 0 . Nevertheless, all four effects were shown to be due to a single gene. Mei-3 is not allelic with previously reported uv-sensitive mutants. Two other results were obtained that are not necessarily due to mei-3: (1) a cross involving mei-3 produced a new unlinked meiotic mutant, mei-4, which is not sensitive to uv or histidine, and (2) a burst of several new mutants occurred in a different mei-3 stock, including a partial revertant to mei-3. Mei-3 has previously been shown to cause frequent complete loss of a terminal duplicate segment, beginning exactly at the original rearrangement breakpoint. Possible mechanisms are discussed by which a uv-sensitive mutant could cause such precise deletions

The Text of the Fifth Agreement to Extend the 1987 Regional Cooperative Agreement for Research, Development and Training Related to Nuclear Science and Technology (RCA). Extension of Agreement. Latest Status

International Nuclear Information System (INIS)

2012-01-01

The Text of the Fifth Agreement to Extend the 1987 Regional Cooperative Agreement for Research, Development and Training Related to Nuclear Science and Technology (RCA). Extension of Agreement. Latest Status [es

Identification of a Gravitropism-Deficient Mutant in Rice

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He Yan

2017-03-01

Full Text Available A gravitropism-deficient mutant M96 was isolated from a mutant bank, generated by ethyl methane sulfonate (EMS mutagenesis of indica rice accession ZJ100. The mutant was characterized as prostrate growth at the beginning of germination, and the prostrate growth phenotype ran through the whole life duration. Tiller angle and tiller number of M96 increased significantly in comparison with the wild type. Tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96. Genetic analysis and gene mapping showed that M96 was controlled by a single recessive nuclear gene, tentatively termed as gravitropism-deficient M96 (gdM96, which was mapped to a region of 506 kb flanked by markers RM5960 and InDel8 on the long arm of chromosome 11. Sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840. Additionally, real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96. Furthermore, the expression level was more than four times in M96 stem than in the wild type stem. Our results suggested that the mutant gene was likely a new allele to the reported gene LAZY1. Isolation of this new allele would facilitate the further characterization of LAZY1.

Ectopic Expression of CsCTR1, a Cucumber CTR-Like Gene, Attenuates Constitutive Ethylene Signaling in an Arabidopsis ctr1-1 Mutant and Expression Pattern Analysis of CsCTR1 in Cucumber (Cucumis sativus

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Beibei Bie

2014-09-01

Full Text Available The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1 is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1 was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.

Neurodegeneration in drop-dead mutant drosophila melanogaster is associated with the respiratory system but not with Hypoxia.

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Christine Lynn Sansone

Full Text Available Mutations in the gene drop-dead (drd cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.

Relative codon adaptation: a generic codon bias index for prediction of gene expression.

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Fox, Jesse M; Erill, Ivan

2010-06-01

The development of codon bias indices (CBIs) remains an active field of research due to their myriad applications in computational biology. Recently, the relative codon usage bias (RCBS) was introduced as a novel CBI able to estimate codon bias without using a reference set. The results of this new index when applied to Escherichia coli and Saccharomyces cerevisiae led the authors of the original publications to conclude that natural selection favours higher expression and enhanced codon usage optimization in short genes. Here, we show that this conclusion was flawed and based on the systematic oversight of an intrinsic bias for short sequences in the RCBS index and of biases in the small data sets used for validation in E. coli. Furthermore, we reveal that how the RCBS can be corrected to produce useful results and how its underlying principle, which we here term relative codon adaptation (RCA), can be made into a powerful reference-set-based index that directly takes into account the genomic base composition. Finally, we show that RCA outperforms the codon adaptation index (CAI) as a predictor of gene expression when operating on the CAI reference set and that this improvement is significantly larger when analysing genomes with high mutational bias.

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

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)

Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression.

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Yue, Xuetian; Zhang, Cen; Zhao, Yuhan; Liu, Juan; Lin, Alan W; Tan, Victor M; Drake, Justin M; Liu, Lianxin; Boateng, Michael N; Li, Jun; Feng, Zhaohui; Hu, Wenwei

2017-08-15

Tumor suppressor p53 is frequently mutated in human cancer. Mutant p53 often promotes tumor progression through gain-of-function (GOF) mechanisms. However, the mechanisms underlying mutant p53 GOF are not well understood. In this study, we found that mutant p53 activates small GTPase Rac1 as a critical mechanism for mutant p53 GOF to promote tumor progression. Mechanistically, mutant p53 interacts with Rac1 and inhibits its interaction with SUMO-specific protease 1 (SENP1), which in turn inhibits SENP1-mediated de-SUMOylation of Rac1 to activate Rac1. Targeting Rac1 signaling by RNAi, expression of the dominant-negative Rac1 (Rac1 DN), or the specific Rac1 inhibitor NSC23766 greatly inhibits mutant p53 GOF in promoting tumor growth and metastasis. Furthermore, mutant p53 expression is associated with enhanced Rac1 activity in clinical tumor samples. These results uncover a new mechanism for Rac1 activation in tumors and, most importantly, reveal that activation of Rac1 is an unidentified and critical mechanism for mutant p53 GOF in tumorigenesis, which could be targeted for therapy in tumors containing mutant p53. © 2017 Yue et al.; Published by Cold Spring Harbor Laboratory Press.

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

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Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

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Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

Intact interval timing in circadian CLOCK mutants.

Science.gov (United States)

Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

Science.gov (United States)

Nagahara, Yuki; Shimazawa, Masamitsu; Ohuchi, Kazuki; Ito, Junko; Takahashi, Hitoshi; Tsuruma, Kazuhiro; Kakita, Akiyoshi; Hara, Hideaki

2017-08-01

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Radiation-induced mutagenicity in repair deficient Chinese hamster ovary (CHO) mutants

International Nuclear Information System (INIS)

Tesmer, J.G.; Saunders, E.H.; Chen, D.J.

1987-01-01

To determine if there is a relationship between DNA double-strand break repair and mutagenicity the authors utilized two x-ray sensitive mutants of Chinese hamster ovary cells along with the parental line K1. The two mutant lines xrs-5 and xrs-6, which have different DSB repair capabilities, were used to determine cell killing and 6-thioguanine resistance (6TG/sup r/) mutation frequencies induced by either x-rays of α-particles, x-ray survival data indicated the two mutant lines have similar sensitivity and are 5-7 fold more sensitive than the parental line K1. The mutant lines are also sensitive to α-particles but to a lesser extent. The authors' 6TG mutation data indicated that the two mutant lines are hypermutable. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in mutant cell population than in parental K1 cells. Their results support the notion that repair of DSB play an important role in the expression of radiation-induced cell killing and mutagenicity

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: a review.

Science.gov (United States)

Salinas, Thalia; Larosa, Véronique; Cardol, Pierre; Maréchal-Drouard, Laurence; Remacle, Claire

2014-05-01

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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...... of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. Conclusions The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation...... is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems....

Specific TP53 Mutants Overrepresented in Ovarian Cancer Impact CNV, TP53 Activity, Responses to Nutlin-3a, and Cell Survival

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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.

Automatd assessment of the state of central hemodynamics using a radiocardioanalyser RCA 3-01

International Nuclear Information System (INIS)

Sivachenko, T.P.; Zozulya, A.A.; Sribnaya, A.F.; Ratmanskij, A.Yu.; Tishchenko, F.M.; Kurenya, A.G.; Krupka, I.N.; Kulagin, S.N.; Protsenko, A.S.

1987-01-01

The authors presented the results of a clinical use of a new radiocardioanalyzer RCA 3-01 (manufactured in the USSR) providing for automated measurements and computations of indices of the central hemodynamics during studies using a method of dilution of radioactive nuclides. The main technical potentialities of the device designed on the basis of microprocessor technology, were described. The authors also provided the results of automated computation of the circulating blood volume and the main hemodynamic indices in a group of patients. Values of the hemodynamic indices were shown to correspond to actual ones and comparable to control ones and clinical evidence. The comparison of automated processing with a manual method of calculation indicated a significant coincidence of index values. The clinical use of the radiocardioanalyzer RGA 3-01 for automated assessment of the indices of the cardiovascular system was shown to hold promise among various groups of patients

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

Science.gov (United States)

Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

2016-03-10

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

Aplicación de la tribología y el análisis de la causa raíz (RCA en motores de combustión interna.

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F. Martínez Pérez

2008-09-01

Full Text Available En el trabajo, se hace empleo de la herramienta de trabajo Análisis de la Causa Raíz (RCA para llegar a unas primeras conclusiones acerca de fallos producidos en el nudo tribológico válvula – guía de válvula en un motor de combustión interna, y así pasar a hallar la causa final y dar solución al problema. El método permite arribar a conclusiones en cuanto a la causa del problema.Application of tribology and root cause analysis (RCA on diesel engine.It is used the process root cause in the analysis of a problem in the tribological pair valve- valve guide of an internal combustion engine. Using this process was possible to have the first conclusions in the problem and finally to arrive to a final conclusion about the cause of the problem.

A detailed study of gerJ mutants of Bacillus subtilis.

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Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O

1986-08-01

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.

Connexin mutants and cataracts

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Eric C Beyer

2013-04-01

Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants1

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Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-01-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cβ. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units. PMID:12805615

Defective glycinergic synaptic transmission in zebrafish motility mutants

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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.

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

The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-fucose Transporter*

Science.gov (United States)

Geisler, Christoph; Kotu, Varshika; Sharrow, Mary; Rendić, Dubravko; Pöltl, Gerald; Tiemeyer, Michael; Wilson, Iain B. H.; Jarvis, Donald L.

2012-01-01

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency. PMID:22745127

Alterations in biochemical and physiological characters in radiation-induced mutants of grain legumes

International Nuclear Information System (INIS)

Mueller, H.P.

1984-01-01

Selected examples from different grain legumes are studied. The biochemically and physiologically detectable alterations in distintc characters as caused by the action of mutant genes are presented comparatively. The interactions between different mutant genes in order to evaluated the influence of the genotypic constitution on the expression of mutated genes are emphasized. (M.A.C.) [pt

Genetic background of Prop1(df) mutants provides remarkable protection against hypothyroidism-induced hearing impairment.

Science.gov (United States)

Fang, Qing; Giordimaina, Alicia M; Dolan, David F; Camper, Sally A; Mustapha, Mirna

2012-04-01

Hypothyroidism is a cause of genetic and environmentally induced deafness. The sensitivity of cochlear development and function to thyroid hormone (TH) mandates understanding TH action in this sensory organ. Prop1(df) and Pou1f1(dw) mutant mice carry mutations in different pituitary transcription factors, each resulting in pituitary thyrotropin deficiency. Despite the same lack of detectable serum TH, these mutants have very different hearing abilities: Prop1(df) mutants are mildly affected, while Pou1f1(dw) mutants are completely deaf. Genetic studies show that this difference is attributable to the genetic backgrounds. Using embryo transfer, we discovered that factors intrinsic to the fetus are the major contributor to this difference, not maternal effects. We analyzed Prop1(df) mutants to identify processes in cochlear development that are disrupted in other hypothyroid animal models but protected in Prop1(df) mutants by the genetic background. The development of outer hair cell (OHC) function is delayed, but Prestin and KCNQ4 immunostaining appear normal in mature Prop1(df) mutants. The endocochlear potential and KCNJ10 immunostaining in the stria vascularis are indistinguishable from wild type, and no differences in neurofilament or synaptophysin staining are evident in Prop1(df) mutants. The synaptic vesicle protein otoferlin normally shifts expression from OHC to IHC as temporary afferent fibers beneath the OHC regress postnatally. Prop1(df) mutants exhibit persistent, abnormal expression of otoferlin in apical OHC, suggesting delayed maturation of synaptic function. Thus, the genetic background of Prop1(df) mutants is remarkably protective for most functions affected in other hypothyroid mice. The Prop1(df) mutant is an attractive model for identifying the genes that protect against deafness.

Overexpression of mutant ataxin-3 in mouse cerebellum induces ataxia and cerebellar neuropathology.

Science.gov (United States)

Nóbrega, Clévio; Nascimento-Ferreira, Isabel; Onofre, Isabel; Albuquerque, David; Conceição, Mariana; Déglon, Nicole; de Almeida, Luís Pereira

2013-08-01

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominant neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neuronal degeneration. Currently, there is no therapy able to modify disease progression. In the present study, we aimed at investigating one of the most severely affected brain regions in the disorder--the cerebellum--and the behavioral defects associated with the neuropathology in this region. For this purpose, we injected lentiviral vectors encoding full-length human mutant ataxin-3 in the mouse cerebellum of 3-week-old C57/BL6 mice. We show that circumscribed expression of human mutant ataxin-3 in the cerebellum mediates within a short time frame--6 weeks, the development of a behavioral phenotype including reduced motor coordination, wide-based ataxic gait, and hyperactivity. Furthermore, the expression of mutant ataxin-3 resulted in the accumulation of intranuclear inclusions, neuropathological abnormalities, and neuronal death. These data show that lentiviral-based expression of mutant ataxin-3 in the mouse cerebellum induces localized neuropathology, which is sufficient to generate a behavioral ataxic phenotype. Moreover, this approach provides a physiologically relevant, cost-effective and time-effective animal model to gain further insights into the pathogenesis of MJD and for the evaluation of experimental therapeutics of MJD.

Experience with WASP and MAED among IAEA Member States participating in the Regional Co-operative Agreement (RCA) in Asia and the Pacific Region

International Nuclear Information System (INIS)

1989-10-01

The report includes the proceedings and papers presented during the workshop on the experience with WASP/MAED computer programs among IAEA Member States participating in the regional co-operative agreement (RCA) in Asia and the Pacific Region, organized by the IAEA and held in Kuala Lumpur (Malaysia) between 5-9 December 1988. A separate abstract was prepared for each of the 14 papers presented. Refs, figs and tabs

Detection of DNA polymorphisms in Dendrobium Sonia White mutant lines

International Nuclear Information System (INIS)

Affrida Abu Hassan; Putri Noor Faizah Megat Mohd Tahir; Zaiton Ahmad; Mohd Nazir Basiran

2006-01-01

Dendrobium Sonia white mutant lines were obtained through gamma ray induced mutation of purple flower Dendrobium Sonia at dosage 35 Gy. Amplified Fragment Length Polymorphism (AFLP) technique was used to compare genomic variations in these mutant lines with the control. Our objectives were to detect polymorphic fragments from these mutants to provide useful information on genes involving in flower colour expression. AFLP is a PCR based DNA fingerprinting technique. It involves digestion of DNA with restriction enzymes, ligation of adapter and selective amplification using primer with one (pre-amplification) and three (selective amplification) arbitrary nucleotides. A total number of 20 primer combinations have been tested and 7 produced clear fingerprint patterns. Of these, 13 polymorphic bands have been successfully isolate and cloned. (Author)

Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats : preferential survival of transduced astroglial cells in nude rats

NARCIS (Netherlands)

Hermens, W.T.J.M.C.; Verhaagen, J

1997-01-01

In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

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Saeed Akhtar

2015-01-01

Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

Science.gov (United States)

Kaurilind, Eve; Brosché, Mikael

2017-01-01

Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

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Guocai Li

Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.

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Field, H J; Darby, G; Wildy, P

1980-07-01

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

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Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J; Aghera, Nilesh; Varadarajan, Raghavan

2016-11-01

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

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Najafzadeh, M J; Vicente, V A; Feng, Peiying; Naseri, A; Sun, Jiufeng; Rezaei-Matehkolaei, A; de Hoog, G S

2018-03-05

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants.

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Kailiang Sun

2012-02-01

Full Text Available miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

Mutation induction and evaluation of high yield rice mutants

International Nuclear Information System (INIS)

Abdul Rahim Harun; Sobri Husein; Rusli Ibrahim

2006-01-01

The successful use of plant breeding for improving crops requires the existence of genetic variation of useful traits. Unfortunately, the desired variation is often lacking. However, radiation has been used to induce mutations and thereby generate genetic variation from which desired mutants may be selected. Mutation induction has become a proven way of creating variation within a crop variety. It offers the possibility of inducing desired attributes that either cannot be expressed in nature or have been lost during evolution. Rice is security food crop in Malaysia. Efforts were undertaken to enhance rice yield from 4.0 tones per hectare in 1995 to 5.5 tones per hectare in 2010. Proper management and good varieties are two factors that require for enhancing yield of rice. In this research, purified seeds of MR211 and MR219 were gamma irradiated at 100 to 400 Gray and sown for planting as M1 generation at MARDI experimental plot. The M2 population was sown in bulk with population size around 15,000 to 20,000 plants. Individual plant selection was carried out at maturity and each selected plant became a mutant line of M3 generation. Agronomic trial of M3 mutants lines were conducted in Mardi, Tanjung Karang, Selangor. About 115 of selected mutant lines were evaluated. Each row of those mutant lines were planted in two rows at planting distance of 25cm within and between rows. These mutant lines were visually observed and data were recorded in each of every mutant line. (Author)

Human liver cell trafficking mutants: characterization and whole exome sequencing.

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Fei Yuan

Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

A preliminary study on induction and identification of chlorophyll mutants of indica type temperature sensitive genie male-sterile rice

International Nuclear Information System (INIS)

Xia Yingwu; Liu Guifu; Shu Qingyao; Jiang Ronghua; Xie Jiahua

1995-01-01

Chlorophyll mutants of different type were obtained from indica type temperature sensitive genie male-sterile rice (cv. 2177s) by using 60 Co γ-rays irradiation. The total chlorophyll mutation frequency reached to 0.26% in M 2 generation. However only about 4.50% of these mutants could survived. Among them, 33 heritable chlorophyll mutant lines were easily distinguished, and were screened and studied. The mutants either showed chlorosis or yellowing or expressed only at seedling period or persisted all growth cycle. The expression of mutant character was stable under different environment. It is suggested that they are useful as the marker traits in two-line hybrid rice. Moreover, the agronomic traits of most of these lines changed in different levels compared with the parent line 2177S. Every mutation line seemed to be controlled by one recessive gene as the F 1 plants of reciprocal crosses between mutant and 2177S showed normal leaf color. And the ratio of green plants/mutant plants was 3:1 in the segregated F 2 population

Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

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Dri, A M; Rouviere-Yaniv, J; Moreau, P L

1991-01-01

Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes. Images PMID:2019558

The Text of the Fourth Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training related to Nuclear Science and Technology (RCA). Extension of Agreement. Latest Status

International Nuclear Information System (INIS)

2007-01-01

The text of the Fourth Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology, 'the 1987 RCA', is reproduced herein for the information of all Members

Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.

Science.gov (United States)

Cullen, Kirsty S; Matschinsky, Franz M; Agius, Loranne; Arden, Catherine

2011-12-01

The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non-β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non-β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.

Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.

Science.gov (United States)

Oshima, Taku; Aiba, Hirofumi; Masuda, Yasushi; Kanaya, Shigehiko; Sugiura, Masahito; Wanner, Barry L; Mori, Hirotada; Mizuno, Takeshi

2002-10-01

We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components).

Global Expression in Sorghum Brown Midrib Mutants to Improve Biomass for Biofuels

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Brown midrib (bmr) mutants are being investigated for their ability to increase the conversion efficiency of sorghum biomass for lignocellulosic bioenergy. Brown midrib 6 and 12 (bmr6 and 12) are impaired the last two steps of monolignol biosynthesis resulting in reduced lignin content and altered ...

Identification of differentially expressed genes in sorghum (Sorghum bicolor) brown midrib mutants

Science.gov (United States)

Sorghum (Sorghum bicolor L.), with a high biomass yield and excellent tolerance to drought and low nutrition, has been recommended as one of the most competitive bioenergy crops. Brown midrib (bmr) mutant sorghum with reduced lignin content showed a high potential for the improvement of bioethanol ...

Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

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Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

2018-06-08

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Hypermutability of a UV-sensitive aphidicolin-resistant mutant of Chinese hamster fibroblasts

International Nuclear Information System (INIS)

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

1982-01-01

An ultraviolet light (UV)-sensitive thymidine auxotroph of Chinese hamster V79 cells that exhibits pleiotropic effects such as a high level of deoxycytidine triphosphate, slow growth, sensitivity to cytidine, and high frequencies of site-specific bromodeoxyuridine-dependent chromosomal aberrations was selected by its resistance to aphidicolin. The UV-induced mutability of this mutant and one of its revertants, which retains some of the phenotypes listed above, was studied in 3 mutation assay systems. The results showed that the mutant was hypermutable for ouabain and diphtheria-toxin-resistant mutations compared to wild-type V79 cells at the same UV dose or the same survival level. The mutant exhibits a delayed expression of maximal frequency of induced 6-thioguanine-resistant mutants. When maximal frequencies are compared at the same UV dose, the mutant also has higher mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase locus. The revertant was similar to the wild-type in UV sensitivity and mutability. (orig./AJ)

The Text of the Fourth Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training related to Nuclear Science and Technology (RCA). Extension of Agreement. Latest Status

International Nuclear Information System (INIS)

2007-01-01

The text of the Fourth Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology, 'the 1987 RCA', is reproduced herein for the information of all Members [es

Normal aging modulates the neurotoxicity of mutant huntingtin.

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Elsa Diguet

Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

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.

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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.

Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

Science.gov (United States)

Mei, Wenhan; Wang, Kemin; Huang, Jian; Zheng, Xinmin

2016-01-01

Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.

The Text of the Third Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training related to Nuclear Science and Technology (RCA). Latest Status. Extension of Agreement

International Nuclear Information System (INIS)

2002-01-01

The text of the Third Agreement to Extend the 1987 Regional Co-operative Agreement for Research, Development and Training Related to Nuclear Science and Technology, t he 1987 RCA , is reproduced herein for the information of all Members [es

A mutant of a mutant of a mutant of a ...: Irradiation of progressive radiation-induced mutants in a mutation-breeding programme with Chrysanthenum morifolium RAM

International Nuclear Information System (INIS)

Broertjes, C.; Koene, P.; Veen, J.W.H. van.

1980-01-01

Radiation-induced sports in Chrysanthemum morifolium RAM. have been reported for several years. It has become an everyday practice to produce flower-colour mutants from outstanding cross-breeding products, even before they are distributed for the commercial production of cut flowers. One of the most successful and recent examples is that of cv. Horim, of which hundreds of mutants were produced by successive use of radiation-induced mutants in the mutation-breeding programme. Over about 4 years a variety of flower-colour mutants was obtained, not only largely including the outstanding characteristics of the original cultivar but sometimes even with an appreciable improvement in quality and yield. It is expected that the latter types, the Miros group, will soon completely supersede the spontaneous or raditation-induced Horim sports and mutants and take over the leading position of the Horim group in the production of all-year-round (AYR) cut-flowers. (orig.)

Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

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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

A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

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Man Hei Cheng

Full Text Available Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg was investigated by sequencing. A single base deletion (299delG in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the

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...

Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis.

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Adriana Rodríguez-Marí

2010-07-01

Full Text Available The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53 mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

Genome-wide analysis of the Pho regulon in a pstCA mutant of Citrobacter rodentium.

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Catherine Cheng

Full Text Available The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein and the tad focus (which encodes type IVb pili in Yersinia enterocolitica. Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized

Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53

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Arindam Datta

2016-06-01

Full Text Available Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR treated SW480 cells expressing mutant p53R273H (GEO#: GSE77533. We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.

Construction of an Unmarked Zymomonas mobilis Mutant Using a Site-Specific FLP Recombinase

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Shao-Lan Zou

2012-01-01

Full Text Available Flippase expression was carried out in Zymomonas mobilis strain ZM4. The FRT-flanked selection marker gene was first integrated into the ZM4 chromosome by homologous recombination. The Saccharomyces cerevisiae flp gene was then introduced under the control of the ZM4 gap gene promoter (Pgap, encoding glyceraldehyde-3-phosphate dehydrogenase or the λ bacteriophage cI857-pR contained in the broad-host-range cloning vector pBBR1-MCS-2. This study demonstrated that flp was expressed and that the deletion frequency of the FRT-flanked marker gene was very high (approx. 100 %. In addition, the flp gene expression vector could be conveniently removed from the resulting unmarked Z. mobilis mutants by serially transferring the cells three times into antibiotic-free medium, thereby establishing an efficient method for constructing unmarked Z. mobilis mutants.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

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Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

Transcriptional Responses of the Bdtf1-Deletion Mutant to the Phytoalexin Brassinin in the Necrotrophic Fungus Alternaria brassicicola

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Yangrae Cho

2014-07-01

Full Text Available Brassica species produce the antifungal indolyl compounds brassinin and its derivatives, during microbial infection. The fungal pathogen Alternaria brassicicola detoxifies brassinin and possibly its derivatives. This ability is an important property for the successful infection of brassicaceous plants. Previously, we identified a transcription factor, Bdtf1, essential for the detoxification of brassinin and full virulence. To discover genes that encode putative brassinin-digesting enzymes, we compared gene expression profiles between a mutant strain of the transcription factor and wild-type A. brassicicola under two different experimental conditions. A total of 170 and 388 genes were expressed at higher levels in the mutants than the wild type during the infection of host plants and saprophytic growth in the presence of brassinin, respectively. In contrast, 93 and 560 genes were expressed, respectively, at lower levels in the mutant than the wild type under the two conditions. Fifteen of these genes were expressed at lower levels in the mutant than in the wild type under both conditions. These genes were assumed to be important for the detoxification of brassinin and included Bdtf1 and 10 putative enzymes. This list of genes provides a resource for the discovery of enzyme-coding genes important in the chemical modification of brassinin.

Lifespan decrease in a Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory neurons.

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Miranda-Vizuete, Antonio; Fierro González, Juan Carlos; Gahmon, Gabriele; Burghoorn, Jan; Navas, Plácido; Swoboda, Peter

2006-01-23

Thioredoxins are a class of small proteins that play a key role in regulating many cellular redox processes. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The Caenorhabditis elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ ciliated sensory neurons, and to some extent also in the posterior-most intestinal cells. TRX-1 is active at reducing protein disulfides in the presence of a heterologous thioredoxin reductase. A mutant worm strain carrying a null allele of the trx-1 gene displays a reproducible decrease in both mean and maximum lifespan when compared to wild-type. The identification and characterization of TRX-1 paves the way to use C. elegans as an in vivo model to study the role of thioredoxins in lifespan and nervous system physiology and pathology.

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

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Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi

2016-10-01

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Transcriptome Analysis of a New Peanut Seed Coat Mutant for the Physiological Regulatory Mechanism Involved in Seed Coat Cracking and Pigmentation.

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Wan, Liyun; Li, Bei; Pandey, Manish K; Wu, Yanshan; Lei, Yong; Yan, Liying; Dai, Xiaofeng; Jiang, Huifang; Zhang, Juncheng; Wei, Guo; Varshney, Rajeev K; Liao, Boshou

2016-01-01

Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts ( Arachis hypogaea L.). With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa) was identified from an EMS treated mutant population and designated as "peanut seed coat crack and brown color mutant line ( pscb )." The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly shorter than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyanidin content, and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40). Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1), c40902_g2 (kinesin) , and c33560_g1 (MYB3) were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color.

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

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Whitt, M.A.; Chong, L.; Rose, J.K.

1989-01-01

The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding

A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

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Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

2005-01-01

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

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Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

Enhanced Thermostability of Arabidopsis Rubisco Activase Improves Photosynthesis and Growth Rates under Moderate Heat Stress[OA

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Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M.; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W.; Zhu, Genhai

2007-01-01

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Δrca) line. In a long-term growth test at either constant 26°C or daily 4-h 30°C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions. PMID:17933901

Studies on induced partially resistant mutants of barley against powdery mildew

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Roebbelen, G.; Abdel-Hafez, A.G.; Reinhold, M.; Kwon, H.J.; Neuhaus-Steinmetz, J.P.; Heun, M.

1983-01-01

After mutagenic seed treatment of three partially resistant cultivars of spring barley with EMS and NaN 3 , 45 mutants in a first and 16 in a second experiment were selected in the M 2 -M 4 generations. The screening was done alternatively under natural infection in the field or controlled infection with a single pathotype in the greenhouse. These mutants exhibited a higher resistance and a higher susceptibility, respectively, than the initial cultivars Asse, Bomi and Vada. Some mutants expressed their altered resistance behaviour particularly during certain stages of development. High-level resistance was conditioned by mutation in the ml-o locus in three cases. For several Bomi mutants pathotype specificity with and without reversed ranking was proven as well as pathotype non-specificity in comparison with the reaction of the original cultivar. In 14 cases studied the inheritance of the involved mutants was monogenic recessive. The laevigatum locus responsible for the intermediate mildew resistance of Bomi was not affected by the mutations. Detection of groups of allelic mutants showed that there are at least two regions in the barley genome in which mutations for mildew resistance can occur rather frequently. In total, the past ten years of this mutation research have given convincing evidence that the strategies of mutant screening applied have yielded promising new material both for breeding and for progress in basic understanding of host-pathogen interactions. (author)

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

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Zhang Xue

2006-07-01

Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

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

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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.

Expression of Ricinus communis receptors on epithelial cells in oral carcinomas and oral wounds.

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Dabelsteen, E; Mackenzie, I C

1978-12-01

The histological distribution of receptors for Ricinus communis Fraction 1 (RCA1) in oral carcinomas and in oral epithelial cells during wound healing has been studied by use of fluorescein-tagged RCA1. Biopsies from 15 human oral carcinomas and adjacent normal mucosa showed RCA1 receptors at the cell membranes in the basal and spinous layer of the normal epithelium, whereas receptors could not be demonstrated in invading islands of the tumors. In healing oral wounds from eight humans and three monkeys, RCA1 receptors were demonstrated both in normal epithelium adjacent to the wounds and in the epithelial outgrowth from the wound margin. Titrations, however, showed that the epithelial outgrowth reacted more weakly than did the normal adjacent epithelium. These results support previous in vitro studies showing changes in carbohydrate composition of moving normal cells and of malignant cells, a finding that may be of interest in relation to formation of metastases.

Mutant DnaAs of Escherichia coli that are refractory to negative control.

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Chodavarapu, Sundari; Felczak, Magdalena M; Simmons, Lyle A; Murillo, Alec; Kaguni, Jon M

2013-12-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

Low-energy N-ion beam biotechnology application in the induction of Thai jasmine rice mutant with improved seed storability

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Semsang, Nuananong; Techarang, Jiranat; Yu, Liangdeng; Phanchaisri, Boonrak

2018-06-01

Low-energy heavy-ion beam is a novel biotechnology used for mutation induction in plants. We used a low-energy N-ion beam to induce mutations in Thai jasmine rice (Oryza sativa L. cv. KDML 105) to improve the yield and seed quality. Seeds of BKOS6, a Thai jasmine rice mutant previously induced by ion beams, were re-bombarded with 60-kV-accelerated N-ions (N++N2+) to fluences of 1-2 × 1016 ions/cm2. The resulting mutant, named HyKOS21, exhibited photoperiod insensitivity, semi-dwarfness, and high yield potential. Seed storability of the mutant was studied in natural and accelerated ageing conditions and compared to that of KDML 105 and six other Thai rice varieties. In both testing conditions, HyKOS21 mutant had the highest seed storability among the tested varieties. After storage in the natural condition for 18 months, HyKOS21 had a seed germination percentage nearly two times as that of the original KDML 105. Biochemical analysis showed that the lipid peroxidation level of the mutant seeds was the lowest among those of the tested varieties. Furthermore, an expression analysis of genes encoding lipoxygenase isoenzyme (lox1, lox2, and lox3) revealed that the mutant lacked expression of lox1 and lox2 and expressed only lox3 in seeds. These results may explain the improved seed longevity of the mutant after storage. This work provides further evidence of the modification of biological materials using a low-energy ion beam to produce rice mutants with improved yield and seed storability. The benefits of this technology, to create new varieties with improved values, could serve for local economic development.

Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.

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Wei, Yujun; Chen, Jianhua; Jia, Ruibo; Wang, Min; Wu, Wutong

2008-07-01

The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.

Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

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Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

2008-01-01

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models

EMS mutant spectra generated by multi-parameter flow cytometry

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Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H., E-mail: michael.fox@colostate.edu [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States)

2009-12-01

The CHO A{sub L} cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59{sup -} regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59{sup -} clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata.

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Fan, Lili; Fu, Kehe; Yu, Chuanjin; Ma, Jia; Li, Yaqian; Chen, Jie

2014-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.

Data on quantification of signaling pathways activated by KIT and PDGFRA mutants

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Christelle Bahlawane

2016-12-01

Full Text Available The present data are related to the article entitled “Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling” (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016 [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells. Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf. We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

Evidence for a Ustilago maydis Steroid 5α-Reductase by Functional Expression in Arabidopsis det2-1 Mutants1

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Basse, Christoph W.; Kerschbamer, Christine; Brustmann, Markus; Altmann, Thomas; Kahmann, Regine

2002-01-01

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5α-steroid reductases. Udh1 differs from those of known 5α-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5α-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5α-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins. PMID:12068114

Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities.

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Garrett, Matthew; Sperry, Jantzen; Braas, Daniel; Yan, Weihong; Le, Thuc M; Mottahedeh, Jack; Ludwig, Kirsten; Eskin, Ascia; Qin, Yue; Levy, Rachelle; Breunig, Joshua J; Pajonk, Frank; Graeber, Thomas G; Radu, Caius G; Christofk, Heather; Prins, Robert M; Lai, Albert; Liau, Linda M; Coppola, Giovanni; Kornblum, Harley I

2018-01-01

There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13 C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable

Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase

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Wang Yuxia; Ticu Boeck, Andreea; Duysen, Ellen G.; Van Keuren, Margaret; Saunders, Thomas L.; Lockridge, Oksana

2004-01-01

Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 μg/ml of human G117H BChE in plasma as well as 2 μg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 μg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal

Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.

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Di Pietro, Chiara; Marazziti, Daniela; La Sala, Gina; Abbaszadeh, Zeinab; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

2017-01-01

Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.

Rescue of glaucoma-causing mutant myocilin thermal stability by chemical chaperones

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Burns, J. Nicole; Orwig, Susan D.; Harris, Julia L.; Watkins, J. Derrick; Vollrath, Douglas; Lieberman, Raquel L.

2010-01-01

Mutations in myocilin cause an inherited form of open angle glaucoma, a prevalent neurodegenerative disorder associated with increased intraocular pressure. Myocilin forms part of the trabecular meshwork extracellular matrix presumed to regulate intraocular pressure. Missense mutations, clustered in the olfactomedin (OLF) domain of myocilin, render the protein prone to aggregation in the endoplasmic reticulum of trabecular meshwork cells, causing cell dysfunction and death. Cellular studies have demonstrated temperature-sensitive secretion of myocilin mutants, but difficulties in expression and purification have precluded biophysical characterization of wild-type (wt) myocilin and disease-causing mutants in vitro. We have overcome these limitations by purifying wt and select glaucoma-causing mutant (D380A, I477N, I477S, K423E) forms of the OLF domain (228–504) fused to maltose binding protein (MBP) from E. coli. Monomeric fusion proteins can be isolated in solution. To determine the relative stability of wt and mutant OLF domains, we developed a fluorescence thermal stability assay without removal of MBP, and provide the first direct evidence that mutated OLF is folded but less thermally stable than wt. We tested the ability of seven chemical chaperones to stabilize mutant myocilin. Only sarcosine and trimethylamine N-oxide were capable of shifting the melting temperature of all mutants tested to near that of wt OLF. Our work lays the foundation for the identification of tailored small molecules capable of stabilizing mutant myocilin and promoting secretion to the extracellular matrix, to better control intraocular pressure and ultimately delay the onset of myocilin glaucoma. PMID:20334347

Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state.

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Pamela J Lescault

2010-12-01

Full Text Available Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states--genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.

Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts

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Satoru Noguchi

2014-01-01

Full Text Available Ullrich congenital muscular dystrophy (UCMD is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

Purification and characterization of mutant miniPlasmin for thrombolytic therapy

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Lin Xiaotao

2013-01-01

Full Text Available Abstract Background Previous animal studies by us and others have indicated that catheter-administered plasmin or its des-kringle derivatives may be more appropriate alternatives to plasminogen activators for treating thrombolytic diseases, since it has a very short serum half-life and therefore does not result in hemorrhaging. We have previously produced recombinant miniPlasmin (mPlasmin that was proven suitable for treating peripheral arterial occlusion in animal models. However, our previous results showed that non-specific cleavage at position K698 of mPlasmin during activation hindered the further development of this promising therapeutic candidate. In order to minimize or eliminate the non-specific cleavage problem, we performed saturation mutagenesis at the K698 position to develop a mutant form of mPlasmin for thrombolytic therapy. Methods We changed K698 to 16 other amino acids, with preferred E. coli codons. Each of these mutants were expressed in E. coli as inclusion bodies and then refolded, purified, and subsequently characterized by detailed kinetic assays/experiments/studies which identified highly active mutants devoid of non-specific cleavage. Results Activation studies indicated that at those conditions in which the wild type enzyme is cut at the non-specific position K698, the active mutants can be activated without being cleaved at this position. Conclusions From the above results, we selected two mutants, K698Q and K698N, as our lead candidates for further thrombolytic drug developments. The selected mutants are potentially better therapeutic candidates for thrombolytic therapy.

Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.

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Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W; Zhu, Genhai

2007-10-01

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

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Peter Novodvorsky

Full Text Available The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2 transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development.Using Transcription Activator-Like Effector Nucleases (TALEN we generated a klf2a mutant (klf2ash317 with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl, a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17 in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Transcriptome Analysis of a New Peanut Seed Coat Mutant for the Physiological Regulatory Mechanism Involved in Seed Coat Cracking and Pigmentation

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Wan, Liyun; Li, Bei; Pandey, Manish K.; Wu, Yanshan; Lei, Yong; Yan, Liying; Dai, Xiaofeng; Jiang, Huifang; Zhang, Juncheng; Wei, Guo; Varshney, Rajeev K.; Liao, Boshou

2016-01-01

Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts (Arachis hypogaea L.). With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa) was identified from an EMS treated mutant population and designated as “peanut seed coat crack and brown color mutant line (pscb).” The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly shorter than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyanidin content, and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40). Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1), c40902_g2 (kinesin), and c33560_g1 (MYB3) were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color. PMID

Sustracted library obtained from mutant sugarcane variety B 4362 resistant to rust

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María I. Oloriz

2002-07-01

Full Text Available The hypersensitive response is one of the most powerful mechanisms for which the plants resist pathogen attack. Mutations carried out previously on the variety B4362, of sugarcane, originated five mutants that express this mechanism towards the attack of rust (Puccinia melanocephala Syd.. By means of a subtractive hybridization among the cDNA obtained starting from the resistant clone inoculated with rust and a pool of cDNA of the susceptible variety (B4362 inoculated and of the resistant clone not inoculated, it was possible to reduce the number of genes expressed during the infection with the fungus. A subtractive library was carried out where we hope that most of the genes are involved in the hypersensitive response that present these mutants towards the infection of the pathogen. Key words: Subtractive hybridization, hypersensitive response, Puccinia melanocephala Syd.

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.

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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.

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.

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Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

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.

Differential gene expression by RamA in ciprofloxacin-resistant Salmonella Typhimurium.

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Jie Zheng

Full Text Available Overexpression of ramA has been implicated in resistance to multiple drugs in several enterobacterial pathogens. In the present study, Salmonella Typhimurium strain LTL with constitutive expression of ramA was compared to its ramA-deletion mutant by employing both DNA microarrays and phenotype microarrays (PM. The mutant strain with the disruption of ramA showed differential expression of at least 33 genes involved in 11 functional groups. The study confirmed at the transcriptional level that the constitutive expression of ramA was directly associated with increased expression of multidrug efflux pump AcrAB-TolC and decreased expression of porin protein OmpF, thereby conferring multiple drug resistance phenotype. Compared to the parent strain constitutively expressing ramA, the ramA mutant had increased susceptibility to over 70 antimicrobials and toxic compounds. The PM analysis also uncovered that the ramA mutant was better in utilization of 10 carbon sources and 5 phosphorus sources. This study suggested that the constitutive expression of ramA locus regulate not only multidrug efflux pump and accessory genes but also genes involved in carbon metabolic pathways.

An extra early mutant of pigeonpea

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Ravikesavan, R.; Kalaimagal, T.; Rathnaswamy, R.

2001-01-01

The redgram (Cajanus cajan (L.) Huth) variety 'Prabhat DT' was gamma irradiated with 100, 200, 300 and 400 Gy doses. Several mutants have been identified viz., extra early mutants, monostem mutants, obcordifoliate mutants and bi-stigmatic mutants. The extra early mutant was obtained when treated with 100 Gy dose. The mutant was selfed and forwarded from M 2 to M 4 generation. In the M 4 generation the mutant line was raised along with the parental variety. Normal cultural practices were followed and the biometrical observations were recorded. It was observed that for the characters viz., total number of branches per plant, number of pods per plants, seeds per pod, 100 seed weight and seed yield per plant there was no difference between the mutant and parent variety. Whereas, regarding the days to flowering and maturity the mutants were earlier than the parents. The observation was recorded from two hundred plants each. The mutant gives the same yield in 90 days as that of the parent variety in 107 days, which make it an economic mutant

Molecular Basis Underlying Leaf Variegation of a Moth Orchid Mutant (Phalaenopsis aphrodite subsp. formosana

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Chi-Chu Tsai

2017-07-01

Full Text Available Leaf variegation is often the focus of plant breeding. Here, we studied a variegated mutant of Phalaenopsis aphrodite subsp. formosana, which is usually used as a parent of horticultural breeding, to understand its anatomic and genetic regulatory mechanisms in variegation. Chloroplasts with well-organized thylakoids and starch grains were found only in the mesophyll cells of green sectors but not of yellow sectors, confirming that the variegation belongs to the chlorophyll type. The two-dimensional electrophoresis and LC/MS/MS also reveal differential expressions of PsbP and PsbO between the green and yellow leaf sectors. Full-length cDNA sequencing revealed that mutant transcripts were caused by intron retention. When conditioning on the total RNA expression, we found that the functional transcript of PsbO and mutant transcript of PsbP are higher expressed in the yellow sector than in the green sector, suggesting that the post-transcriptional regulation of PsbO and PsbP differentiates the performance between green and yellow sectors. Because PsbP plays an important role in the stability of thylakoid folding, we suggest that the negative regulation of PsbP may inhibit thylakoid development in the yellow sectors. This causes chlorophyll deficiency in the yellow sectors and results in leaf variegation. We also provide evidence of the link of virus CymMV and the formation of variegation according to the differential expression of CymMV between green and yellow sectors.

Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants.

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Verma, Manish; Callio, Jason; Otero, P Anthony; Sekler, Israel; Wills, Zachary P; Chu, Charleen T

2017-11-15

Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis. SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are

Gene expression profiling of loss of TET2 and/or JAK2V617F mutant hematopoietic stem cells from mouse models of myeloproliferative neoplasms

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Takuro Kameda

2015-06-01

Full Text Available Myeloproliferative neoplasms (MPNs are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and the gradual expansion of malignant intramedullary/extramedullary hematopoiesis. In MPNs mutations in JAK2 MPL or CALR are detected mutually exclusive in more than 90% of cases [1,2]. Mutations in them lead to the abnormal activation of JAK/STAT signaling and the autonomous growth of differentiated cells therefore they are considered as “driver” gene mutations. In addition to the above driver gene mutations mutations in epigenetic regulators such as TET2 DNMT3A ASXL1 EZH2 or IDH1/2 are detected in about 5%–30% of cases respectively [3]. Mutations in TET2 DNMT3A EZH2 or IDH1/2 commonly confer the increased self-renewal capacity on normal hematopoietic stem cells (HSCs but they do not lead to the autonomous growth of differentiated cells and only exhibit subtle clinical phenotypes [4,6–8,5]. It was unclear how mutations in such epigenetic regulators influenced abnormal HSCs with driver gene mutations how they influenced the disease phenotype or whether a single driver gene mutation was sufficient for the initiation of human MPNs. Therefore we focused on JAK2V617F and loss of TET2—the former as a representative of driver gene mutations and the latter as a representative of mutations in epigenetic regulators—and examined the influence of single or double mutations on HSCs (Lineage−Sca-1+c-Kit+ cells (LSKs by functional analyses and microarray whole-genome expression analyses [9]. Gene expression profiling showed that the HSC fingerprint genes [10] was statistically equally enriched in TET2-knockdown-LSKs but negatively enriched in JAK2V617F–LSKs compared to that in wild-type-LSKs. Double-mutant-LSKs showed the same tendency as JAK2V617F–LSKs in terms of their HSC fingerprint genes but the expression of individual genes differed between the two groups. Among 245 HSC fingerprint genes 100 were more

highroad Is a Carboxypetidase Induced by Retinoids to Clear Mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa Models

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Huai-Wei Huang

2018-02-01

Full Text Available Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.

Mutant heterosis in rice

International Nuclear Information System (INIS)

1987-01-01

In the variety TKM6 a high yielding semidwarf mutant has been induced. This TKM6 mutant was used in test crosses with a number of other varieties and mutants to examine the extent of heterosis of dwarfs in rice and to select superior crosses. An excerpt of the published data is given. It appears from the backcross of the mutant with its original variety, that an increase in number of productive tillers occurs in the hybrid, leading to a striking grain yield increase, while the semi-dwarf culm length (the main mutant character) reverts to the normal phenotype. In the cross with IR8 on the other hand, there is only a minimal increase in tiller number but a substantial increase in TGW leading to more than 30% yield increase over the better parent

Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

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Hama, S; Kimura, G

1980-01-01

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

Impacts of using spectral nudging on regional climate model RCA4 simulations of the Arctic

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P. Berg

2013-06-01

Full Text Available The performance of the Rossby Centre regional climate model RCA4 is investigated for the Arctic CORDEX (COordinated Regional climate Downscaling EXperiment region, with an emphasis on its suitability to be coupled to a regional ocean and sea ice model. Large biases in mean sea level pressure (MSLP are identified, with pronounced too-high pressure centred over the North Pole in summer of over 5 hPa, and too-low pressure in winter of a similar magnitude. These lead to biases in the surface winds, which will potentially lead to strong sea ice biases in a future coupled system. The large-scale circulation is believed to be the major reason for the biases, and an implementation of spectral nudging is applied to remedy the problems by constraining the large-scale components of the driving fields within the interior domain. It is found that the spectral nudging generally corrects for the MSLP and wind biases, while not significantly affecting other variables, such as surface radiative components, two-metre temperature and precipitation.

Transcriptome Changes Associated with Delayed Flower Senescence on Transgenic Petunia by Inducing Expression of etr1-1, a Mutant Ethylene Receptor

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Lin, Jing; Liu, Gang; Zhang, Zhen; Chang, Youhong; Reid, Michael S.; Jiang, Cai-Zhong

2013-01-01

Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1), a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX). Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, ‘the regulation of transcription’ was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death. PMID:23874385

Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor.

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Hong Wang

Full Text Available Flowers of ethylene-sensitive ornamental plants transformed with ethylene-insensitive 1-1(etr1-1, a mutant ethylene receptor first isolated from Arabidopsis, are known to have longer shelf lives. We have generated petunia plants in which the etr1-1 gene was over-expressed under the control of a chemically-inducible promoter, which would allow expression of etr1-1 to be initiated at the desired time and stage of development. Here, we showed that transgenic plants grew and developed normally without a chemical inducer. Semi-quantitative RT-PCR demonstrated that the abundance of transcripts of Arabidopsis etr1-1 gene was substantially induced in flowers with 30 μM dexamethasone (DEX. Consequently, t he life of the flowers was almost doubled and the peak of ethylene production was delayed. We compared gene expression changes of petals with DEX to those without DEX at 24 h and 48 h by microarray. Our results indicated that transcripts of many putative genes encoding transcription factors were down-regulated by etr1-1 induced expression at the early stage. In addition, putative genes involved in gibberellin biosynthesis, response to jasmonic acid/gibberellins stimulus, cell wall modification, ethylene biosynthesis, and cell death were down-regulated associating with etr1-1 induced expression. We investigated time-course gene expression profiles and found two profiles which displayed totally opposite expression patterns under these two treatments. In these profiles, 'the regulation of transcription' was predominant in GO categories. Taking all results together, we concluded those transcription factors down-regulated at early stage might exert a major role in regulating the senescence process which were consequently characterized by cell wall modification and cell death.

A thermo-sensitive purple leaf rice mutant--PLM12 and its genetical study

International Nuclear Information System (INIS)

Wu Guanting; Wang Xianyu; Jin Wei

2001-01-01

PLM12 was a thermo-sensitive purple leaf mutant selected from Indica rice variety Luqingzao 1 treated with pingyangmycin in combination with γ-rays, and for display of its mutant character, a relatively high temperature was required. Compared with its original parent, many major agronomic traits of PLM12 changed to varied extents. Based on spikelet cutting experiment, it was believed that significant decreases in number of filled grains per panicle, fertility, and 1000-grain weight in PLM12 resulted mainly from a great decline in photosynthetic capacity and serious lack of photosynthate in purple leaves. It was indicated by genetic analysis that expression of the mutant phenotype in PLM12 was conditioned by a single recessive major nuclear gene and modified by several minor genes

Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

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Seok Joo Park

2014-09-01

Full Text Available BackgroundIt has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH/insulin-like growth factor-1 (IGF-1.MethodsIn this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice.ResultsThe GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt/phospho-glycogen synthase kinase3β (p-GSK3β, phospho-extracellular signal-related kinase (p-ERK, and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK, Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist.ConclusionThe results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation.

Mig6 Puts the Brakes on Mutant EGFR-Driven Lung Cancer | Center for Cancer Research

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Lung cancer is the most common cause of cancer-related death worldwide. These cancers are often induced by mutations in the epidermal growth factor receptor (EGFR), resulting in constitutive activation of the protein’s tyrosine kinase domain. Lung cancers expressing these EGFR mutants are initially sensitive to tyrosine kinase inhibitors (TKIs), such as erlotinib, but often become resistant by developing compensatory mutations in EGFR or other growth-promoting pathways. To better understand how mutant EGFR initiates and maintains tumor growth in the hopes of identifying novel targets for drug development, Udayan Guha, M.D., Ph.D., of CCR’s Thoracic and Gastrointestinal Oncology Branch, and his colleagues examined the landscape of proteins phosphorylated in EGFR wild type and mutant cells. One protein hyper-phosphorylated in mutant EGFR cells was Mig6, a putative tumor suppressor.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

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Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

2016-01-01

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

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Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

2016-01-01

Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation.

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Kupfer, G; Naf, D; Garcia-Higuera, I; Wasik, J; Cheng, A; Yamashita, T; Tipping, A; Morgan, N; Mathew, C G; D'Andrea, A D

1999-04-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A>C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wild-type FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway.

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

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Lauren E Hudson

Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

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Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

2014-01-01

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

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Kenta Wada

Full Text Available Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3 as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of

Morphogenes bolA and mreB mediate the photoregulation of cellular morphology during complementary chromatic acclimation in Fremyella diplosiphon.

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Singh, Shailendra P; Montgomery, Beronda L

2014-07-01

Photoregulation of pigmentation during complementary chromatic acclimation (CCA) is well studied in Fremyella diplosiphon; however, mechanistic insights into the CCA-associated morphological changes are still emerging. F. diplosiphon cells are rectangular under green light (GL), whereas cells are smaller and spherical under red light (RL). Here, we investigate the role of morphogenes bolA and mreB during CCA using gene expression and gene function analyses. The F. diplosiphon bolA gene is essential as its complete removal from the genome was unsuccessful. Depletion of bolA resulted in slow growth, morphological defects and the accumulation of high levels of reactive oxygen species in a partially segregated ΔbolA strain. Higher expression of bolA was observed under RL and was correlated with lower expression of mreB and mreC genes in wild type. In a ΔrcaE strain that lacks the red-/green-responsive RcaE photoreceptor, the expression of bolA and mre genes was altered under both RL and GL. Observed gene expression relationships suggest that mreB and mreC expression is controlled by RcaE-dependent photoregulation of bolA expression. Expression of F. diplosiphon bolA and mreB homologues in Escherichia coli demonstrated functional conservation of the encoded proteins. Together, these studies establish roles for bolA and mreB in RcaE-dependent regulation of cellular morphology. © 2014 John Wiley & Sons Ltd.

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

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

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

Science.gov (United States)

Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

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.

Root-Expressed Maize Lipoxygenase 3 Negatively Regulates Induced Systemic Resistance to Colletotrichum graminicola in Shoots

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Nasie eConstantino

2013-12-01

Full Text Available We have previously reported that disruption of a maize root-expressed 9-lipoxygenase (9-LOX gene, ZmLOX3, results in dramatic increase in resistance to diverse leaf and stalk pathogens. Despite evident economic significance of these findings, the mechanism behind this increased resistance remained elusive. In this study, we show that increased resistance of the lox3-4 mutants is due to constitutive activation of induced systemic resistance (ISR signaling. We showed that ZmLOX3 lacked expression in leaves in response to anthracnose leaf blight pathogen Colletotrichum graminicola, but was expressed constitutively in the roots, thus prompting our hypothesis: the roots of lox3-4 mutants are the source of increased resistance in leaves. Supporting this hypothesis, treatment of wild-type plants (WT with xylem sap of lox3-4 mutant induced resistance to C. graminicola to the levels comparable to those observed in lox3-4 mutant. Moreover, treating mutants with the sap collected from WT plants partially restored the susceptibility to C. graminicola. lox3-4 mutants showed primed defense responses upon infection, which included earlier and greater induction of defense-related PAL and GST genes compared to WT. In addition to the greater expression of the octadecanoid pathway genes, lox3-4 mutant responded earlier and with a greater accumulation of H2O2 in response to C. graminicola infection or treatment with alamethicin. These findings suggest that lox3-4 mutants display constitutive ISR-like signaling. In support of this idea, root colonization by Trichoderma virens strain GV29-8 induced the same level of disease resistance in WT as the treatment with the mutant sap, but had no additional resistance effect in lox3-4 mutant. While treatment with T. virens GV29 strongly and rapidly suppressed ZmLOX3 expression in hydroponically grown WT roots, T. virens Δsml mutant, which is deficient in ISR induction, was unable to suppress expression of ZmLOX3, thus

Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

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McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martín; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

1998-01-01

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′. PMID:9721309

Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

International Nuclear Information System (INIS)

Ugai, Hideyo; Inabe, Kumiko; Yamasaki, Takahito; Murata, Takehide; Obata, Yuichi; Hamada, Hirofumi; Yokoyama, Kazunari K.

2005-01-01

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions

Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose

DEFF Research Database (Denmark)

Hong, Kuk-Ki; Nielsen, Jens

2013-01-01

the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from...... the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants...

Effect of hypergravity on lignin formation and expression of lignin-related genes in inflorescence stems of an ethylene-insensitive Arabidopsis mutant ein3-1

Science.gov (United States)

Karahara, Ichirou; Kobayashi, Mai; Tamaoki, Daisuke; Kamisaka, Seiichiro

Our previous studies have shown that hypergravity inhibits growth and promotes lignin forma-tion in inflorescence stems of Arabidopsis thaliana by up-regulation of genes involved in lignin biosynthesis (Tamaoki et al. 2006, 2009). In the present study, we have examined whether ethylene is involved in these responses using an ethylene-insensitive Arabidopsis mutant ein3-1. Our results revealed that hypergravity treatment at 300 G for 24 h significantly inhibited growth of inflorescence stems, promoted both deposition of acetyl bromide extractable lignin and gene expression involved in lignin formation in inflorescence stems of wild type plants. Growth inhibition of inflorescence stems was also observed in ein3-1. However, the effects of hypergravity on the promotion of the deposition of acetyl bromide lignin and the expression of genes involved in lignin formation were not observed in ein3-1, indicating that ethylene sig-naling is involved in the up-regulation of the expression of lignin-related genes as well as the promotion of deposition of lignin by hypergravity in Arabidopsis inflorescence stems.

Role of Myofibril-Inducing RNA in cardiac TnT expression in developing Mexican axolotl

Science.gov (United States)

Sferrazza, Gian-Franco; Zhang, Chi; Jia, Pingping; Lemanski, Sharon L.; Athauda, Gagani; Stassi, Alyssa; Halager, Kristine; Maier, Jennifer A.; Rueda-de-Leon, Elena; Gupta, Amit; Dube, Syamalima; Huang, Xupei; Prentice, Howard M.; Dube, Dipak K.; Lemanski, Larry F.

2007-01-01

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene “c”, for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts.. Myofibril-Inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac Troponin-T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart PMID:17408593

Recombination Phenotypes of Escherichia coli greA Mutants

Directory of Open Access Journals (Sweden)

Poteete Anthony R

2011-03-01

Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

NARCIS (Netherlands)

Huang, M.; Jongejan, L.; Zheng, H.; Zhang, L.; Bol, J. F.

2001-01-01

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype.

Science.gov (United States)

Hogan, Alison L; Don, Emily K; Rayner, Stephanie L; Lee, Albert; Laird, Angela S; Watchon, Maxinne; Winnick, Claire; Tarr, Ingrid S; Morsch, Marco; Fifita, Jennifer A; Gwee, Serene S L; Formella, Isabel; Hortle, Elinor; Yuan, Kristy C; Molloy, Mark P; Williams, Kelly L; Nicholson, Garth A; Chung, Roger S; Blair, Ian P; Cole, Nicholas J

2017-07-15

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume.

Science.gov (United States)

Grassini, Daniela R; Lagendijk, Anne K; De Angelis, Jessica E; Da Silva, Jason; Jeanes, Angela; Zettler, Nicole; Bower, Neil I; Hogan, Benjamin M; Smith, Kelly A

2018-05-11

Atrial natriuretic peptide ( nppa/anf ) and brain natriuretic peptide ( nppb/bnp ) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy however their genomic location in cis has impeded formal analysis. Using genome-editing, we generated mutants for nppa and nppb and found single mutants indistinguishable from wildtype whereas nppa / nppb double mutants display heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4 , tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for Hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirm cardiac jelly expansion in nppa / nppb double mutants. Finally, bmp4 knockdown rescues the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber. © 2018. Published by The Company of Biologists Ltd.

Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

Science.gov (United States)

Einav, Tal; Duque, Julia; Phillips, Rob

2018-02-01

Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

Site-directed mutagenesis of HIV-1 vpu gene demonstrates two clusters of replication-defective mutants with distinct ability to down-modulate cell surface CD4 and tetherin

Directory of Open Access Journals (Sweden)

Masako Nomaguchi

2010-11-01

Full Text Available HIV-1 Vpu acts positively on viral infectivity by mediating CD4 degradation in endoplasmic reticulum and enhances virion release by counteracting a virion release restriction factor, tetherin. In order to define the impact of Vpu activity on HIV-1 replication, we have generated a series of site-specific proviral vpu mutants. Of fifteen mutants examined, seven exhibited a replication-defect similar to that of a vpu-deletion mutant in a lymphocyte cell line H9. These mutations clustered in narrow regions within transmembrane domain (TMD and cytoplasmic domain (CTD. Replication-defective mutants displayed the reduced ability to enhance virion release from a monolayer cell line HEp2 without exception. Upon transfection with Vpu expression vectors, neither TMD mutants nor CTD mutants blocked CD4 expression at the cell surface in another monolayer cell line MAGI. While TMD mutants were unable to down-modulate cell surface tetherin in HEp2 cells, CTD mutants did quite efficiently. Confocal microscopy analysis revealed the difference of intracellular localization between TMD and CTD mutants. In total, replication capability of HIV-1 carrying vpu mutations correlates well with the ability of Vpu to enhance virion release and to impede the cell surface expression of CD4 but not with the ability to down-modulate cell surface tetherin. Our results here suggest that efficient viral replication requires not only down-regulation of cell surface tetherin but also its degradation.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

Science.gov (United States)

Inga, Alberto; Nahari, Dorit; Velasco-Miguel, Susana; Friedberg, Errol C; Resnick, Michael A

2002-08-22

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 2. Comparison of Various Mutants

International Nuclear Information System (INIS)

Balchiuniene, L.

1995-01-01

Spontaneous and gamma-induced mutability was compared in two groups of genetically unstable barley ear structure mutants - tweaky spike (tw) and branched ear (be). Instability in different loci causes different levels of spontaneous and gamma-induced mutability. A high spontaneous level of chlorophyll mutations is peculiar to be-ust mutants. It is suggested that the high level of induced chlorophyll mutations in allelic tw mutants is a result of better surviving of chlorophyll mutation carriers in the genotypical-physiological environment created by mutant tw alleles. (author). 6 refs., 2 tabs

Identification of genes associated with growth cessation and bud dormancy entrance using a dormancy-incapable tree mutant.

Science.gov (United States)

Jiménez, Sergio; Li, Zhigang; Reighard, Gregory L; Bielenberg, Douglas G

2010-02-09

In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points. We identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure. The use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.

Promising rice mutants

International Nuclear Information System (INIS)

Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

1988-01-01

Two induced mutants namely, Mut NS 1 (tall) and Mut NS 5 (semi-dwarf) derived from rice variety Nizersail were evaluated for various agronomic characters at four locations in Bangladesh. Both the mutants matured about three weeks earlier and yielded significantly higher than the parent variety Nizersail. (author). 3 tabs., 9 refs

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

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Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S; Wentzell, Jill S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

2012-03-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. Copyright © 2011 Elsevier Inc. All rights reserved.

Genetic study of Pea (Pisum sativum L.) mutants with changed shape and/or dentation of leaves

International Nuclear Information System (INIS)

Naidenova, N.

2001-01-01

The purpose of this study is to describe the morphological differences between normal plants and mutants (due to irradiation) with different shape and/or dentation of leaflets and to evaluate their productivity and genetic potential. Dry seeds have been submitted to gamma irradiation with doses 100 Gy, 150 Gy and 200 Gy.The mutants studies in this research introduce an important source for further investigation of genetics of the mutant traits - dentation of leaflets, shape and especially flowering time that is controlled by genetically determined responses to photo period and temperature. Due to the clear phenotypic expression of the shape and leaves in some plants it is considered that the development of the leaves mutants is and important finding for pea genetics making tham valuable morphological markers for genetic investigations

KRAS Mutant Status, p16 and β-catenin Expression May Predict Local Recurrence in Patients Who Underwent Transanal Endoscopic Microsurgery (TEMS) for Stage I Rectal Cancer.

Science.gov (United States)

Sideris, Michail; Moorhead, Jane; Diaz-Cano, Salvador; Bjarnason, Ingvar; Haji, Amyn; Papagrigoriadis, Savvas

2016-10-01

Transanal endoscopic microsurgery (TEMS) is emerging as an alternative treatment for rectal cancer Stage I. There remains a risk of local recurrence. The Aim of the study was to study the effect of biomarkers in local recurrence for Stage I rectal cancer following TEMS plus or minus radiotherapy. This is a case control study where we compared 10 early rectal cancers that had recurred, against 19 cases with no recurrence, total 29 patients (age=28.25-86.87, mean age=67.92 years, SD=14.91, Male, N=18, Female, N=11). All patients underwent TEMS for radiological Stage I rectal cancer (yT1N0M0 or yT2N0M0) established with combination of magnetic resonance imaging (MRI) and endorectal ultrasound. We prospectively collected all data on tumour histology, morphological features, as well as follow-up parameters. Molecular analysis was performed to identify their status on BRAF, KRAS, p16 O 6 -methylguanine-DNA methyltransferase (MGMT) and β-catenin. Out of 29 specimens analyzed, 19 were KRAS wild type (65.9%) and 10 mutant (34.5%). Recurrence of the tumour was noted in 10 cases (34.5%) from which 60% were pT1 (N=6) and 40% pT2 (N=4). There was a statistically significant association between KRAS mutant status and local recurrence (N=6, p=0.037). P16 expression greater than 5% (mean=10.8%, min=0, max=95) is linked with earlier recurrence within 11.70 months (N=7, p=0.004). Membranous β-catenin expression (N=12, 48%) was also related with KRAS mutant status (p=0.006) but not with survival (p>0.05). BRAF gene was found to be wild type in all cases tested (N=23). KRAS/p16/β-catenin could be used as a combined biomarker for prediction of local recurrence and stratification of the risk for further surgery. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Flower morphology of Dendrobium Sonia mutants

International Nuclear Information System (INIS)

Sakinah Ariffin; Azhar Mohamad; Affrida Abu Hassan; Zaiton Ahmad; Mohd Nazir Basiran

2010-01-01

Dendrobium Sonia is a commercial hybrid which is popular as cut flower and potted plant in Malaysia. Variability in flower is important for new variety to generate more demands and choices in selection. Mutation induction is a tool in creating variability for new flower color and shape. In vitro cultures of protocorm-like bodies (PLBs) were exposed to gamma ray at dose 35 Gy. Phenotypic characteristics of the flower were observed at fully bloomed flower with emphasis on shape and color. Approximately 2000 regenerated irradiated plants were observed and after subsequent flowering, 100 plants were finally selected for further evaluation. Most of the color and shape changes are expressed in different combinations of petal, sepal and lip of the flower. In this work, 11 stable mutants were found different at flower phenotype as compared to control. Amongst these, four mutant varieties with commercial potential has been named as Dendrobium 'SoniaKeenaOval', Dendrobium 'SoniaKeenaRadiant', Dendrobium 'SoniaKeenaHiengDing' and Dendrobium 'Sonia KeenaAhmadSobri'. In this paper, variations in flower morphology and flower color were discussed, giving emphasis on variations in flower petal shape. (author)

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

Science.gov (United States)

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

[Construction of FANCA mutant protein from Fanconi anemia patient and analysis of its function].

Science.gov (United States)

Chen, Fei; Zhang, Ke-Jian; Zuo, Xue-Lan; Zeng, Xian-Chang

2007-11-01

To study FANCA protein expression in Fanconi anemia patient's (FA) cells and explore its function. FANCA protein expression was analyzed in 3 lymphoblast cell lines derived from 3 cases of type A FA (FA-A) patients using Western blot. Nucleus and cytoplasm localization of FANCA protein was analyzed in one case of FA-A which contained a truncated FANCA (exon 5 deletion). The FANCA mutant was constructed from the same patient and its interaction with FANCG was evaluated by mammalian two-hybrid (M2H) assay. FANCA protein was not detected in the 3 FA-A patients by rabbit anti-human MoAb, but a truncated FANCA protein was detected in 1 of them by mouse anti-human MoAb. The truncated FANCA could not transport from cytoplasm into nucleus. The disease-associated FANCA mutant was defective in binding to FANCG in M2H system. FANCA proteins are defective in the 3 FA-A patients. Disfunction of disease-associated FANCA mutant proved to be the pathogenic mutations in FANCA gene. Exon 5 of FANCA gene was involved in the interaction between FANCA and FANCG.

Traits and meiosis in mutant of impatiens balsamina induced by space treatment

International Nuclear Information System (INIS)

Tang Zesheng; Yang Jun; Zhao Yan; Yuan Haiyun

2004-01-01

A mutant of Impatiens balsamina was obtained after space induction, and its traits and meiosis were investigated. Characters such as color and form of the mutant expressed great variation. Observation of meiosis showed that most of pollen mother cells were normal in meiosis phase I, except the disproportion of chromosomal segregation, lagging chromosome and dispersal chromosome in anaphase I. Most pollen mother cells developed into microspores tetrad after meiosis, but paraspores also appeared. The number of chromosome in microspore varied from 1 to 21, even more than 30. The shape and size of the microspores fluctuated apparently, and the size of the microspores was in positive correlation to chromosome number. When staining with iodic solution, most of the pollens showed sterility, which was in consistence with the low setting percentage of the mutant plant. It was thought that space induction caused the variation of size, fertility and the abnormal meiosis

Identification, cloning and characterization of sis7 and sis10 sugar-insensitive mutants of Arabidopsis

Directory of Open Access Journals (Sweden)

Biddle Kelly D

2008-10-01

Full Text Available Abstract Background The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways. Results A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis mutants, both of which are also involved in abscisic acid (ABA biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination. Conclusion Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in

Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

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Mariana Vieira Tomazett

Full Text Available Abstract Carbonic anhydrases (CA belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4. In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1 and CA4 (rCA4, with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class and rCA4 (α-class were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration, arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Science.gov (United States)

Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

Directory of Open Access Journals (Sweden)

Peng Li

2017-09-01

Full Text Available Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the

[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

Science.gov (United States)

Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

2014-03-01

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

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Dudu Alkoby

Full Text Available pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M and F267C on Na+ (0.6 M. Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs).

Science.gov (United States)

Hon, Shuen; Lanahan, Anthony A; Tian, Liang; Giannone, Richard J; Hettich, Robert L; Olson, Daniel G; Lynd, Lee R

2016-12-01

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE . To explore the effects of overexpressing wild-type, mutant, and exogenous adhE s, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum . As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

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

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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.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs

Directory of Open Access Journals (Sweden)

Shuen Hon

2016-12-01

Full Text Available Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results. Keywords: Clostridium Thermocellum, Plasmid, adhE, Structural stability, Gene expression

The mitochondrial inner membrane AAA metalloprotease family in metazoans.

Science.gov (United States)

Juhola, M K; Shah, Z H; Grivell, L A; Jacobs, H T

2000-09-15

Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.

Gamma ray induced mutants in Coleus

Energy Technology Data Exchange (ETDEWEB)

Vasudevan, K; Jos, J S [Central Tuber Crops Research Institute, Trivandrum, Kerala (India)

1988-07-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M{sub 1}V{sub 1} generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m{sup 2} area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing.

Gamma ray induced mutants in Coleus

International Nuclear Information System (INIS)

Vasudevan, K.; Jos, J.S.

1988-01-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M 1 V 1 generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m 2 area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing

Deep Sequencing of Porphyromonas gingivalis and comparative transcriptome analysis of a LuxS mutant

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Takanoi eHirano

2012-06-01

Full Text Available Porphyromonas gingivalis is a major etiological agent and chronic and aggressive forms of periodontal disease. The organism is an assacharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve noncoding RNAs were identified, including 11 small RNAs and one cobalamine riboswitch. Fifty seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

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Zhimin Lao

2012-08-01

Full Text Available Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination, which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26MASTR was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26MASTR mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression.

Science.gov (United States)

Keck, Mathilde; Andrini, Olga; Lahuna, Olivier; Burgos, Johanna; Cid, L Pablo; Sepúlveda, Francisco V; L'hoste, Sébastien; Blanchard, Anne; Vargas-Poussou, Rosa; Lourdel, Stéphane; Teulon, Jacques

2013-09-01

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance. © 2013 WILEY PERIODICALS, INC.

Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

NARCIS (Netherlands)

Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

2013-01-01

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

Isolation and characterization of Candida albicans morphological mutants derepressed for the formation of filamentous hypha-type structures

International Nuclear Information System (INIS)

Gil, C.; Pomes, R.; Nombela, C.

1990-01-01

Several Candida albicans morphological mutants were obtained by a procedure based on a combined treatment with nitrous acid plus UV irradiation and a double-enrichment step to increase the proportion of mutants growing as long filamentous structures. Altered cell morphogenesis in these mutants correlated with an altered colonial phenotype. Two of these mutants, C. albicans NEL102 and NEL103, were selected and characterized. Mutant blastoconidia initiated budding but eventually gave rise to filamentous hypha-type formations. These filaments were long and septate, and they branched very regularly at positions near septa. Calcofluor white (which is known to bind chitin-rich areas) stained septa, branching zones, and filament tips very intensely, as observed under the fluorescence microscope. Wild-type hybrids were obtained by fusing protoplasts of strain NEL102 with B14, another morphological mutant previously described as being permanently pseudomycelial, indicating that genetic determinants responsible for the two altered phenotypes are different. The mutants characterized in this work seemed to sequentially express the morphogenic characteristics of C. albicans, from blastoconidia to hyphae, in the absence of any inducer. Further characterization of these strains could be relevant to gain understanding of the genetic control of dimorphism in this species

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.

Science.gov (United States)

Chen, Longzheng; Li, Wei; Katin-Grazzini, Lorenzo; Ding, Jing; Gu, Xianbin; Li, Yanjun; Gu, Tingting; Wang, Ren; Lin, Xinchun; Deng, Ziniu; McAvoy, Richard J; Gmitter, Frederick G; Deng, Zhanao; Zhao, Yunde; Li, Yi

2018-01-01

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium -mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase ( PDS ) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

Functional verification of a porcine myostatin propeptide mutant.

Science.gov (United States)

Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

The Swedish mutant barley collection

Energy Technology Data Exchange (ETDEWEB)

NONE

1989-07-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

The Swedish mutant barley collection

International Nuclear Information System (INIS)

1989-01-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

Mutants of alfalfa mosaic virus

International Nuclear Information System (INIS)

Roosien, J.

1983-01-01

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

Science.gov (United States)

Young, Douglas; Mayer, Franziska; Vidotto, Nella; Schweizer, Tatjana; Berth, Ramon; Abramowski, Dorothee; Shimshek, Derya R.; van der Putten, P. Herman; Schmid, Peter

2013-01-01

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model. PMID:24086450

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

Science.gov (United States)

Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-06-22

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

A METABOLIC SIGNATURE FOR LONG-LIFE IN THE C. ELEGANS MIT MUTANTS

Science.gov (United States)

Butler, Jeffrey A.; Mishur, Robert J.; Bhaskaran, Shylesh; Rea, Shane L.

2012-01-01

SUMMARY Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent upon concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer’s disease. When the expression of DLD in wild type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened but, at higher doses, it was significantly lengthened, suggesting DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. PMID:23173729

Cerebrovascular defects in Foxc1 mutants correlate with aberrant WNT and VEGF-A pathways downstream of retinoic acid from the meninges.

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Mishra, Swati; Choe, Youngshik; Pleasure, Samuel J; Siegenthaler, Julie A

2016-12-01

Growth and maturation of the cerebrovasculature is a vital event in neocortical development however mechanisms that control cerebrovascular development remain poorly understood. Mutations in or deletions that include the FOXC1 gene are associated with congenital cerebrovascular anomalies and increased stroke risk in patients. Foxc1 mutant mice display severe cerebrovascular hemorrhage at late gestational ages. While these data demonstrate Foxc1 is required for cerebrovascular development, its broad expression in the brain vasculature combined with Foxc1 mutant's complex developmental defects have made it difficult to pinpoint its function(s). Using global and conditional Foxc1 mutants, we find 1) significant cerebrovascular growth defects precede cerebral hemorrhage and 2) expression of Foxc1 in neural crest-derived meninges and brain pericytes, though not endothelial cells, is required for normal cerebrovascular development. We provide evidence that reduced levels of meninges-derived retinoic acid (RA), caused by defects in meninges formation in Foxc1 mutants, is a major contributing factor to the cerebrovascular growth defects in Foxc1 mutants. We provide data that suggests that meninges-derived RA ensures adequate growth of the neocortical vasculature via regulating expression of WNT pathway proteins and neural progenitor derived-VEGF-A. Our findings offer the first evidence for a role of the meninges in brain vascular development and provide new insight into potential causes of cerebrovascular defects in patients with FOXC1 mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

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Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

2017-07-01

Thiamin diphosphate (TPP, vitamin B 1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Radio-sensitivity of the cells from amyotrophic lateral sclerosis model mice transfected with human mutant SOD1

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Wate, Reika; Ito, Hidefumi; Kusaka, Hirofumi; Takahashi, Sentaro; Kubota, Yoshihisa; Suetomi, Katsutoshi; Sato, Hiroshi; Okayasu, Ryuichi

2005-01-01

In order to clarify the possible involvement of oxidative damage induced by ionizing radiation in the onset and/or progression of familial amyotrophic lateral sclerosis (ALS), we studied radio-sensitivity in primary cells derived from ALS model mice expressing human mutant Cu/Zn superoxide dismutase (SOD1). The primary mouse cells expressed both mouse and the mutant human SOD1. The cell survival of the transgenic mice (with mutant SOD1), determined by counting cell numbers at a scheduled time after X-irradiation, is very similar to that of cells from wild type animals. The induction and repair of DNA damage in the transgenic cells, measured by single cell gel electrophoresis and pulsed field gel electrophoresis, are also similar to those of wild type cells. These results indicate that the human mutant SOD1 gene does not seem to contribute to the alteration of radio-sensitivity, at least in the fibroblastic cells used here. Although it is necessary to consider the difference in cell types between fibroblastic and neuronal cells, the present results may suggest that ionizing radiation is not primarily responsible for the onset of familial ALS with the SOD1 mutation, and that the excess risks are probably not a concern for radiation diagnosis and therapy in familial ALS patients. (author)

Morphological Characterization and Assessment of Genetic Variability, Character Association, and Divergence in Soybean Mutants

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M. A. Malek

2014-01-01

Full Text Available Genetic diversity is important for crop improvement. An experiment was conducted during 2011 to study genetic variability, character association, and genetic diversity among 27 soybean mutants and four mother genotypes. Analysis of variance revealed significant differences among the mutants and mothers for nine morphological traits. Eighteen mutants performed superiorly to their mothers in respect to seed yield and some morphological traits including yield attributes. Narrow differences between phenotypic and genotypic coefficients of variation (PCV and GCV for most of the characters revealed less environmental influence on their expression. High values of heritability and genetic advance with high GCV for branch number, plant height, pod number, and seed weight can be considered as favorable attributes for soybean improvement through phenotypic selection and high expected genetic gain can be achieved. Pod and seed number and maturity period appeared to be the first order traits for higher yield and priority should be given in selection due to their strong associations and high magnitudes of direct effects on yield. Cluster analysis grouped 31 genotypes into five groups at the coefficient value of 235. The mutants/genotypes from cluster I and cluster II could be used for hybridization program with the mutants of clusters IV and V in order to develop high yielding mutant-derived soybean varieties for further improvement.

Combined BRAF and HSP90 inhibition in patients with unresectable BRAF V600E mutant melanoma.

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Eroglu, Zeynep; Chen, Yian Ann; Gibney, Geoffrey T; Weber, Jeffrey S; Kudchadkar, Ragini R; Khushalani, Nikhil I; Markowitz, Joseph; Brohl, Andrew S; Tetteh, Leticia F; Ramadan, Howida; Arnone, Gina; Li, Jiannong; Zhao, Xiuhua; Sharma, Ritin; Darville, Lancia N F; Fang, Bin; Smalley, Inna; Messina, Jane L; Koomen, John M; Sondak, Vernon K; Smalley, Keiran S M

2018-04-19

BRAF inhibitors are clinically active in patients with advanced BRAF V600 -mutant melanoma, although acquired resistance remains common. Preclinical studies demonstrated that resistance could be overcome using concurrent treatment with the HSP90 inhibitor XL888. Vemurafenib (960 mg PO BID) combined with escalating doses of XL888 (30, 45, 90 or 135 mg PO twice weekly) was investigated in 21 patients with advanced BRAF V600 -mutant melanoma. Primary endpoints were safety and determination of a maximum tolerated dose. Correlative proteomic studies were performed to confirm HSP inhibitor activity. Objective responses were observed in 15/20 evaluable patients (75%; 95% CI: 51-91%), with 3 complete and 12 partial responses. Median progression-free and overall survival were 9.2 months (95% CI: 3.8-not reached) and 34.6 months (6.2-not reached), respectively. The most common grade 3/4 toxicities were skin toxicities such as rash (n=4, 19%) and cutaneous squamous cell carcinomas (n=3, 14%), along with diarrhea (n=3, 14%). Pharmacodynamic analysis of patients' PBMCs showed increased day 8 HSP70 expression compared to baseline in the three cohorts with XL888 doses ≥45 mg. Diverse effects of vemurafenib-XL888 upon intratumoral HSP-client protein expression were noted, with the expression of multiple proteins (including ERBB3 and BAD) modulated on therapy. XL888 in combination with vemurafenib has clinical activity in patients with advanced BRAF V600 -mutant melanoma, with a tolerable side-effect profile. HSP90 inhibitors warrant further evaluation in combination with current standard-of-care BRAF plus MEK inhibitors in BRAF V600 -mutant melanoma. Copyright ©2018, American Association for Cancer Research.

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

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Orue, Andrea; Chavez, Valery; Strasberg-Rieber, Mary; Rieber, Manuel

2016-11-18

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.

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Chris Jopling

2007-12-01

Full Text Available Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

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Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

2016-09-12

Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic fingerprinting of mutant rose cultivars

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Kumar, S; Prasad, K V; Singh, K P; Singh, A.P. [Division of Floriculture and Landscaping, Indian Agricultural Research Institute, Pusa, New Delhi (India)], E-mail: kvprasad66@gmail.com

2008-07-01

Six rose mutants evolved at the Indian Agricultural Research Institute, New Delhi from four parent cultivars were characterized based on RAPD markers. Contrary to the earlier findings our effort has conclusively proven that the RAPD markers are indeed robust tools to discern the mutants from their parents. Among 40 primers screened, 7 primers produced inconsistent banding pattern. The number of polymorphic bands varied between 4 (OPA 14) and 10 (OPA1) with an average of 6.5 bands per primer. The percentage polymorphism ranged from 62.5 (OPM 9) to 100 percent (OPA 1). Most of the primers produced monomorphic bands between parent and mutant rose cultivars. When primer OPA 2 was used a specific band of 2.5 kb was noticed in mutant cv. Pusa Urmil and cv. Pusa Abhishek but was absent in parent cv. Jantar Mantar. A polymorphic band of 750 bp was noticed in the parent Kiss of Fire and helped in differentiating the parent from its mutant when amplified with OPK 3. Primer OPS 16 produced discriminatory band of 800 bp in mutant cv. Pink Sport of Montezuma while it was absent in its parent cv. Montezuma. Another specific band of 650 bp was present in parent cv. Montezuma and absent in its mutant cv. Pink Sport of Montezuma signifying the uniqueness of the mutant. Primer OPM 5 brought out distinct polymorphism among the parent Jantar Mantar and its three mutants with absence of a specific band of 1.5 kb in the parent. The four parents and 6 mutants were divided into four distinct groups in the Dendogram constructed by UPGMA method. The most genetically similar cultivar among the 10 cultivars analyzed are Montezuma and its pink sport of Montezuma whereas Abhisarika a mutant of cv. Kiss of Fire was distinctly different and formed a separate cluster. (author)

Paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor.

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An Ye

Full Text Available The expression of two adjacent imprinted genes, Peg3 and Zim1, is inversely correlated: down-regulation of Peg3 coinciding with up-regulation of Zim1. The current study characterized this inverse correlation using a mutant allele targeting Peg3. According to the results, the mutation on the paternal allele of Peg3 resulted in a dramatic increase in the transcription levels of the maternal allele of Zim1, suggesting the involvement of unknown trans factors in this trans-allelic event. Subsequent ChIP experiments revealed that the protein encoded by Peg3 itself binds to the zinc finger exon of Zim1, which is modified with the repression mark H3K9me3. Interestingly, the levels of H3K9me3 on Zim1 are also reduced in the mutant cells lacking the protein PEG3, suggesting potential roles for PEG3 in establishing H3K9me3 on Zim1. Reintroducing PEG3 into the mutant cell restored down-regulation of Zim1, confirming the predicted repressor role for Peg3 on Zim1. Overall, these results demonstrated that paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor. The current study also provides the first case for the trans-allelic interaction of two oppositely imprinted genes through their gene products.

From one body mutant to one cell mutant. A progress of radiation breeding in crops

International Nuclear Information System (INIS)

Nagatomi, Shigeki

1996-01-01

An effective method was established to obtain non-chimeral mutants with wide spectrum of flower colors, regenerated from floral organs on which mutated sectors were come out on chronic irradiated plants. By this way, six mutant varieties of flower colors have been selected from one pink flower of chrysanthemum, and cultivated for cut-flower production. By the same method, 3 mutant varieties with small and spray type flowers were selected in Eustoma. Mutant varieties such as a rust disease resistant in sugarcane, 6 dwarfs in Cytisus and pure-white mushroom in velvet shank have been selected successively for short period. (J.P.N.)

Map-based cloning and expression analysis of BMR-6 in sorghum.

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Li, Jieqin; Wang, Lihua; Zhang, Qiuwen; Liu, Yanlong

2015-09-01

Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which shows reddish pigment in the midrib and stem after the fifth-leaf stage. Compared to wild type, Kalson lignin content of bmr-6 is decreased significantly. We used histological analysis to determine that the mutant exhibited a modified pattern of lignin staining and found an increased polysaccharide content. We cloned BMR-6 gene, a gene encoded a cinnamyl alcohol dehydrogenase (CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient expression assays in Nicotiana benthamiana leaves demonstrated cytomplasmic localization of BMR-6. We found that the expression level of bmr-6 was significantly decreased in the mutant but expression of SbCAD3 and SbCAD5 were significantly increased. Our results indicate that BMR-6 not only affects the distribution of lignin but also the biosynthesis of lignin in sorghum.

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine.

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Ivanov, Ana; Kameka, Alexander; Pajak, Agnieszka; Bruneau, Luanne; Beyaert, Ronald; Hernández-Sebastià, Cinta; Marsolais, Frédéric

2012-06-01

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

A novel mouse Fgfr2 mutant, hobbyhorse (hob, exhibits complete XY gonadal sex reversal.

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Pam Siggers

Full Text Available The secreted molecule fibroblast growth factor 9 (FGF9 plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob, which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6 genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected.

Matching NLR Immune Receptors to Autoimmunity in camta3 Mutants Using Antimorphic NLR Alleles

DEFF Research Database (Denmark)

Lolle, Signe; Greeff, Christiaan; Petersen, Klaus

2017-01-01

result from inappropriate NLR activation because mutations in plant guardees could trigger corresponding NLR guards. To explore these hypotheses, we expressed 108 dominant-negative (DN) Arabidopsis NLRs in various lesion mimic mutants, including camta3, which exhibits autoimmunity. CAMTA3 was previously...

Induction of Mutants in Durum Wheat

International Nuclear Information System (INIS)

AL-Ubaidi, M.; Ibrahim, I.; AL-Hadithi, A.

2002-01-01

This investigation presents a breeding program for induction and development of a new genotype of durum wheat, resistant to lodging with high yield, by irradiation durum wheat hybrids (F2) with gamma rays 100 Gy, during 1990-1997 cultivation seasons. This program involves: induction of variability, selection evaluation of the mutants at three locations: Twaitha (Baghdad) Latifya ( Babylon) and Swari (Kutt). All mutants showed resistance to lodging and there was a significant reduction in plant height. Mutant SIXIZ-22 surpassed other mutants and its origin in lodging resistance and plant height (83.5,82.8 and 89.4 cm) in the three locations at generation M5 and M6, respectively. Also, there were significant differences between mutant and their origin in the number of spikes/M 2 and grain yild during the two successive generation. On the other hand, mutant IZxCO-105 surpassed other mutants in the number of spikes/M 2 (231.8,242.3 and 292) and grain yield (4336,3376 and 5232 kg/ha) in all testing location, respectively . (authors) 14 refs., 4 tabs

Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

International Nuclear Information System (INIS)

Seigne, Christelle; Fontanière, Sandra; Carreira, Christine; Lu, Jieli; Tong, Wei-Ming; Fontanière, Bernard; Wang, Zhao-Qi; Zhang, Chang Xian; Frappart, Lucien

2010-01-01

Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1 +/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Six Men1 +/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland

Distinct expression profile in fumarate-hydratase-deficient uterine fibroids

DEFF Research Database (Denmark)

Vanharanta, S; Pollard, PJ; Lehtonen, HJ

2006-01-01

-related genes. Other significantly up-regulated gene categories in FH mutants were, for example, iron ion homeostasis and oxidoreduction. Genes with lower expression in FH-mutant fibroids belonged to groups such as extracellular matrix, cell adhesion, muscle development and cell contraction. We show that FH...

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

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Chen, Weiwei; Yu, Hongwei; Ye, Lidan

2016-07-01

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.

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

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

2017-08-01

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

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

Science.gov (United States)

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-08-15

To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Reduced anti-oxidative stress activities of DJ-1 mutants found in Parkinson's disease patients

International Nuclear Information System (INIS)

Takahashi-Niki, Kazuko; Niki, Takeshi; Taira, Takahiro; Iguchi-Ariga, Sanae M.M.; Ariga, Hiroyoshi

2004-01-01

DJ-1 is a multi-functional protein that plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in onset of Parkinson's disease. We have previously reported that L166P, a mutant DJ-1 found in Parkinson's disease patients, had no activity to prevent hydrogen peroxide (H 2 O 2 )-induced cell death. In this study, we analyzed other mutants of DJ-1 found in Parkinson's disease patients, including M26I, R98Q, and D149A, as well as L166P. We first found that all of the mutants made heterodimers with wild-type DJ-1, while all of the mutants except for L166P made homodimers. We then found that M26I and L166P, both of which are derived from homozygous mutations of the DJ-1 gene, were unstable and that their stabilities were recovered, in part, in the presence of proteasome inhibitor, MG132. NIH3T3 cell lines stably expressing these mutants of DJ-1 showed that cell lines of L166P and C106S, a mutant for protease activity (-) of DJ-1, had no activity to scavenge even endogenously producing reactive oxygen species. These cell lines also showed that all of the mutants had reduced activities to eliminate exogenously added H 2 O 2 and that these activities, except for that of D149A, were parallel to those preventing H 2 O 2 -induced cell death