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Sample records for mutant mouse lines

  1. The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations

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    Helmut Fuchs

    2016-12-01

    Full Text Available The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein family consists of three independent members, Scube1–3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K, which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC. Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB, associated with the chromosomal region of human SCUBE3. In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

  2. The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations.

    Science.gov (United States)

    Fuchs, Helmut; Sabrautzki, Sibylle; Przemeck, Gerhard K H; Leuchtenberger, Stefanie; Lorenz-Depiereux, Bettina; Becker, Lore; Rathkolb, Birgit; Horsch, Marion; Garrett, Lillian; Östereicher, Manuela A; Hans, Wolfgang; Abe, Koichiro; Sagawa, Nobuho; Rozman, Jan; Vargas-Panesso, Ingrid L; Sandholzer, Michael; Lisse, Thomas S; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Calzada-Wack, Julia; Ehrhard, Nicole; Elvert, Ralf; Gau, Christine; Hölter, Sabine M; Micklich, Katja; Moreth, Kristin; Prehn, Cornelia; Puk, Oliver; Racz, Ildiko; Stoeger, Claudia; Vernaleken, Alexandra; Michel, Dian; Diener, Susanne; Wieland, Thomas; Adamski, Jerzy; Bekeredjian, Raffi; Busch, Dirk H; Favor, John; Graw, Jochen; Klingenspor, Martin; Lengger, Christoph; Maier, Holger; Neff, Frauke; Ollert, Markus; Stoeger, Tobias; Yildirim, Ali Önder; Strom, Tim M; Zimmer, Andreas; Wolf, Eckhard; Wurst, Wolfgang; Klopstock, Thomas; Beckers, Johannes; Gailus-Durner, Valerie; Hrabé de Angelis, Martin

    2016-12-07

    The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3 N294K/N294K ), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3 N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3 N294K/N294K mice. The Scube3 N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function. Copyright © 2016 Fuchs et al.

  3. Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

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    Raffaele ed'Isa

    2011-12-01

    Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

  4. Grain product of 34 soya mutant lines

    International Nuclear Information System (INIS)

    Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E.; Cervantes S, T.; De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A.

    2009-01-01

    This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R 4 M 18 ) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co 60 gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L 25 and L 32 produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

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

    DEFF Research Database (Denmark)

    Jensen, Helle Lone; Norrild, Bodil

    2003-01-01

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

  6. Characteristics of mutant lines of sweet potato flour

    International Nuclear Information System (INIS)

    Aryanti

    2012-01-01

    Research on mutation induction of sweet potato Sari variety has been conducted. Flour mutant lines were obtained from selection of M1V5 tubers irradiated by gamma rays at the dose of 10 Gy. Flour was made by peeling of tubers, then dried, blended and sieved. The quality test of flour have been done by measuring degree of whiteness, proximate, amylose contents, water content, soluble water, swelling power, and flour characteristics. The result of this work showed that flour of C6.26.13 mutant line had higher protein content than the parent plant with concentration of 3.62 % and its amylose content was also higher than the other mutant lines. The soluble water value of mutant lines were significant different compared to the parent plant from 1.82 to 2.25 % and swelling power from 4.28 to 5.55 %. The flour granule of the mutant line was different compared to the parent plant. (author)

  7. Photosynthetic and nitrogen fixation capability in several soybean mutant lines

    International Nuclear Information System (INIS)

    Gandanegara, S.; Hendratno, K.

    1987-01-01

    Photosynthetic and nitrogen fixation capability in several soybean mutant lines. A greenhouse experiment has been carried out to study photosynthetic and nitrogen fixation capability of five mutant lines and two soybean varieties. An amount of 330 uCi of 14 CO 2 was fed to the plants including of the non-fixing reference crop (Chippewa non-nodulating isoline). Nitrogen fixation measurements was carried out using 15 N isotope dilution technique according to A-value concept. Results showed that beside variety/mutant lines, plant growth also has important role in photosynthetic and N fixing capability. Better growth and a higher photosynthetic capability in Orba, mutant lines nos. 63 and 65 resulted in a greater amount of N 2 fixed (mg N/plant) than other mutant lines. (author). 12 refs.; 5 figs

  8. 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

  9. Differences in heavy-ion-induced DNA double-strand breaks in a mouse DNA repair-deficient mutant cell line (SL3-147) before and after chromatin proteolysis

    International Nuclear Information System (INIS)

    Murakami, Masahiro; Eguchi-Kasai, Kiyomi; Sato, Koki; Minohara, Shinichi; Kanai, Tatsuaki; Yatagai, Fumio.

    1995-01-01

    DNA double-strand breaks induced by X- or neon beam-irradiation in a DNA double-strand break-repair-deficient mutant cell line (SL3-147) were examined. The increase in the number of DNA double-strand breaks was dose-depend after irradiation with X-rays and neon beams and was enhanced by chromatin-proteolysis treatment before irradiation. These results suggest that the induction of DNA double-strand breaks by ionizing radiation, including heavy-ions, is influenced by the chromatin structure. (author)

  10. Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

    International Nuclear Information System (INIS)

    Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

    1982-01-01

    We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

  11. A preliminary yield trial of some soybean mutant lines

    International Nuclear Information System (INIS)

    Ratma, Rivaie

    1985-01-01

    A preliminary yield trial of some soybean mutant lines, derived from irradiated Orba variety with dose of 0.40 kGy, were carried out during the wet and dry season in 1979-1982 in Muara and Citayam, Bogor. The result obtained showed that yield potential of mutant lines no. M6/40/10 was higher than that of the control in dry season in 1979 as well as in the wet season of 1979/80 in Muara. Whereas, the yield potential of the mutant lines no. M6/40/8 and no. M6/40/14 were higher than that of the control only in the wet season. The yield potential of semi dwarf mutant lines no. M6/40/68 was highly significant compared to that of the control in dry season in Muara and the wet season in 1981/82 in Citayam. Whereas, the yield potential of the mutant lines no. M6/40/69 was higher yield compared to that of the control in dry season in 1981 in Muara. (author). 10 refs

  12. 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)

  13. 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

  14. Plants Regeneration Derived From Various on Peanut on Mutant Lines

    International Nuclear Information System (INIS)

    Dewi, Kumala; Masrizal; Mugiono

    1998-01-01

    The study of calli, greenspot formation and shoot regeneration on peanut mutant lines has ben conducted by MS media. Three explants derived from shoot tips, embryo and seeding root of two mutant lines a/20/3 and D/25/3/2 were used in this experiment. the explants were cultured on modified MS media enriched by vitamins, growth regulation, amino acids for fourth teen calli were transferred on regeneration media. The ability of calli formation and plant regeneration of each explant and genotypes of plants was varied. Greenspot and shoot formation were observed seventh days after the calli transferred on regeneration media. It is shown that the ability of calli, greenspot and shoot formation of each explants and genotypes was varied. the high ability of calli, greenspot and shoot formation were found in explant derived from shoot tip and embryo. Seedling root explant has lower ability in calli formation, while greenspot and shoot was formatted. The ability of calli, greenspot and shoot formation on A/20/3 mutant line was better than D/25/3/2 mutant line. (author)

  15. Semi-dwarf mutant lines of hexaploid triticale

    International Nuclear Information System (INIS)

    Pidra, M.

    1989-01-01

    A spring form of hexaploid secondary triticale ADD 143/71, bred by MOGILEVA at the Plant Breeding Station at Uhretice was used for the mutagen treatment. The mutation experiment started in 1979. Seeds were treated with a 0.8 mM water solution of N-methyl-N-nitrosourea (MNH) (CETL and RELICHOVA, unpublished). From 180 M 1 plants, one spike was harvested per plant. A random sample of these seeds was sown as M 2 in 1980 and several plants with shorter main culm were selected. Selfed progenies of eight mutant plants designated ADD 143-m1, ADD 143-m2, ADD 143-m3 etc. were further tested in M 3 and M 4 . There were significant differences in culm length and in some other characters between the original line and the mutant lines. Especially the line m8 looks like a promising source of semi-dwarfness for breeding programmes of hexaploid triticale. During 1985-1987 genetic analysis was performed on the ADD 143/71 and the mutant lines m2, m6, m7 and m8, which suggest that their mutant genes are allelic and recessive

  16. Biochemical characteristics of mutant lines of currant tomato

    International Nuclear Information System (INIS)

    Gorbatenko, I.Yu.; Khrustaleva, V.V.; Shcherbakov, V.K.

    1988-01-01

    The currant tomato is used in breeding for fruit quality. It contains up to 50 mg% ascorbic acid, a large quantity of sugar and 8-10% of dry matter. The weight of the fruit, however, does not exceed 1.2-1.5 g. The plants have long, spreading and very branchy stems. Gamma ray induced mutants of currant tomato were used, as initial material in breeding for fruit quality in varieties suitable for mechanized harvesting. The research was carried out mainly at the Department of Vegetable Growing Ukrainian Scientific Research Institute of Irrigation Farming. The regional variety Lebyazhinskij (suitable for mechanized harvesting) was adopted as the standard. Its fruits contain: 5.6% dry matter, 2.7% sugars, 0.543% titrated acidity, 26.6 mg/100 g ascorbic acid, 0.425 mg% carotene and 0.35% cellulose. The biochemical characteristics of the tomato mutants are shown. In terms of fruit dry matter, all mutants surpassed the standard. The acidity and the ascorbic acid content varied considerably. Most noteworthy in terms of carotene were the lines GP-5, GP-9 and GP-12. An important factor in the production of tomato paste is the fruit cellulose content. The lowest cellulose content is found in mutant GP-3. As shown, all of the mutants were early ripening. The mutants surpassed the standard in simultaneous fruit ripening. Mutant lines GP-3, GP-6, GP-9 and GP-12 will be used in the breeding programme for improving fruit quality of varieties suitable for mechanized harvesting

  17. Creating Sunflower Mutant Lines (Helianthus Annuus L.) Using Induced Mutagenesis

    International Nuclear Information System (INIS)

    Encheva, J.

    2009-01-01

    Immature sunflower zygotic embryos of sunflower fertility restorer line 374 R were treated with ultrasound and gamma radiation before plating embryos to culture medium. All plants were isolated and self-pollinated for several generations. New sunflower forms with inherited morphological and biochemical changes were obtained. The genetic changes occurring during the mutation procedure included fourteen morphological and biochemical characters. In comparison to the check line 374 R, decreasing of the mean value of the indexes was registered for 33 % of the total number of characters and vise verse, significant increasing was observed for 60 %. Mutation for resistance to the local population of Orobanche cumana race A-E was obtained from the susceptible Bulgarian control line 374 R. Two investigated mutant lines possessed 100 % resistance to Orobanche and stable inheritance in the next generations. Our results showed that induced mutagenesis in sunflower can be successfully used to develop new lines useful for heterosis breeding

  18. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

    NARCIS (Netherlands)

    Korstanje, Ron; Desai, Jigar; Lazar, Gloria; King, Benjamin; Rollins, Jarod; Spurr, Melissa; Joseph, Jamie; Kadambi, Sindhuja; Li, Yang; Cherry, Allison; Matteson, Paul G.; Paigen, Beverly; Millonig, James H.

    Korstanje R, Desai J, Lazar G, King B, Rollins J, Spurr M, Joseph J, Kadambi S, Li Y, Cherry A, Matteson PG, Paigen B, Millonig JH. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects. Physiol Genomics 35:

  19. Yield of two mutant lines of soybean for human consumption

    International Nuclear Information System (INIS)

    Salmeron E, J.; Mastache L, A. A.; Diaz V, G. E.; Valencia E, F.; Ranfla C, R.; Melendez P, M.; Cervantes S, T.; De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.

    2009-01-01

    The present work has the objective of to evaluate the yield and the agronomic behavior of 2 mutant lines of soybean for human consumption, obtained by means of a process of recurrent irradiation of soybean seed ISAAEG-BM 2 with gammas of Co 60 and selection in the generation R 4 M 18 . For the variable yield significant statistical differences were not observed, but considering the rest of the evaluated agronomic characteristics the mutant lines L 6 and Bombona they were excellent with values of 3,934.6 and 3,806.8 Kg ha- 1 to 15% of grain humidity, they also possess excellent genetic characteristics result of the irradiations and selections of these new genetic materials. (Author)

  20. Evaluation of artemisia mutant lines conducted from gamma irradiation treatment

    International Nuclear Information System (INIS)

    Ragapadmi Purnamaningsih; EG Lestari; M Syukur

    2010-01-01

    Cases of Malaria diseases attack in Indonesia has been increasing. Plasmodium falciparum the cause of malaria disease is now resistant to the usual medicine. One of malaria medicine which recommended by WHO is artemisinine compound extracted from Artemisia annua L plant. Low artemisinine content is one problem of Artemisia development in Indonesia. Increasing genetic variation using gamma irradiation is one alternative method to improve artemisinin content. In 2007, induce mutation had been done to artemisia seeds using gamma irradiation at dosage of 10-100 Gy. The good rooting planlet was regenerated and acclimatized in the green house, and then the seedling (M0 generation) was planted in the field at 1545 m asl. Plants derived from seeds without gamma irradiation treatment and cultured in vitro (in vitro control) were used as control. The result showed there were some morphological variations between the mutant lines (plant height, shape of the leaves and time of flowering). Ten mutant lines were selected based on biomass yield and analyzed for the artemisinine content.The result showed that artemisinine content of the mutant lines ranged from 0.44 - 1.41%, and it was significantly higher than that of in vitro control (0.43%). (author)

  1. Creation and evaluation of best cotton mutant lines

    International Nuclear Information System (INIS)

    Rastegari, S. J.; Hosseini, Z.

    2001-01-01

    During (1997-1999) a study was carried out to recognize the best mutant lines, which were already obtained from a mutation breeding project. A Triple Rectangular Latis Design (8 7) in form of randomized complete blocks (RCB) with fifty- six treatments and three replications, were used in Estahban, Kordkouy and Varamin, under different ecological conditions, rainfall (Kordkouy) desert (Varamin) hot and dry (Estahban). During growing season some important morphological characteristics were recorded. Some lines had specific characters, for example: line 3191 (Chirpan 150 gray) had a low leaf number per plant, line 3169 (Bakhtegan 200 gray) plants were clustered. The results of the data in Varamin station showed that Bakhtegan irradiated with 150 gray line 3485 and Tashkand with 300 gray line 3451 compared to check (Varamin with 4373 kg/ha) had highest yield with 4942 kg/ha, and 4871 kg/ha respectively. In view of boll weight line 3405 of Sahel irradiated with 200 gray had highest boll weight (6.5 g/boll). In Kordkouy station the best mutant line was Chirpan irradiated with 250 gray, line 3208, with 20% yield increase compared to Sahel and 30% yield increase compared to original Chirpan. In respect to irradiation effect on lint percentage and fiber quality, the results showed; there was a positive effect on lint percentage of all varieties, especially in Tashkant, Bakhtegan and Chirpan which are inherently weak in lint percentage. As a whole gamma radiation did not have any negative effect on fiber quality. Even in Estahban 1.6 to 2.4 mm fiber increase were observed in some Chirpan irradiated material (C150-3516) and (C200-3523)

  2. Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

    International Nuclear Information System (INIS)

    Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

    1985-01-01

    Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

  3. Selection and agronomic evaluation of induced mutant lines of sesame

    International Nuclear Information System (INIS)

    Hoballah, A.A.

    2001-01-01

    Station yield trial: Three high yielding mutants (8, 48, and EFM92) with better and stable performance were developed in our breeding programme and submitted for registration to the Agricultural Research Center (ARC), Egyptian Ministry of Agriculture and Land Reclamation. Multi-location yield trials indicated that mutant line EFM92 ranked first in all locations; significant yield increases recorded for it ranged from 14.7 to 74.0% over the check variety. Moreover, it was 15-20 days earlier than the check and/or other mutants. Mutant lines 8 and 48 produced higher seed yields than the check at two different locations. These mutants can probably be grown and produce more yield than the check variety at the low yielding environments. Seed quality assay: During 1996 and 1997, 15 promising lines of sesame including mutants and hybrid populations as well as the local variety were evaluated for seed protein, oil content and fatty acid composition. The protein content varied from 20.6 to 26.7%; hybrid population EXM90 gave the highest value. About 85% of the total fatty acids in the oil are unsaturated (oleic and linoleic) and 15% saturated, mainly palmitic and stearic. Linoleic acid ranged from 41.8 to 47.9%. Mutant lines 6, 9, and EFM92, which gave high oil content (54-55.5%) together with high linoleic acid values (45.2-47.8%), are recommended for breeding for seed oil quality. Heterosis, combining ability and type of gene action in sesame: A half diallel set of crosses involving seven parents was used to study heterosis and combining ability in the F 1 generation as well as the nature of gene action controlling seed yield and its contributing traits in both F 1 and F 2 in order to identify the most efficient breeding methods leading to rapid genetic improvement. The expressions of heterosis varied with the crosses and characters investigated. The maximal significant positive useful heterosis was observed for branches/plant (52.9%) followed by seed yield/plant (38

  4. Temperature-Sensitive Mutants of Mouse Hepatitis Virus Strain A59: Isolation, Characterization and Neuropathogenic Properties.

    NARCIS (Netherlands)

    M.J.M. Koolen (Marck); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert); M.C. Horzinek; B.A.M. van der Zeijst (Ben)

    1983-01-01

    textabstractTwenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the

  5. Evaluation of the combining ability of mutant maize lines

    Directory of Open Access Journals (Sweden)

    V. Valkova

    2016-09-01

    Full Text Available Abstract. The study shows the results of a preliminary evaluation of the combining ability for grain yield of 17 mutant maize lines. For the purpose the top cross method for early testing and the mathematical model of Savchenko for analysis of the general and the specific combining ability were used. The lines were tested on three testers with high general combining ability that belong to two genetic groups: K 46 52 and XM 552 from SSS and N 192 – Lancaster. For the purposes of evaluation of the productive abilities of the received top cross two preliminary varietal experiments were carried out at the experimental field of Maize Research Institute, Knezha As a result of the conducted experimental work and the analysis it was found that the highest general combining ability have lines XM 11 6 and XM 12 1. These lines can be included as components of high-yielding synthetics or as testers in analyzing crosses to determine general combining ability in early stages of the selection process. The above lines with high specific combining ability – XM 11 13 and XM 11 46 are suitable for inclusion in combinations to develop high-yielding hybrids. Three of the tested lines XM 11 7 11 XM 10 and XM 11 11 have both high GCA and SCA. These lines can be used in corresponding breeding in the selection programs.

  6. Intra lines uniformity and inter lines variation of rice mutants resulting from irradiation of South Kalimantan local varieties

    International Nuclear Information System (INIS)

    Raihani Wahdah; Gusti Rumayadi; Rahmi Zulhidiani

    2016-01-01

    The preference of farmer in tidal swamp on local rice varieties are quite high, but local varieties have a long life and low yield characters, so it needs to be improved for the trait. This study is part of activities of the local rice varieties improvement to generate promising lines were short-moderate aged, but the slimming and pera (high amylose content) grains maintained. The aims of this study were to determine the intra lines uniformity and the inter lines variation of M5 generation of rice mutant lines. The experiment was carried out in the Experimental Station of Agriculture Faculty, Lambung Mangkurat University from March to September 2014. The experiment used 150 earliest flowering lines of 300 M5 mutant lines that were planted. Intra lines uniformity were analysed by comparing the variance of each mutant lines with variance of its parent, while the variation among lines were analyzed by comparing the variance of all lines with variance of its parent. More than 85 % M5 mutant lines from Siam Harli as parent and > 79 % of Siam Kuatek as parent are uniform. The uniform character at all M5 mutant lines, both of Siam Harli or Siam Kuatek parent are the harvest age, the filled grains number, and the empty grains number. There is no variability between M5 mutant lines, but some of M5 mutant lines from Siam Harli and Siam Kuatek have some better characters than their parents, so there is an opportunity for selection. (author)

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

    Science.gov (United States)

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

    2017-04-05

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

  8. Application Of Database Program in selecting Sorghum (Sorghum bicolor L) Mutant Lines

    International Nuclear Information System (INIS)

    H, Soeranto

    2000-01-01

    Computer database software namely MSTAT and paradox have been exercised in the field of mutation breeding especially in the process of selecting plant mutant lines of sorghum. In MSTAT, selecting mutant lines can be done by activating the SELECTION function and then followed by entering mathematical formulas for the selection criterion. Another alternative is by defining the desired selection intensity to the analysis results of subprogram SORT. Including the selected plant mutant lines in BRSERIES program, it will make their progenies be easier to be traced in subsequent generations. In paradox, an application program for selecting mutant lines can be made by combining facilities of Table, form and report. Selecting mutant lines with defined selection criterion can easily be done through filtering data. As a relation database, paradox ensures that the application program for selecting mutant lines and progeny trachings, can be made easier, efficient and interactive

  9. Reaction of some soybean mutant lines to natural rust fungus caused by (phakopsora pachyrhizi syd)

    International Nuclear Information System (INIS)

    Ratma, R.

    1988-01-01

    Reaction of some soybean mutant lines to natural rust fungus caused by (phakopsora pachyhizi syd). Eleven soybean mutant lines of orba variety derived from gamma fungus disease in the wet season 1985/86 at the experimental station of Citayam, Bogor. Based on IWGSR rating system, soybean mutant lines No 18/PsJ was moderately resistant to rust fungus disease. The other mutant lines, 14/PsJ, 15/PsJ, 20/PsJ, 102/PsJ, 106/PsJ, 111/PsJ, 118/PsJ, 119/PsJ and 220/PsJ were susceptible. (author). 4 figs.; 8 refs

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

    Directory of Open Access Journals (Sweden)

    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.

  11. The circling mutant Pcdh15roda is a new mouse model for hearing loss.

    Science.gov (United States)

    Torres, Adriana Amorim; Rzadzinska, Agnieszka K; Ribeiro, Andrea Frozino; Silva, Daniel Almeida da Silva E; Guénet, Jean-Louis; Massironi, Sílvia Maria Gomes; Godard, Ana Lúcia Brunialti

    2013-01-01

    Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles. Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear. The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay. In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. 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

  13. Evaluation of Promising Mutant Lines of Canola Grown under New Reclamation Lands (Harsh Lands)

    International Nuclear Information System (INIS)

    Amer, I.M.; Farrag, M.E.; Soliman, S.S.; Hassan, A.A.

    2017-01-01

    Canola seed lots of four varieties (Serow4, Serow6, Pactol as local cultivars and Evita as exotic variety) were treated with gamma rays at four doses (0, 100, 400 and 600 Gy). The present study aims to evaluate useful mutations in canola which possess high seed yield and oil content under new reclamation desert land at Ras-Suder-Sinai (saline) and Inshas (harsh and poor fertility) in M 4 and M 5 generations. The results at M 4 and M 5 generations showed that the 13-selected mutant lines on the bases of number of pods and seed yield/plant differed in their yield response according to environmental conditions. Over the two locations, the highest number of pods plant and seed yield was found at line 75 (M4) and line 11 for seed yield and line 78 for number of pods in M5 compared to other genotypes. More over, all the mutant lines compared to their parents showed significant or insignificant increases for all studies traits during the two successive generations. Over the two generations, the highest mean value compared to all genotypes was found in line 22 for plant height at Sudr and line 11 at Inshas, for fruiting zone length, the highest value was noticed in line 18 at Sudr and line 75 at Inshas, for the highest number of pods, (125/plant) it was found in line 63 at Sudr and (193/plant) in line 75 at Inshas which reflected the highest seed yield ( 8 g/plant).The highest mean value compared to all genotypes was found for 100 seed-weight in line 8 at Sudr and line 11 at Inshas which appeared the highest seed yield at Suder. Over all studied conditions, the mutant line 75 derived from Evita variety was characterized by the highest mean values for fruiting zone length of plant and number of pods/plant, reflecting a high seed yield (6.47 g/plant ) or about 83.87% over its parent. The increase of seed yield/plant for mutant line 11 over its parent was about 68.8% followed by line 8 surpassed its parent for seed yield by about 60.2 %. The oil content of canola seeds in

  14. Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Gloria C.; Franco, Ricardo; Lu, Yi; Ma, Jian-Guo; Shelnutt, John A.

    1999-05-19

    Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has

  15. Impaired Eye-Blink Conditioning in waggler, a Mutant Mouse With Cerebellar BDNF Deficiency

    OpenAIRE

    Bao, Shaowen; Chen, Lu; Qiao, Xiaoxi; Knusel, Beat; Thompson, Richard F.

    1998-01-01

    In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expressi...

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

    OpenAIRE

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu, Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslink...

  17. Soybean promising mutant lines super early maturity Q-298 and 4-Psj

    International Nuclear Information System (INIS)

    Arwin; Mulyana, H.I.; Tarmizi; Masrizal; Faozi, K.; Adie, M.

    2012-01-01

    One of the efforts to increase the national soybean (Glycine max L. Merr.) production is by growing super early maturity with high yielding varieties, so that the planting time can be shortened to fill out the cropping pattern of ''rice-rice-soybean''. Such varieties can be developed through mutation breeding method using γ ray irradiation. In this research the seeds of Tidar variety were irradiated by 200 Gy γ ray from 60 Co. Irradiated seeds were planted in the field and selections with emphasis on early maturing character were conducted in M 2 generation. Selected plants were purified to M 7 generation and selected pure mutant lines were subjected to preliminary and advanced yield trials. Based on these results 5 promising mutant lines were selected to continue in multi location yield trials. A set of lines for multi location yield trials consist of 14 lines included 5 mutant lines from this experiment, 5 lines from UNSUD, 3 national leading varieties, Argomulyo, Gorobogan, Burangrang, as national control varieties and Tidar as an original of mutant lines. Based on the result of multi location yield trials, 2 mutant lines, Q-298 dan 4-Psj, have significant high productivities compared to productivities of other lines and varieties. The growth duration of these lines were only 66 days and 68 days, respectively with average productivities were 2.41 tons / ha and 2.42 tons / ha, respectively. Index stability of Q-298 and 4-Psj mutant lines were 0.84 and 0.79, respectively, it means that the productivities of these two lines were stable in all tested locations. Based on the results, the Q-298 and 4-Psj mutant lines were proposed to be released as new varieties with the names of Gamasugen 1 and Gamasugen 2, respectively. (author)

  18. 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.

  19. Silencing neuronal mutant androgen receptor in a mouse model of spinal and bulbar muscular atrophy.

    Science.gov (United States)

    Sahashi, Kentaro; Katsuno, Masahisa; Hung, Gene; Adachi, Hiroaki; Kondo, Naohide; Nakatsuji, Hideaki; Tohnai, Genki; Iida, Madoka; Bennett, C Frank; Sobue, Gen

    2015-11-01

    Spinal and bulbar muscular atrophy (SBMA), an adult-onset neurodegenerative disease that affects males, results from a CAG triplet repeat/polyglutamine expansions in the androgen receptor (AR) gene. Patients develop progressive muscular weakness and atrophy, and no effective therapy is currently available. The tissue-specific pathogenesis, especially relative pathological contributions between degenerative motor neurons and muscles, remains inconclusive. Though peripheral pathology in skeletal muscle caused by toxic AR protein has been recently reported to play a pivotal role in the pathogenesis of SBMA using mouse models, the role of motor neuron degeneration in SBMA has not been rigorously investigated. Here, we exploited synthetic antisense oligonucleotides to inhibit the RNA levels of mutant AR in the central nervous system (CNS) and explore its therapeutic effects in our SBMA mouse model that harbors a mutant AR gene with 97 CAG expansions and characteristic SBMA-like neurogenic phenotypes. A single intracerebroventricular administration of the antisense oligonucleotides in the presymptomatic phase efficiently suppressed the mutant gene expression in the CNS, and delayed the onset and progression of motor dysfunction, improved body weight gain and survival with the amelioration of neuronal histopathology in motor units such as spinal motor neurons, neuromuscular junctions and skeletal muscle. These findings highlight the importance of the neurotoxicity of mutant AR protein in motor neurons as a therapeutic target. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Agronomic performance of rape seed (brassica napus L.) mutant lines under drought conditions

    International Nuclear Information System (INIS)

    Shah, S.A.; Ali, I.; Shah, S.J.A.; Rehman, K.; Rashid, A.

    1995-01-01

    Oil seed forms of Brassica napus are not well adapted to drought and the warner environments of Pakistan. Induced mutations were, therefore, utilized for improving drought tolerance efficiency of two napus cultivars. Induction of genetic variability, selection of desirable mutants and stabilization of mutants in acceptable agronomic background were carried out during 1988-1991. Fourteen promising mutants each of cv. Pak-cheen and Tower were evaluated for different agronomic characters in separate yield trials, under extremely drought conditions. The results demonstrated that yield potential of some mutants was very high and 9 mutants of cv. Pak-cheen and 8 mutants of cv. Tower significantly (P<0.05) out yield the local commercial cultivar. Eleven mutants in both the trials matured significantly earlier than the check. Nevertheless, more extensive testing of the drought tolerant lines under diversified environs of the country will help confirm these findings. (author)

  1. Genetic characterization of glossy-leafed mutant broccoli lines

    Science.gov (United States)

    Glossy mutants of Brassica oleracea L. have reduced or altered epicuticular wax on the surface of their leaves as compared to wild-type plants, conveying a shiny green appearance. Mutations conferring glossiness are common and have been found in most B. oleracea crop varieties, including cauliflower...

  2. Grain product of 34 soya mutant lines;Rendimiento de grano de 34 lineas mutantes de soya

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Mastache L, A. A.; Valencia E, F.; Diaz V, G. E. [Colegio Superior Agropecuario del Estado de Guerrero, Vicente Guerrero No. 81, Col. Centro, 40000 Iguala, Guerrero (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad, Colegio de Posgraduados, Carretera Mexico-Texcoco Km. 36.5, Montecillo, 56230 Texcoco, Estado de Mexico (Mexico); De la Cruz T, E.; Garcia A, J. M.; Falcon B, T.; Gatica T, M. A. [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2009-07-01

    This work was development with the objective of obtaining information of the agronomic behavior of 34 soya mutant lines (R{sub 4}M{sub 18}) for human consumption and this way to select the 2 better lines. The genetic materials were obtained starting from the variety ISAAEG-B M2 by means of the application of recurrent radiation with Co{sup 60} gammas, to a dose of 350 Gray for the first two generations and both later to 200 Gray and selection during 17 cycles, being obtained the 34 better lines mutants with agronomic characteristic wanted and good flavor. The obtained results were that the mutant lines L{sub 25} and L{sub 32} produced the major quantity in branches/plant number with 7.5 and 7.25, pods/plant number with 171.25 and 167, grains/plant number with 350.89 and 333.07 and grain product (ton/ha) to 15% of humidity 5.15 and 4.68 ton/ha, respectively. (Author)

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    International Nuclear Information System (INIS)

    Cuthill, S.; Poellinger, L.

    1988-01-01

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

  6. Assessment of Genetic diversity in mutant cowpea lines using ...

    African Journals Online (AJOL)

    FKOLADE

    2016-11-09

    Nov 9, 2016 ... DNA extraction. The seeds of the mutants and their parents were planted out in pots in the screen house, and young leaves were harvested from them ... The PCR was done using a modified touch down progam as follows: 94°C for 2 min, 12 cycles of 2 min at 94°C, one min at 65°C. (-0.7°C per cycle) and 1 ...

  7. Analysis of the albino-locus region of the mouse. II. Mosaic mutants

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    Among 119 mutations involving the c locus that were recovered in the course of mouse specific-locus experiments with external radiations, 16 were found in mosaic, or fractional, mutants. The number of additional c-locus fractionals that could have occurred in these experiments and, for a variety of reasons, might not have been clearly identified, probably does not exceed the present number. There was no evidence for radiation induction of the fractionals, and even those occurring in the irradiated groups may thus be assumed to be of spontaneous origin. Since only two mutations in the control groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mutations are fractionals. None of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except for one subvital and one not tested. Genetic tests of the fractionals indicated no major selection against the new mutations, either gametically or in the progeny. For the group of fractionals as a whole, about one-half of the germinal tissue carried the mutation, indicating that the fractionals came from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a daughter chromosome derived from pronuclear DNA synthesis of the zygote, or in one of the first two blastomeres prior to replication. Since the mouse embryo does not stem from all of the cleavage products of the zygote, the frequency of fractionals observeed underestimates the frequency of mutational events that result in two types of blastomeres

  8. Autophagy and UPR in alpha-crystallin mutant knock-in mouse models of hereditary cataracts.

    Science.gov (United States)

    Andley, Usha P; Goldman, Joshua W

    2016-01-01

    Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C+/- heterozygotes develop cataracts by 1-2months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens. Wild-type, heterozygote (αA-R49C+/-), and homozygote (αA-R49C+/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting). Morphology was altered by embryonic day 17 in R49C+/+ lenses; R49C+/- lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G+/+ mice, suggesting its role in lens opacification. Mutated crystallins alter lens morphology, autophagy, and stress responses. Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Modified Starch of Sorghum Mutant Line Zh-30 For High Fiber Muffin Products

    International Nuclear Information System (INIS)

    Santosa, D. D. S; Human, S

    2009-01-01

    Sorghum mutant line Zh-30 is a breeding line developed at the Center for the Application of Isotope and Radiation Technology, BATAN by using mutation techniques. Gamma irradiation with the dose of 300 Gy was used to induce sorghum genetic variation. Through selection processes in several generations, the mutant line Zh-30 was identified to have better agronomic characteristics, better grain quality and higher grain yield than the original variety. Research on modified starch quality of this mutant line was done to identify its potential use in food industry. Functionality of pregelatinized, hydroxypropyl and crosslinked starch of this mutant line (Mutant TexInstant 30) has been studied for its use in high fiber muffin products. Characteristics of high fiber muffins containing 1.50; 3.50 and 5.50% of Mutant Tex-Instant 30 replacement levels to wheat flour were evaluated using both sensory panel and physical test methods. With regard to the sensory parameters, the high fiber muffins containing 1.50 - 5.50 % Mutant Tex-Instant 30 in general were not significantly different compared to the standard reference muffin. Results of physical evaluations showed that all Mutant Tex-Instant 30 containing products retained more moisture during baking than the standard reference. Tenderness of all products decreased at similar rate following 24 and 48 hr of room temperature storage and seven days at freezer temperature. These results suggested that sorghum mutant line Zh-30 starch could be modified and potentially used in food industry as a subtitute of wheat flour (author)

  10. Modified Starch of Sorghum Mutant Line Zh-30 for High Fiber Muffin Products

    Directory of Open Access Journals (Sweden)

    D.D.S. Santosa

    2009-01-01

    Full Text Available Sorghum mutant line Zh-30 is a breeding line developed at the Center for the Application of Isotope and Radiation Technology, BATAN by using mutation techniques. Gamma irradiation with the dose of 300 Gy was used to induce sorghum genetic variation. Through selection processes in several generations, the mutant line Zh-30 was identified to have better agronomic characteristics, better grain quality and higher grain yield than the original variety. Research on modified starch quality of this mutant line was done to identify its potential use in food industry. Functionality of pregelatinized, hydroxypropyl and crosslinked starch of this mutant line (Mutant TexInstant 30 has been studied for its use in high fiber muffin products. Characteristics of high fiber muffins containing 1.50; 3.50 and 5.50% of Mutant Tex-Instant 30 replacement levels to wheat flour were evaluated using both sensory panel and physical test methods. With regard to the sensory parameters, the high fiber muffins containing 1.50 - 5.50 % Mutant Tex-Instant 30 in general were not significantly different compared to the standard reference muffin. Results of physical evaluations showed that all Mutant Tex-Instant 30 containing products retained more moisture during baking than the standard reference. Tenderness of all products decreased at similar rate following 24 and 48 hr of room temperature storage and seven days at freezer temperature. These results suggested that sorghum mutant line Zh-30 starch could be modified and potentially used in food industry as a subtitute of wheat flour.

  11. Evaluation of Mungbean Mutant Lines to Drought Stress and Their Genetic Relationships Using SSR Markers

    Directory of Open Access Journals (Sweden)

    Yuliasti

    2015-12-01

    Full Text Available Development of mungbean cultivarstolerant to drought stress through mutation breeding approach would enable us to anticipate the crop yield-reducing effects of climate changes. The objective of this research was to evaluate the yield performance of mungbean mutant lines that showed tolerance to drought stress, and to analyze their genetic diversity and relationship among mutant lines using SSR markers. The study was conducted during the dry season of 2012 in the Muneng experimental farm, Probolinggo, East Java. The experiment was laid out in a randomized block design with four replications. Five mutant lines and two parental lines as control were tested for evaluation of yield and drought tolerance under twoenvironments of two irrigation systems as treatment. The two environmental conditions consisted of optimal irrigation (at least three times: at planting, flowering and during pod filling and suboptimal irrigation (two times at planting and flowering. To evaluate genetic variation among selected mutant lines and their discrimination from parental lines in molecular level, a cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA in the NTSYS software. The results showed that three mutant lines, including PsJ30, PsJ31, PsJ32 produced the highest grain yields of 1.17, 1.01, and 1.04 ton/ha, respectively, compared to the other mutant lines and the parents Gelatik (0.85 ton/ha and Perkutut (0.87 ton/ha as control check. Of those mutant lines, PSJ31 was the most tolerant to drought with sensitivity index value of 0.47. The PSJ31 has now been officially released as a new variety ( 2013, named as Muri which was identified to have high yield and tolerant to drought. Based on 23 SSR markers used for clustering analysis of those 3 selected mutant lines,9SSR markers (MBSS R033; satt137; MBSSR008; MBSSR203; MBSSR013; MBSSR021; MBSSR016; MBSSR136; and DMBSSR013 were successfully identified the three mungbean mutant

  12. Resistance of some early mutant lines of soybean to rust fungus (Phakospora pachyrhizi Syd)

    International Nuclear Information System (INIS)

    Ratma, Rivaie

    1984-01-01

    A trial for resistance to rust fungus (Phakospora pachyrhizi Syd.) was conducted on 11 early mutant lines of soybean M6 (derived from Orba variety with a dose of 0.4 kGy of Co-60) at Citayam Experimental Station, Bogor, in the wet season of 80/81. Based on IWGSR rating system, soybean mutant lines number M6/40/6 was moderately susceptible to rust fungus (Phakospora pachyrhizi Syd). While 10 other soybean mutant lines M6/40/1, M6/40/2, M6/40/3, M6/40/4, M6/40/5, M6/40/7, M6/40/8, M6/40/9, M6/40/10 and M6/40/11 were susceptible to rust fungus. Significant differences in yield were observed between the early mutant lines M6/40/6 (moderate susceptible), 10 other mutant lines (susceptible) and ringgit variety (susceptible). However, a significant lower yield was produced by those mutant lines compared with the yield of orba variety. (author)

  13. Mutant lines of currant tomato, valuable germplasm with multiple disease resistance

    International Nuclear Information System (INIS)

    Govorova, G.F.; Khrustaleva, V.V.; Shcherbakov, V.K.

    1987-01-01

    Studies were carried out for two years on eight mutant lines of currant tomato at the Krymsk Experimental Breeding Station of the N.I. Vavilov All-Union Scientific Research Institute of Plant-Growing (VIR). The station is situated in an area of commercial field tomato growing (Krasnodar region). The mutant lines of currant tomato (VIR specimen No. k-4053) were obtained through chronic gamma-irradiation. A disease resistance evaluation of the mutants was carried out for Verticillium wilt (Verticillium albo-atrum Rein. and Berth.), for black bacterial spotting (Xanthomonas vesicatoria Dows.), for tobacco mosaic virus Nicotiana 1 Smith), for streak virus (Nicotiana 1), for the combination TMV with X and Y potato viruses, for cucumber virus (Cucumis 1), and also for top rot. Fifty plants of each mutant line were evaluated and checks were made three times in each season. A comparison of the currant tomato mutants with the standard tomato varieties demonstrates the better resistance shown by the mutant germplasm to the main pathogens. The degree to which some currant tomato mutants were affected by Verticillium was lower than that of the most VerticiIlium-resistant samples of tomato evaluated between 1975 and 1981. The mutants of currant tomato should therefore be of interest as germplasm in breeding tomatoes for improved multiple disease resistance

  14. Evaluation of rice mutant lines for resistance to brown planthopper, nilaparvata lugens stall

    International Nuclear Information System (INIS)

    Mugiono

    1985-01-01

    The most important and common insect in rice cultivation in South East Asia is brown planthopper, nilaparvata lugens stall. Seven rice mutant lines produced by the National Atomic Energy Agency, Indonesia, were tested at IRRI, the Philippines for resistance to brown planthopper. Those mutant lines were Atomita 1, 627/10-3/PsJ, Atomita 2 and 627/4-E/PsJ originated from Pelita 1/1 which was irradiated with 0.2 kGy of gamma rays and A227/2/PsJ, A227/3/PsJ and A227/5/PsJ, originated from early maturing mutant A23/PsJ/72K from irradiated Pelita 1/1 which was irradiated with 0.1 kGy of gamma rays. Evaluation of resistance was carried out by seedling bulk screening, honeydew excretion, survival and population build up tests by using brown planthopper biotype 1, 2 and 3. Results of these tests showed that the seven tested mutant lines were resistant to biotype 1 but susceptible to biotype 2. Reaction to biotype 3 showed that six mutant lines tested were moderately resistant and only one mutant of 627/4-E/PsJ was susceptible. Reactions of the mutant lines to biotype 1, 2 and 3 were different from the resistant varieties, Mudgo or ASD-7. This indicated that mutant lines might have gene(s) for resistance which differed from those of resistant varieties. The results showed that resistance to brown planthopper is possible to be introduced in Indonesian rice varieties by means of mutations. (author)

  15. Nutrient Changes and in Vitro Digestibility in Generative Stage of M10-BMR Sorghum Mutant Lines

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    R. Sriagtula

    2017-08-01

    Full Text Available The objective of this research was to investigate the influences of generative stage on crude protein, crude fiber, ash, and crude fat contents as well as in-vitro dry matter and organic matter digestibilities of M-10 BMR sorghum mutant lines. This research was arranged into a randomized block design with 2 factors. The first factor was M-10 BMR sorghum mutant lines (Patir 3.1, Patir 3.2 and Patir 3.7 and the second factor was generative stages (flowering, soft dough and hard dough phase. The observed variables were proximate contents of stem, leaves and panicle of sorghum plant and in-vitro digestibility of whole plant. The results showed that leaves crude protein (CP was more influenced by M-10 BMR sorghum mutant lines. Stems and panicles CP were influenced by the interaction between M-10 BMR sorghum mutant lines and generative stages. Further generative stage reduced stems CP but increased panicles CP. Crude fiber (CF, ash, and ether extract (EE in leaves were not influenced by generative stages. Stems CF was influenced by M-10 BMR sorghum mutant lines and generative stages, while stems EE was more influenced by generative stages. Stems ash content was influenced by the interaction between M-10 BMR sorghum mutant lines and generative stages while panicles ash content was more influenced by generative stages. M-10 BMR sorghum mutant lines and hard dough phase increased in-vitro dry matter and organic matter digestibilities. Based on those findings, it can be concluded that the increased maturity reduces CP and CF contents so it increases in-vitro digestibilities.

  16. Meiosis in gamma-ray induced tomato mutants of line XXIV-a

    International Nuclear Information System (INIS)

    Zagorcheva, L.; Jordanov, M.

    1976-01-01

    Results are reported of investigations on meiosis in tomato mutants obtained by gamma-irradiation ( 60 Co) of seeds from line XXIV-a with doses of 20 and 30 krad. Two genome mutants (one a triploid and the other a tetraploid form) as well as a chromosome aberration of the translocation type, were selected in the course of the investigations and their meiosis is described. Meiosis in the initial form (line XXIV-a) was also studied. About 16% of the initial line XXIV-a plants proved to be trisomic forms. (author)

  17. Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

    Full Text Available An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn, inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

  18. SRAP analysis for space induced mutant line of maize (Zea mays L.)

    International Nuclear Information System (INIS)

    Du Wenping; Yu Guirong; Song Jun; Xu Liyuan

    2011-01-01

    In order to detect the effects of space mutation on maize, 16 SRAP primers were applied for the discrimination of the maize inbred line '968' and its 93 mutant materials, 154 polymorphic fragments were amplified. The average of polymorphic bands detected by per SRAP primer combination was 9.6 with a range from 5 to 18. Genetic similarities among the 94 materials ranged from 0.481 to 1.000 with an average of 0.903, and the largest genetic distance was found between mutant line 37 and control. The 94 materials were divided into six groups with the similarity coefficient of 0.732. The phylogenetic analysis showed distinct variation among the mutants. The results indicated that SRAP markers could be used for analyzing genetic variation of mutants. (authors)

  19. Evaluation of yield and N2 fixation of mutant lines of groundnut in Malaysia

    International Nuclear Information System (INIS)

    Rusli, I.; Harun, A.R.; Rahman, K.A.; Shamsuddin, S.; Rahim, K.A.; Danso, S.K.A.

    1998-01-01

    The 15 N-dilution technique was used to evaluate N 2 fixation in groundnut (Arachis hypogaea L.) in three field trials of cultivars Matjan and V-13 (parents), their selected mutant lines, and a other local and foreign genotypes. Matjan mutant MJ/40/42 consistently produced the highest pod yields, at above 4 t ha -1 , 14-22% higher yields than the parent. In contrast, none of the V-13 mutants had consistently better yields than the parent. The mutant lines did not show consistent agronomic performance from year to year. Total dry matter yield did not correlate with pod yield, and pod yield did not correlate with amount of N fixed

  20. Development of One mutant line with Improved Quantitative and Qualitative Traits through Induced Mutation

    International Nuclear Information System (INIS)

    Saif, A. A.; Al-kibssi, M; Al-Shamiri, A; Kassem, R

    2008-01-01

    A field experiment was conducted in three consecutive seasons 2005, 2006 and 2007 for evaluating five mutant lines derived from Gemiza-9 variety. Gemiza-9 and Shibam-8 were used as a checks for yellow rust resistance and some agronomic characters. The mutant lines were planted in Al-erra research farm and farmer's field under rainfed condition, in particularly at Shibam and Bani-Mater regions. Results showed that the MS-5 and MS-9 mutant lines were earlier than the others and the checks. They matured on 102 - 105 days compared with 111 - 118 days for the other lines including the original variety and the Shibam-8 variety. These two mutant lines showed not only early maturing but also resistance to yellow rust disease, they scored R20% -R30%, while the all material were medium resistance including the checks. With respect to yield, the MS -5 mutant had a significant high yield (3963 kg/ha) compared with the others including the Gemiza-9 and Shibam-8 variety amounting to 35.5 % and 32.2 % for the two checks respectively. (author)

  1. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  3. Characterization of Boerhavia diffusa L. mutant lines by RAPD and isozyme, selected for agronomically valuable traits

    International Nuclear Information System (INIS)

    Shukla, N.; Sangwan, N.S.; Misra, H.O.; Sangwan, R.S.

    2004-01-01

    Boerhavia diffusa is a medicinally important plant and finds extensive uses in traditional herbal drug preparations. For the development of improved varieties in terms of superior yield and quality of herb/root of B. diffusa, mutation breeding was attempted. Mutants generated by physical and chemical mutagenic treatments were screened for yield and quality parameters of the root/herb up to three consecutive generations. The selected-screened lines generated by physical and chemical mutagenic treatments on two selected genotypes I and II were molecularly analyzed using eight isozymes and eleven RAPD primers producing good amplification. Mutants from BD10 (selected genotype I) were distinct, while, in case of BD22 (selected genotype II), only one mutant BDMu7 was recorded distinct by isozyme analysis. The wild mutant (BDMu16, with maximum height and mouve coloured flower) was distinct in RAPD banding pattern. Isozymes differentiated the mutants from their respective controls, whereas RAPD differentiated the mutants and controls and also distinguished the mutants. The RAPD analysis was found to be better suited than isozymes for detecting genetic differences among controls and their mutants. However, both RAPD and isozyme analyses gave similar patterns of genetic relationships [it

  4. Stability Test For Sorghum Mutant Lines Derived From Induced Mutations with Gamma-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    S. Human

    2011-12-01

    Full Text Available Sorghum breeding program had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN. Plant genetic variability was increased through induced mutations using gamma-ray irradiation. Through selection process in successive generations, some promising mutant lines had been identified to have good agronomic characteristics with high grain yield. These breeding lines were tested in multi location trials and information of the genotypic stability was obtained to meet the requirements for officially varietal release by the Ministry of Agriculture. A total of 11 sorghum lines and varieties consisting of 8 mutant lines derived from induced mutations (B-100, B-95, B-92, B-83, B-76, B-75, B-69 and Zh-30 and 3 control varieties (Durra, UPCA-S1 and Mandau were included in the experiment. All materials were grown in 10 agro-ecologically different locations namely Gunungkidul, Bantul, Citayam, Garut, Lampung, Bogor, Anyer, Karawaci, Cianjur and Subang. In each location, the local adaptability test was conducted by randomized block design with 3 replications. Data of grain yield was used for evaluating genotypic stability using AMMI approach. Results revealed that sorghum mutation breeding had generated 3 mutant lines (B-100, B-76 and Zh-30 exhibiting grain yield significantly higher than the control varieties. These mutant lines were genetically stable in all locations so that they would be recommended for official release as new sorghum varieties to the Ministry of Agriculture

  5. Responses of Soybean Mutant Lines to Aluminium under In Vitro and In Vivo Condition

    International Nuclear Information System (INIS)

    Yuliasti; Sudarsono

    2011-01-01

    The main limited factors of soybean plants expansion in acid soil are Aluminium (Al) toxicity and low pH. The best approach to solve this problem is by using Al tolerance variety. In vitro or in vivo selections using selective media containing AlCl 3 and induced callus embryonic of mutant lines are reliable methods to develop a new variety. The objectives of this research are to evaluate response of soybean genotypes against AlCl 3 under in vitro and in vivo condition. Addition of 15 part per million (ppm) AlCl 3 into in vitro and in vivo media severely affected plant growth. G3 soybean mutant line was identified as more tolerant than the control soybean cultivar Tanggamus. This mutant line was able to survive under more severe AlCl 3 concentrations (15 ppm) under in vitro conditions. Under in vivo conditions, G1 and G4 mutants were also identified as more tolerant than Tanggamus since they produced more pods and higher dry seed weigh per plant. Moreover, G4 mutant line also produced more dry seed weight per plant than Tanggamus when they were grown on soil containing high Al concentration 8.1 me/100 gr = 81 ppm Al +3 . (author)

  6. A novel mouse Fgfr2 mutant, hobbyhorse (hob, exhibits complete XY gonadal sex reversal.

    Directory of Open Access Journals (Sweden)

    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.

  7. p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer

    Science.gov (United States)

    McFadden, David G.; Vernon, Amanda; Santiago, Philip M.; Martinez-McFaline, Raul; Bhutkar, Arjun; Crowley, Denise M.; McMahon, Martin; Sadow, Peter M.; Jacks, Tyler

    2014-01-01

    Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. PMID:24711431

  8. An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

    Science.gov (United States)

    Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

    2017-12-01

    Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    International Nuclear Information System (INIS)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan; Hasty, Paul

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-01

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

  11. Quality characteristics of soybean pasted (Doenjang) manufactured with 2 soybean mutant lines derived from cv. baekwon

    International Nuclear Information System (INIS)

    Lee, Kyung Jun; Kang, Si Yong; Choi, Hong Il; Kim, Jin Baek

    2016-01-01

    In order to identification of the possibility of manufacturing soybean paste (doenjang) with soybean mutant lines induced from gamma-ray mutagenesis, this study was performed to investigate the quality characteristics of doenjang using two soybean mutant lines, Baekwon-1 (BW-1) and Baekwon-2 (BW-2) and their original cultivar (cv. Baekwon, BW) for 8 weeks. The BW and two mutant lines (BW-1 and BW-2) were showed higher content of amino type nitrogen than control (cv. Taegwang). The pH decreased and the titratable acidity increased all the samples during aging period. The lightness, redness and yellowness of doenjang were the lowest in BW. Total free sugar content of doenjang was the highest in control (10.43%) after 4 weeks and composed mainly fructose and glucose. The order of the free amino acid content was Glutamic acid>Leucine>Lysine>Phenylalanine>Aspartic acid in control, Glutamic acid>Leucine >Arginine>Lysine>Phenylalanine in BW, Glutamic acid>Lysine>Phenylalanine>Aspartic acid>Valine in BW-1 and Glutamic acid>Arginine>Lysine>Phenylalanine>Aspartic acid in BW-2, respectively. Our results showed that it is possible to increase the quality of doenjang using soybean mutant lines in manufacturing soybean paste

  12. Nitrogen Dynamic Study on Rice Mutant Lines Using 15N Isotope Techniques

    International Nuclear Information System (INIS)

    Ahmad Nazrul Abd Wahid; Shyful Azizi Abdul Rahman; Abdul Rahim Harun

    2015-01-01

    Malaysian Nuclear Agency in collaboration with UPM and MARDI has produced two types of rice mutant lines of MR219, viz. MR219-4 and MR219-9 developed under rice radiation mutagenenesis programme for adaptability to aerobic conditions. Aerobic cultivating is rice cultivation system on well drained soil and using minimal water input. At Malaysian Nuclear Agency, a nitrogen fertilization study in aerobic condition for the rice mutant lines was carried out in the shade house and field. The study is intended to examine and assess the dynamics of nitrogen by rice mutant lines through the different soil water management and nitrogen levels. Direct 15 N isotopic tracer method was used in this study, whereby the 15 N labeled urea fertilizer was utilized as a tracer for nitrogen nutrient uptake by the test crops. This paper is intended to highlight the progress that has been made in the study of the nitrogen dynamics on MR219-4 and MR219-9 rice mutant lines. (author)

  13. Genotypic variability in sesame mutant lines in Kenya | Ong'injo ...

    African Journals Online (AJOL)

    Sesame (Sesamum indicum L) is one of the major oil crops with potential for production by small- scale holders in the marginal agro-ecological zones of Kenya. Variability studies on yield and yield components of sesame mutant lines now in M7generation was carried out in two locations for two seasons in Kenya.

  14. Quality characteristics of soybean pasted (Doenjang) manufactured with 2 soybean mutant lines derived from cv. baekwon

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung Jun; Kang, Si Yong; Choi, Hong Il; Kim, Jin Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2016-11-15

    In order to identification of the possibility of manufacturing soybean paste (doenjang) with soybean mutant lines induced from gamma-ray mutagenesis, this study was performed to investigate the quality characteristics of doenjang using two soybean mutant lines, Baekwon-1 (BW-1) and Baekwon-2 (BW-2) and their original cultivar (cv. Baekwon, BW) for 8 weeks. The BW and two mutant lines (BW-1 and BW-2) were showed higher content of amino type nitrogen than control (cv. Taegwang). The pH decreased and the titratable acidity increased all the samples during aging period. The lightness, redness and yellowness of doenjang were the lowest in BW. Total free sugar content of doenjang was the highest in control (10.43%) after 4 weeks and composed mainly fructose and glucose. The order of the free amino acid content was Glutamic acid>Leucine>Lysine>Phenylalanine>Aspartic acid in control, Glutamic acid>Leucine >Arginine>Lysine>Phenylalanine in BW, Glutamic acid>Lysine>Phenylalanine>Aspartic acid>Valine in BW-1 and Glutamic acid>Arginine>Lysine>Phenylalanine>Aspartic acid in BW-2, respectively. Our results showed that it is possible to increase the quality of doenjang using soybean mutant lines in manufacturing soybean paste.

  15. Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation?

    Directory of Open Access Journals (Sweden)

    Natascha eSchaefer

    2012-10-01

    Full Text Available Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400 with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans, single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the α (spasmodic, oscillator, cincinnati, Nmf11 or the β (spastic subunit of the GlyR are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABAA receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator, displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation.

  16. Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

    Science.gov (United States)

    Banks, Gareth T; Haas, Matilda A; Line, Samantha; Shepherd, Hazel L; Alqatari, Mona; Stewart, Sammy; Rishal, Ida; Philpott, Amelia; Kalmar, Bernadett; Kuta, Anna; Groves, Michael; Parkinson, Nicholas; Acevedo-Arozena, Abraham; Brandner, Sebastian; Bannerman, David; Greensmith, Linda; Hafezparast, Majid; Koltzenburg, Martin; Deacon, Robert; Fainzilber, Mike; Fisher, Elizabeth M C

    2011-04-06

    The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

  17. Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

    Directory of Open Access Journals (Sweden)

    Mitchell S Turker

    Full Text Available Exposure to a small number of high-energy heavy charged particles (HZE ions, as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

  18. Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

    Science.gov (United States)

    Turker, Mitchell S; Grygoryev, Dmytro; Lasarev, Michael; Ohlrich, Anna; Rwatambuga, Furaha A; Johnson, Sorrel; Dan, Cristian; Eckelmann, Bradley; Hryciw, Gwen; Mao, Jian-Hua; Snijders, Antoine M; Gauny, Stacey; Kronenberg, Amy

    2017-01-01

    Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

  19. New early-ripening wheat mutant lines from the varieties Norman and Avalon

    International Nuclear Information System (INIS)

    Djelepov, K.

    1988-01-01

    The English wheat varieties Norman and Avalon are high-productive, resistant to lodging and to diseases but late-ripening in Bulgaria. They are 10-15 days later than the variety Sadovo 1 and therefore suffer often from dry and hot weather, causing premature ripening and shrivelled seed. Dry seeds from the two varieties were irradiated with 10 and 15 kR 60 Co gamma rays. In M 2 , several earlier ripening forms were selected and they were studied also in M 3 in 1987. In the Table, four early ripening mutant lines and the respective initial varieties are compared. They vary significantly in plant height and grain size. The mutant lines of Norman produce smaller grain but all mutants show a higher hectoliter weight. The mutant lines head and mature 4 to 10 days earlier than the respective initial varieties. Some of them are as productive as the standard and other cultivated varieties. We shall continue testing their productivity and possibilities for their use in the breeding

  20. Water-deficit tolerant classification in mutant lines of indica rice

    Directory of Open Access Journals (Sweden)

    Suriyan Cha-um

    2012-04-01

    Full Text Available Water shortage is a major abiotic stress for crop production worldwide, limiting the productivity of crop species, especially in dry-land agricultural areas. This investigation aimed to classify the water-deficit tolerance in mutant rice (Oryza sativa L. spp. indica genotypes during the reproductive stage. Proline content in the flag leaf of mutant lines increased when plants were subjected to water deficit. Relative water content (RWC in the flag leaf of different mutant lines dropped in relation to water deficit stress. A decrease RWC was positively related to chlorophyll a degradation. Chlorophyll a , chlorophyll b , total chlorophyll , total carotenoids , maximum quantum yield of PSII , stomatal conductance , transpiration rate and water use efficiency in mutant lines grown under water deficit conditions declined in comparison to the well-watered, leading to a reduction in net-photosynthetic rate. In addition, when exposed to water deficit, panicle traits, including panicle length and fertile grains were dropped. The biochemical and physiological data were subjected to classify the water deficit tolerance. NSG19 (positive control and DD14 were identified as water deficit tolerant, and AA11, AA12, AA16, BB13, BB16, CC12, CC15, EE12, FF15, FF17, G11 and IR20 (negative control as water deficit sensitive, using Ward's method.

  1. Evaluation of some mutant lines of rice induced by gamma radiation treatment 1. mean performance of rice mutants in M4 generation

    International Nuclear Information System (INIS)

    El-Banna, M.N.; El-Wakil, H.M.F.; Ebaid, R.A.; Sallam, R.A.

    2009-01-01

    Grains of eight rice mutants; SC 1, SC 6, RTY 1, RTY 3, HY 14, HYI 17, EH 4 and HYPI 22 were secured from Botany Department Faculty of Agriculture Cairo university. The procedures and the methodology for induction these mutants as well as the original mean performance of such mutants are presented else where; Sabbour, (1989) and Sabbour etal. (2002). Grains were sown (M4 generation) at the experimental farm in Itai EI-Baroud Agricultural Research Station Behaira Governorate Agricultural Research Center (ARC) in the summer season (2007). The mean performance of such mutants was studied during M4 generation. The most exciting results were as follows: the selected line SC 1 showed in M4 generation superior agronomic and yield traits. Sc 1 mutant line is not bred truly and it need more generations to reach stability. SC 6 in M4 generation showed considerable number of individuals scored low mean values toward the negative direction and lowering the overall trait mean performance. The rice lines RTY 1 and RTY 3 proved that, the average number of fertile tillers per plant of the selected lines maintained previously recorded mean values of M3 generation in M4. The traits showed significant differences among their progeny that recorded high CV% values as compared with those showed no significant differences. The rice lines HY 14 and HYI 17 showed a true breeding signs and no more breeding generations are required. Rice lines EH 4, showed a considerable reduction in number of days elapsed from date of cultivation till harvest. As, this mutant maintained 86.58 days till heading. Rice mutant line HYPI 22 did not bred truly for the original selected traits (high yield and high protein content) and it still need more generations of selection to reach considerable stability

  2. Case Study: Polycystic Livers in a Transgenic Mouse Line

    Energy Technology Data Exchange (ETDEWEB)

    Lovaglio, Jamie A.; Artwohl, James E.; Ward, Christopher J.; Diekwisch, Thomas G. H.; Ito, Yoshihiro; Fortman, Jeffrey D.

    2014-04-01

    Three mice (2 male, 1 female; age, 5 to 16 mo) from a mouse line transgenic for keratin 14 (K14)-driven LacZ expression and on an outbred Crl:CD1(ICR) background, were identified as having distended abdomens and livers that were diffusely enlarged by numerous cysts (diameter, 0.1 to 2.0 cm). Histopathology revealed hepatic cysts lined by biliary type epithelium and mild chronic inflammation, and confirmed the absence of parasites. Among 21 related mice, 5 additional affected mice were identified via laparotomy. Breeding of these 5 mice (after 5 mo of age) did not result in any offspring; the K14 mice with olycystic livers failed to reproduce. Affected male mice had degenerative testicular lesions, and their sperm was immotile. Nonpolycystic K14 control male mice bred well, had no testicular lesions, and had appropriate sperm motility. Genetic analysis did not identify an association of this phenotype with the transgene or insertion site.

  3. Copper phytoextraction in tandem with oilseed production using commercial cultivars and mutant lines of sunflower.

    Science.gov (United States)

    Kolbas, A; Mench, M; Herzig, R; Nehnevajova, E; Bes, C M

    2011-01-01

    Use of sunflower (Helianthus annuus L.) for Cu phytoextraction and oilseed production on Cu-contaminated topsoils was investigated in afield trial at a former wood preservation site. Six commercial cultivars and two mutant lines were cultivated in plots with and without the addition of compost (5% w/w) and dolomitic limestone (0.2% w/w). Total soil Cu ranged from 163 to 1170 mg kg(-1). In soil solutions, Cu concentration varied between 0.16-0.93 mg L(-1). The amendment increased soil pH, reduced Cu exposure and promoted sunflower growth. Stem length, shoot and capitulum biomasses, seed yield, and shoot and leaf Cu concentrations were measured. At low total soil Cu, shoot Cu mineralomass was higher in commercial cultivars, Le., Salut, Energic, and Countri, whereas competition and shading affected morphological traits of mutants. Based on shoot yield (7 Mg DW ha(-1)) and Cu concentration, the highest removal was 59 g Cu ha(-1). At high total soil Cu, shoot Cu mineralomass peaked for mutants (e.g., 52 g Cu ha(-1) for Mutant 1 line) and cultivars Energic and Countri. Energic seed yield (3.9 Mg air-DW ha(-1)) would be sufficient to produce oil Phenotype traits and shoot Cu removal depended on sunflower types and Cu exposure.

  4. HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

    Directory of Open Access Journals (Sweden)

    Michael T Cheeseman

    2011-10-01

    Full Text Available Otitis media with effusion (OME is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006 and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of

  5. Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy.

    Science.gov (United States)

    Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

    2017-06-01

    Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Assesment of spineless safflower (Carthamus tinctorius, L.) mutant lines for seed oil content and fatty acid profiles

    International Nuclear Information System (INIS)

    Ragab, A.I.; Kassem, M.; Moustafa, H.A.M.

    2008-01-01

    This study was conducted to assess the new spineless mutants that previously induced through gamma radiation and hybridization techniques in the advanced generation for seed oil content and fatty acid profiles The obtained results cleared that oil percentages of all seven safflower mutants were increased than local variety Giza (1) and the new mutant hybrid 2 line (white petals) had the highest increase value of oil percentage (10%) but the mutant line M14 (dark red petals) had the lowest increase value of oil percentage (3.1 %) The mutant line M7 (yellow petals) had the highest value of total saturated fatty acid (40.38%), because it had the highest value of palmitic fatty acid (25.16%), comparing to 10.01% value for local variety Giza (1), followed by mutant line hybrid 2 (white petals) which had (39.88%) because it had the highest value of caprylic, capric, lauric, myristic and stearic fatty acids. All safflower mutant lines had higher value of oleic fatty acid than that of the local variety Giza (1) the two new safflower mutant lines M7 (yellow petals) and hybrid 2 (white petal) had the highest value of oleic fatty acid 41.22% and 39.88% respectively in comparison with 13.5% for local variety Giza (1), the obtained results are indicating to seed oil content negative correlation between oleic and linoleic acids

  7. Mapping of a rice thermosensitive genic male sterility gene from a TGMS mutant line

    Energy Technology Data Exchange (ETDEWEB)

    Vu Duc Quang; Nguyen Van Dong; Pham Ngoc Luong; Tran Duy Quy [Argicultural Genetics Institute, Hanoi (Viet Nam); Nguyen, Henry T. [Texas Tech Univ., Department of Plant and Soil Science, Lubbock TX (United States)

    2001-03-01

    At the Agricultural Genetics Institute (AGI), Hanoi, Vietnam, a number of thermo-sensitive genic male sterility (TGMS) homozygous rice lines have been developed by means of experimental mutagenesis followed by anther culture techniques. One of them (TGMS-1 indica mutant line) was used in this research. The critical temperature (at the period from pollen mother cell formation to the beginning of meiotic division) for TGMS-1 sterility was 24-25degC, below which the plants were fertile and above which the plants became sterile. Segregation analysis showed that the TGMS trait of the TGMS-1 mutant line was controlled by a single recessive gene. An F{sub 2} mapping population from a cross between TGMS-1 mutant line and CH1 (a fertile indica line) was developed for tagging and mapping the TGMS gene. From survey of 200 AFLP primer combinations in a bulked segregant analysis, 4 AFLP markers (E2/M5-200, E3/M16-400, E5/M12-600 and E5/M12-200) linked to TGMS-1 gene were identified and cloned. All except E2/M5-200 were found to be low-copy number sequences. The marker E5/M12-600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64xAzucena and CT9993xIR62666. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12-600, was sequenced so that a PCR marker can be developed for the use in marker-assisted breeding. The application of TGMS genes to the commercial two-line hybrid rice breeding system was discussed. (author)

  8. Mapping of a rice thermosensitive genic male sterility gene from a TGMS mutant line

    International Nuclear Information System (INIS)

    Vu Duc Quang; Nguyen Van Dong; Pham Ngoc Luong; Tran Duy Quy; Nguyen, Henry T.

    2001-01-01

    At the Agricultural Genetics Institute (AGI), Hanoi, Vietnam, a number of thermo-sensitive genic male sterility (TGMS) homozygous rice lines have been developed by means of experimental mutagenesis followed by anther culture techniques. One of them (TGMS-1 indica mutant line) was used in this research. The critical temperature (at the period from pollen mother cell formation to the beginning of meiotic division) for TGMS-1 sterility was 24-25degC, below which the plants were fertile and above which the plants became sterile. Segregation analysis showed that the TGMS trait of the TGMS-1 mutant line was controlled by a single recessive gene. An F 2 mapping population from a cross between TGMS-1 mutant line and CH1 (a fertile indica line) was developed for tagging and mapping the TGMS gene. From survey of 200 AFLP primer combinations in a bulked segregant analysis, 4 AFLP markers (E2/M5-200, E3/M16-400, E5/M12-600 and E5/M12-200) linked to TGMS-1 gene were identified and cloned. All except E2/M5-200 were found to be low-copy number sequences. The marker E5/M12-600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64xAzucena and CT9993xIR62666. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12-600, was sequenced so that a PCR marker can be developed for the use in marker-assisted breeding. The application of TGMS genes to the commercial two-line hybrid rice breeding system was discussed. (author)

  9. Effect of sowing dates on yield and yield components on mutant-cum-hybrid lines of bread wheat

    International Nuclear Information System (INIS)

    Sial, M.A.; Arain, M.A.; Dahot, M.U.; Laghari, K.A.; Naqvi, M.H.; Markhand, G.S.; Mangrio, S.M.; Mirbahar, A.A.

    2010-01-01

    Twenty-one stable wheat mutant lines along with four check varieties viz., Sarsabz, Kiran-95, T.J.83 and Khirman were evaluated under normal and late sowing dates. The observations were recorded on phenological, morphological and meteorological parameters. Higher yield and improvement in various yield components were recorded at normal sowing as compared to late sowing. Six mutant lines showed superiority in yield than check varieties at normal sowings while three mutants produced more yield than check varieties except Sarsabz at late sowings. At normal sowing eleven mutant lines matured earlier than all check varieties including short duration variety T.J-83 whereas two mutant lines were earlier than Sarsabz and Kiran-95 and thirteen than T.J-83 and Khirman. (author)

  10. Resistance to Phytophthora in mutant lines of currant tomato and in their original forms

    International Nuclear Information System (INIS)

    Khrustaleva, V.V.; Shcherbakov, V.K.

    1987-01-01

    Information on the production of currant tomato mutants is contained in a previous report. Evaluation of fruit resistance against Phytophthora infestans (Mont.) de Bary was carried out with pathotypes T 0 and T 1 . For artificial infection we used mainly a culture of T 1 (isolate 275), supplied by the Byelorussian Scientific Research Institute of Potato, Fruit and Vegetable Growing at Samokhvalovich. As inoculum for T 0 , a local population of the potato pathotype from the village of Shebantsevo, Moscow province was used. The standard variety 'Gruntovyj gribovskij 1180' was used as the control. Green fruits were taken from the first or second raceme of 20 plants. They were inoculated by spraying in plastic cuvettes with moist filter paper. The cuvettes were covered with glass and maintained at temperature of 18-20 deg. C. The results were checked 5, 9 and 12 days after inoculation. Under natural conditions, each of the 20 plants was also evaluated. As result, three lines with increased resistance to Phytophthora were selected from the original wild-type of currant tomato. Induced mutant forms were tested in the same way for resistance to Phytophthora. Data is presented from 4 years study. Of 26 mutant lines studied, we identified seven whose fruit displayed a stable and enhanced resistance to Phytophthora under both laboratory and field conditions. With regard to leaf infection of these lines, positive results were not obtained. There appears to be no direct relationship between resistance to Phytophthora of the fruit and the leaves. The mutant lines are of determinate type with early and medium ripening time. The average fruit weight is 5-33 g; in the case of the original specimen, it is only 0.9-1.7 g. The fruits have a pleasant sour-sweet taste and a thick skin. It is noteworthy that the mutant lines selected on the basis of their suitability for cultivation not only showed the resistance selected from the wild-type, but in a number of cases even turned out to

  11. Recombinant lines for less-spininess in steroid-bearing Solanum viarum using induced mutants as parents

    International Nuclear Information System (INIS)

    Krishnan, R.; Nanda Kumar, D.; Subhas Chander, M.

    1988-01-01

    In the domestication of the wild, spinous and steroid-bearing Solanum viarum (syn. S. khasianum var. chatterjeeanum) induced mutations play a major role. The development of Glaxo and BARC mutants catalysed commercial cultivation of this species for its berries containing solasodine, used in steroid industries. The commercially more popular Glaxo mutant population consists predominantly of plants that are totally free of spines in aerial parts except lamina where few straight spines develop. The BARC mutant still possesses spines on aerial parts including the persistent calyx. However, the laminary spines of the BARC mutant are curved and vestigial. Comparative studies on morphology, growth behaviour and agronomic characters of the two mutants, their wild progenitor and their hybrid progenies showed that the three types differ only for spine character. In F 2 generation of a cross involving the Glaxo and BARC mutants, a double mutant recombinant was recovered. The recombinant is devoid of spines in aerial parts like its Glaxo mutant parent, but possesses laminary curved vestigial spines like the BARC parent. The spine characters of the recombinant are inherited double recessive. Three advanced lines of this recombinant type (IIHR 2n - 1,2 and 3) were tested in replicated trials 1985 and 1986. They showed parity in berry yield and solasodine content with the Glaxo mutant and three promising lines evolved elsewhere viz. 'RRL (Bhuhaneswar) Y-14', 'RRL (Jorhat)' and 'Pusa'. The results indicate gainful use of induced mutants in hybridization leading to development of superior less-spiny lines of steroid bearing Solanum viarum

  12. Radiation and thermal characteristics of mouse lymphoma cells and their radiation-sensitive mutant

    International Nuclear Information System (INIS)

    Baba, Yuji; Yasunaga, Tadamasa; Uozumi, Hideaki; Takahashi, Mutsumasa; Sawada, Shozo.

    1988-01-01

    Radiation and thermal characteristics of L5178Y cells and their radiation-sensitive mutant M10 cells were studied by the colony-forming method and the dye-exclusion method using eosin-Y. Although M10 cells were remarkably radiation-sensitive compared with L5178Y cells, it was diffcult to cause interphase death of M10 after a large dose of irradiation. After heat treatments, L5178Y cells revealed more cell destruction and were stained well by eosin-Y, but it was relatively difficult to produce cell destruction of M10 cells, which showed poor staining by eosin-Y. When assayed by the colony-forming method, M10 cells were also heat-resistant compared to L5178Y. The dye-exclusion rate was closely correlated with cell survival after hyperthermia of L5178Y cells, suggesting that this is a simple method of detecting the thermosensitivity and thermotolerance of cancer cells. The difference in survival of L5178Y cells and M10 cells after combined treatment with gamma irradiation and hyperthermia was smaller than with gamma irradiation alone. It was also found that there was a relationship between radiation-induced interphase death and hyperthermia-induced interphase death, and that interphase death accounted for a major part of cell death caused by hyperthermia in mouse leukemia cells. (author)

  13. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

    OpenAIRE

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2016-01-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

  14. The first mouse mutants of D14Abb1e (Fam208a) show that it is critical for early development

    Czech Academy of Sciences Publication Activity Database

    Harten, S.K.; Bruxner, T.J.; Bharti, V.; Blewitt, M.; Nguyen, T.M.T.; Whitelaw, E.; Epp, Trevor

    2014-01-01

    Roč. 25, 7-8 (2014), s. 293-303 ISSN 0938-8990 Institutional support: RVO:68378050 Keywords : embryogenesis * forward genetics * mouse mutant Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.068, year: 2014

  15. Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism.

    Science.gov (United States)

    Ju, Anes; Hammerschmidt, Kurt; Tantra, Martesa; Krueger, Dilja; Brose, Nils; Ehrenreich, Hannelore

    2014-08-15

    Neuroligin-4 (Nlgn4) is a member of the neuroligin family of postsynaptic cell adhesion molecules. Loss-of-function mutations of NLGN4 are among the most frequent, known genetic causes of heritable autism. Adult Nlgn4 null mutant (Nlgn4(-/-)) mice are a construct valid model of human autism, with both genders displaying a remarkable autistic phenotype, including deficits in social interaction and communication as well as restricted and repetitive behaviors. In contrast to adults, autism-related abnormalities in neonatal and juvenile Nlgn4(-/-) mice have not been reported yet. The present study has been designed to systematically investigate in male and female Nlgn4(-/-) pups versus wildtype littermates (WT, Nlgn4(+/+)) developmental milestones and stimulus-induced ultrasound vocalization (USV). Neonatal development, followed daily from postnatal days (PND) 4 to 21, including physical development, neurological reflexes and neuromotor coordination, did not yield any differences between Nlgn4(-/-) and their WT littermates. USV in pups (PND8-9) in response to brief separation from their mothers revealed remarkable gender effects, and a genotype influence in females regarding latency to first call. In juveniles (PND22-23), USV monitoring upon exposure to an anesthetized female intruder mouse uncovered a clear genotype effect with reduced USV in Nlgn4(-/-) mice, and again a more prominent phenotype in females. Together, these data support an early manifestation of communication deficits in Nlgn4(-/-) mice that appear more pronounced in immature females with their overall stronger USV as compared to males. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.

    Science.gov (United States)

    Ihara, Tatsuya; Mitsui, Takahiko; Nakamura, Yuki; Kira, Satoru; Miyamoto, Tatsuya; Nakagomi, Hiroshi; Sawada, Norifumi; Hirayama, Yuri; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Yoichi; Yoshiyama, Mitsuharu; Andersson, Karl-Erik; Nakao, Atsuhito; Takeda, Masayuki; Koizumi, Schuichi

    2017-04-01

    The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (Clock Δ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP. Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 Clock Δ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods. No significant differences were observed in behavior patterns between Clock Δ19/Δ19 and WT mice. Clock Δ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in Clock Δ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in Clock Δ19/Δ19 mice than in WT mice. We demonstrated that Clock Δ19/Δ19 mice showed the phenotype of NOC/NP. The Clock Δ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Agronomic and molecular evaluation of induced mutant rice (oryza sativa l.) lines in Egypt

    International Nuclear Information System (INIS)

    Sshehzad, T.; Allah, A.; Aallah, E.A.; Ammar, M.H.; Abdelkhalik, A.H.

    2011-01-01

    The present study was conducted at the farm of the Rice Research and Training Center, Sakha, Kafr El-Sheikh, Egypt, during 2000-2007 rice sowing seasons. Five rice varieties viz., Giza 171, Giza 175, Giza 176, Giza 181 and GZ 1368 were the most widely grown Japonica and Indica types in Egypt during the last period, possesses at that time many positive agronomic characteristics including wide adaptability, high yield potential, tolerance to stresses and good eating quality. But with the passage of time it has lost its vigor. In Rice Research Program, Egypt, dry seeds of the above mentioned varieties were treated with different doses of gamma rays (100, 200, 300, 400, and 500 Gy) for raising M1 generation. M1 plants were established by transplanting in the year 2000 season. One hundred independent lines have been advanced to M5 generation enabling evaluation of quantitative traits by replicated trials and promising lines were selected and tested in multi-location trials as M6, M7 and M8 generations. Morphological variations at vegetative and reproductive stages including plant type and various physiological characters were observed in the five populations. The mutant lines characteristics consisted of better resistance to lodging, blast disease, high yield potential, as well as early maturity. Results from yield trials and molecular assessments indicated that the mutants differed genetically from their parents. So, these mutants could be used as a donor parents in rice breeding program and some of them can be recommended as new rice varieties suitable for rice belt in Egypt. (author)

  18. Productivity and Nutrient Quality of Some Sorghum Mutant Lines at Different Cutting Ages

    Directory of Open Access Journals (Sweden)

    R. E. Puteri

    2015-08-01

    Full Text Available The objective of the study was to explore the appropriate cutting age to produce optimal biomass and good nutrient quality from sorghum mutant lines BMR i.e., PATIR 3.5 M7, PATIR 3.6 M7, and PATIR 3.7 M7, also SAMURAI I (M17. A completely randomized in Split Plot design with 2 factors and 3 replicates was used. The first factor was the type of sorghum (SAMURAI I M17, PATIR 3.5, PATIR 3.6, PATIR 3.7 as the main plot and the second factor was the cutting age (85, 95, 105 as a subplot. Parameters observed were the production of stems, leaves, grains, total biomass production, ash, crude fat, crude fiber, crude protein, NFE, TDN, percentage of DMD, OMD and N-NH3. Data were analyzed by using ANOVA followed by DMRT (Duncan Multiple Range Test. The results showed that there were highly significant interactions (P<0.01 between cutting age and type of sorghum in production of stems, leaves, grains, total biomass production, value of TDN, DMD, OMD, and N-NH3. Increasing cutting age significantly increased the percentage of ash content, crude protein and crude fat. The sorghum type significantly affected crude fat content nonBMR sorghum variety of SAMURAI I (M17 and achieved optimal biomass production and nutrient content at cutting age of 85 d similar to BMR sorghum mutant lines PATIR 3.6 and PATIR 3.5, whereas BMR sorghum mutant lines of PATIR 3.7 achieved optimum production at the age of 95 d of cutting. All types of sorghum varieties was not recommended to be harvested at 105 d. Biomass production increased with the increasing of cutting age, but the nutrient content decreased.

  19. Evaluating Genetic Variability of Sorghum Mutant Lines Tolerant to Acid Soil

    International Nuclear Information System (INIS)

    Puspitasari, W.; Human, S.; Wirnas, D.; Trikoesoemaningtyas

    2012-01-01

    High rainfall in some parts in Indonesia causes soil become acidic. The main constraint of acid soil is phosphor (P) deficiency and aluminum (Al) toxicity which decrease plant productivity. To overcome this problem, it is important to develop a crop variety tolerant to such conditions. Sorghum is probably one of the potential crops to meet that objective. Sorghum has been reported to have wide adaptability to various agro-ecology and can be used as food and animal feed. Unfortunately, sorghum is not Indonesian origin so its genetic variability is still low. From previous breeding works with induced mutation, some promising mutant lines have been developed. These mutant lines were included in the experiment carried out in Tenjo with soil condition was classified as acid soil with pH 4.8 and exchangeable-Al content 2.43 me/100 g. The objectives of this experiment were to study the magnitude of genetic variability of agronomy and grain quality characters in sorghum in order to facilitate the breeding improvement of the species. Plant materials used in this study were ten genotypes, including 6 mutant lines and 4 control varieties. The randomized block design with three replications was used in the experiment. The genetic variabilities of agronomic and grain quality characters existed among genotypes, such as plant height, number of leaves, stalk diameter, biomass weight, panicle length, grain yield per plant, 100 seed weight and tannin content in the grain. The broad sense heritabilities of agronomic characters were estimated ranging from medium to high. Grain yield showed significantly positive correlation with agronomic characters observed, but it was negatively correlated with protein content (author)

  20. Behavioral Actions of Alcohol: Phenotypic Relations from Multivariate Analysis of Mutant Mouse Data

    Science.gov (United States)

    Blednov, Yuri A.; Mayfield, R. Dayne; Belknap, John; Harris, R. Adron

    2012-01-01

    Behavioral studies of genetically diverse mice have proven powerful for determining relationships between phenotypes and have been widely used in alcohol research. Most of these studies rely on naturally occurring genetic polymorphisms among inbred strains and selected lines. Another approach is to introduce variation by engineering single gene mutations in mice. We have tested 37 different mutant mice and their wild type controls for a variety (31) of behaviors and have mined this dataset by K-means clustering and analysis of correlations. We found a correlation between a stress-related response (activity in a novel environment) and alcohol consumption and preference for saccharin. We confirmed several relationships detected in earlier genetic studies including positive correlation of alcohol consumption with saccharin consumption, and negative correlations with conditioned taste aversion and alcohol withdrawal severity. Introduction of single gene mutations either eliminated or greatly diminished these correlations. The three tests of alcohol consumption used (continuous two bottle choice, and two limited access tests: Drinking In the Dark and Sustained High Alcohol Consumption) share a relationship with saccharin consumption, but differ from each other in their correlation networks. We suggest that alcohol consumption is controlled by multiple physiological systems where single gene mutations can disrupt the networks of such systems. PMID:22405477

  1. RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

    International Nuclear Information System (INIS)

    Wang Tiegu; Huang Qunce; Feng Weisen

    2007-01-01

    Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning

  2. RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

    Science.gov (United States)

    Wang, Tiegu; Huang, Qunce; Feng, Weisen

    2007-10-01

    Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

  3. RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

    Energy Technology Data Exchange (ETDEWEB)

    Tiegu, Wang [Henan Provincial Key Laboratory of Ion Beam Bio-Engineering, Zhengzhou University, Zhengzhou 450052 (China); Qunce, Huang [Henan Provincial Key Laboratory of Ion Beam Bio-Engineering, Zhengzhou University, Zhengzhou 450052 (China); Weisen, Feng [Luoyang Institute of Agricultural Science, Luoyang 471022 (China)

    2007-10-15

    Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

  4. Induction of mutations in Thai rice varieties and subsequent selection and testing of beneficial mutant lines

    Energy Technology Data Exchange (ETDEWEB)

    Dasananda, S; Khambanonda, P [Ministry of Agriculture, Bangkok (Thailand)

    1970-03-01

    Ionizing radiations were first used in the Thailand Rice Breeding Program in 1955 when seeds of two recommended varieties were sent to the United States of America for treatment. As a result, five promising mutant lines are at present in regional yield tests where they are being considered for recommendation to rice growers. During the period 1960-1961 an unsuccessful attempt was made to induce resistance to blast in three susceptible varieties by exposing seeds to a local source of ionizing radiation In 1964, after an elapse of about 4 years, another attempt was made to utilize ionizing radiations in the breeding program by treating seeds of two recommended varieties. In 1965, a co-ordinated rice mutation breeding program was initiated under the auspices of the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture which resulted in treating seeds of twelve different rice varieties with both ethyl methane sulphonate and gamma rays from a {sup 60}Co gamma cell. The results so far indicate that mutagenic agents have been successful in producing genetic variability. Differences in heading date, mature plant height and plant type are frequently observed in the M{sub 2} and M{sub 3} generations. Several lines obtained from two of the irradiated varieties have exhibited a higher degree of resistance to blast than the parental material. From 15-kR treatments of non-glutinous varieties, mutants with glutinous endosperm have been obtained. Not all varieties gave the same response to treatment. (author)

  5. The characteristics of high-yield genotype of early-mature mutant lines in barley

    International Nuclear Information System (INIS)

    Chen Xiulan; Han Yuepeng; He Zhentian; Yang Hefeng

    2000-01-01

    The correlation and genetic parameters of eight agronomic traits of 36 early mature mutant lines induced from barley Sunong 9052 were studied by stepwise regression and path analysis. The results showed that: (1) the growing period of early mutants was shortened 2-13 days from that of their parent and the trait of yield had a great mutation range; (2) the number of grain per panicle significantly correlated with the days from sowing to heading; (3) according to direct path coefficients, the main characters related with individual plant-yield were in order of productive panicle per plant > 1000-grain-weight > number of grain per panicle > fertility, the high-yield genotype had more productive panicle and higher 10000-grain-weight, and to increase the yield in the breeding of early mature mutation was to select the lines with more tillers and productive panicles, higher 1000-grain-weight and lower number of grain per panicle; (4) the higher broad-sense heritability and genetic variation coefficient were found in 1000-grain-weight and the days from sowing to heading

  6. Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

    Science.gov (United States)

    Muthusamy, Annamalai; Jayabalan, Narayanasamy

    2013-01-01

    The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

  7. A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

    Science.gov (United States)

    Bouhy, Delphine; Juneja, Manisha; Katona, Istvan; Holmgren, Anne; Asselbergh, Bob; De Winter, Vicky; Hochepied, Tino; Goossens, Steven; Haigh, Jody J; Libert, Claude; Ceuterick-de Groote, Chantal; Irobi, Joy; Weis, Joachim; Timmerman, Vincent

    2018-01-01

    Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces

  8. Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background.

    Science.gov (United States)

    Carstea, Ana Claudia; Pirity, Melinda K; Dinnyes, Andras

    2009-12-31

    In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

  9. Evaluate The Fluctuation Of Phytic Acid Content In Seed From Mutant Lines By Gamma Ray

    International Nuclear Information System (INIS)

    Nguyen Thi Lang; Pham Van Ut

    2011-01-01

    Phytic acid is a molecule composed of myo-inositol 1,2,3,4,5,6 hexakis dihydrogen phosphate (Ins P6), a major component in the source of phosphorus (P) reserves of about 50 plants - 80% of total seed phosphorus (Lott, 1984). At physiological pH in the form of phytic acid have negatively charged ions hold together the complex mineral nutrition creates indigestion. Moreover, phosphorus in the form of phytate or phytic humans and monogastric animals can not absorb, are all discharged polluted environment transitions. In rice OM819, OM4900, OM3536, D4 and D8 are irradiated with gamma rays at 5 doses: 100, 200, 300, 400 and 500 Gy to create mutant strains with low levels of phytic acid. Results in radiation levels may appear 100 Gy line grain phytic acid expression is low. At the level 200 Gy of radiation is three populations OM819, OM4900 and OM3536 with 8 lines for grain phytic acid expression is low. At 300 Gy extent, appeared seven lines with low nuclear expression of phytic acid 4 populations OM819, OM4900, OM3536 and D4. At the level 400 Gy of radiation there are 4 populations appear only 5 lines expressed low phytic acid, with 3-line expression levels 3 and 2 lines with level 4 expression. At the level of radiation 500 Gy only one line appears at level 3 of phytic acid this is OM819 populations. For genotype analysis using marker RM 261 with 66.67% of the rice low phytic acid content of the expression analysis of biochemical polymorphisms. (author)

  10. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

    Science.gov (United States)

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2017-03-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

  11. Identification of the second mutation of BADH2 gene derived from rice mutant lines induced by gamma rays

    International Nuclear Information System (INIS)

    I Ishak

    2016-01-01

    The BADH2 gene acts as suppressor of 2-acetyl-1-pyrolline (2AP) biosynthesis in plants. 2AP is the volatile compound which provides fragrance in rice. Biosynthesis of 2AP occurs when BADH2 loses its function as suppressor gene. Aromatic rice cultivars naturally incur mutation of BADH2 gene at 8 bp. In this experiment, aromatic mutant rice lines derived from irradiation of Sintanur cultivar by gamma rays with dose of 100 Gy were studied in molecular level. These mutant lines were characterized at the M10 plantgeneration under the assumption that genetically these aromatic mutant rice lines were homozygotic. Several primers related to aroma in rice have been used for polymerase chain reaction (PCR) in a thermal cycler instrument. Gel electrophoreses were carried out using 1.5% agarose in TAE buffer. DNA fragments at 254 bp and 355 bp (base pair) were taken and amplified by primer for nucleotide sequencing of these fragments. Molecular identification and characterization after electrophoresis showed that the mutant line from AR1020 can be differentiated from AR.1080 at 254 bp. Nucleotide sequence data from of these DNA fragments showed that point mutations (deletions and substitutions) occurred at the BADH2 gene in exon 7; those are called second mutation and were caused by gamma rays effects. The Sintanur variety was used as check cultivar and its DNA sequence was compared to that of the AR.1020 mutant line. The results from both DNA sequences (from cv. Sintanur and AR.1020) derived from fragments at 254 bp show that point mutations occurred within exon 7 and earlier stop codon occurred in the AR.1020 mutant rice line. Further, the use of EA primer in PCR resulted in detection of deletion and substitution of nucleotides in the AR.1020 mutant line. (author)

  12. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models

    Directory of Open Access Journals (Sweden)

    Sara eAmorim-Vaz

    2015-05-01

    Full Text Available The aim of the present study was to identify C. albicans transcription factors (TF involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens quantified in kidneys. Mutants of unannotated genes which generated a kidney fungal burden significantly different from that of wild-type were selected and individually examined in G. mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25 % of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects, a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching fungal burden phenotypes were observed in 50 % of the cases, highlighting the bias due to host effects. In contrast, 33.4 % concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the pool effect. After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adaptation.

  13. Main agronomic traits and resistance to rice blast of space-induced mutant lines of Zhong-er-ruan-zhan

    International Nuclear Information System (INIS)

    Xiao Wuming; Wang Hui; Liu Yongzhu; Guo Tao; Chen Zhiqiang; Yang Qiyun; Zhu Xiaoyuan

    2012-01-01

    The main agronomic traits and resistance to rice blast of 34 space-induced lines from an elite rice cultivar, Zhong-er-ruan-zhan were evaluated at their SP 4 . The resistance to blast of the mutant lines had been tested by two blast isolates previously. It was found that the mutant lines showed significant difference in plant height, effective panicles, panicle length and grains per panicle etc. from their parent. The range of variation in 1000-grain weight the largest, followed by the seed-setting rate, and that of effective panicles was the least among all the traits. Except for the line Z34, 33 mutant lines had broader resistance spectra than the wild-type based on the test with 38 different blast isolates, and all the 33 lines were also resistant to the panicle blast in the field. The result confirmed that selection for resistant to blast in lower generations was reliable. Taking account of agronomic traits and blast resistance, promising lines with resistance to blast and good agronomic characters could be selected from those mutant lines. Therefore, the elite rice germplasm with enhanced disease resistance can be produced. (authors)

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

    Science.gov (United States)

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

    2015-02-01

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

  15. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  16. Yield of two mutant lines of soybean for human consumption;Rendimiento de dos lineas mutantes de soya para consumo humano

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Mastache L, A. A.; Diaz V, G. E.; Valencia E, F.; Ranfla C, R.; Melendez P, M. [Colegio Superior Agropecuario del Estado de Guerrero, Vicente Guerrero No. 81, Col. Centro, 40000 Iguala, Guerrero (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad, Colegio de Postgraduados, Carretera Mexico-Texcoco Km. 36.5, Montecillo, 56230 Texcoco, Estado de Mexico (Mexico); De la Cruz T, E.; Garcia A, J. M.; Falcon B, T., E-mail: csaegro@prodigy.net.m [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, Ocoyoacac 52750, Estado de Mexico (Mexico)

    2009-07-01

    The present work has the objective of to evaluate the yield and the agronomic behavior of 2 mutant lines of soybean for human consumption, obtained by means of a process of recurrent irradiation of soybean seed ISAAEG-BM{sub 2} with gammas of Co{sup 60} and selection in the generation R{sub 4}M{sub 18}. For the variable yield significant statistical differences were not observed, but considering the rest of the evaluated agronomic characteristics the mutant lines L{sub 6} and Bombona they were excellent with values of 3,934.6 and 3,806.8 Kg ha-{sup 1} to 15% of grain humidity, they also possess excellent genetic characteristics result of the irradiations and selections of these new genetic materials. (Author)

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

    Science.gov (United States)

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

    2012-01-01

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

  18. Imidacloprid does not induce Cyp genes involved in insecticide resistance of a mutant Drosophila melanogaster line.

    Science.gov (United States)

    Kalajdzic, Predrag; Markaki, Maria; Oehler, Stefan; Savakis, Charalambos

    2013-10-01

    Certain xenobiotics have the capacity to induce the expression of genes involved in various biological phenomena, including insecticide resistance. The induction potential of different chemicals, among them different insecticides, has been documented for a number of insect species. In this study, we have analyzed the induction potential of Imidacloprid, a widely used member of the neonicotinoid insecticide family. Genes Cyp6g1 and Cyp6a2, known to be involved in the resistance of mutant Drosophila melanogaster line MiT[W⁻]3R2 to Imidacloprid and DDT were included in the analyzed sample. We find that Imidacloprid does not induce expression of the analyzed genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Multilocation trial of potential selected mutant lines of groundnut (arachis hypogaea) at 3 location in Peninsular Malaysia

    International Nuclear Information System (INIS)

    Abdul Rahim Harun; Rusli Ibrahim; Khairuddin Abdul Rahim; Shuhaimi Shamsuddin

    2002-01-01

    Two fixed mutant lines of groundnut derived from cultivar Matjan were selected for their yield potential at M 1 0 generation. Multilocation trial of these mutants (MJ40/42 and MJ20/165-5) was carried out to evaluate genotype stability at different climate and soil types in Peninsular Malaysia. The mutant lines were planted and compared with their parent (Matjan) and control variety (MKT1). The identified locations were in Taiping (Perak), Machang (Kelantan), and Air Hitam (Johor). The soils at the locations were of the Serdang, Bungor and Rengam series, respectively. The trial was carried out simultaneously in the same year at each location. Mutant MJ20/165-5 showed stable performance at all location compared to other genotypes tested. Its yield was higher than the parent in Kelantan and Johor trial and showed similar performance in Perak. This mutant also showed better yield performance than the control varieties in the Kelantan trial. Meanwhile, mutant line MJ40/42 gave better yield in Kelantan and Johor but did not perform well in Perak as compared to its parent and control varieties. (Author)

  20. The Aβ6w302 gene and molecular mechanisms of resistance to the spread of lymphoma in a mouse mutant, Survivor-27

    International Nuclear Information System (INIS)

    Liu, Y.; Savinov, A.Yu.; McMinimy, D.L.; Kremlev, S.G.; Chapoval, A.I.; Egorov, I.K.

    2000-01-01

    The Aβ6 w302 gene and molecular mechanisms of resistance to the spread of lymphoma induced by γ-radiation ( 60 Co) in a mouse mutant, survivor-27 were studied. Metastatic tumors escape from immune response and spread in the body; survivors are very rare. Novel single exon genes Aβ4-7 and a pseudogene Aβ8Ψ have been cloned from survivors. Their protein coding sequences are similar major histocompatibility complex (MHC) class II β H2-Ab cDNA while their promoter is different from MHC promoters. The Aβ4 protein was demonstrated on macrophages (antigen presenting cells). The Aβ gene family is genetically unstable in germ line and somatic cells of survivors. Mutants S-27 and S-87/1 lost the Aβ5 s5 and acquired the Aβ6 w302 gene; the Ab gene mutated in S-27. The proposed mechanism of resistance is molecular instability of the Aβ gene family resulting in somatic mutations and wandering immune responses that destroy the tumor in the survivor [ru

  1. Evaluation of Drought response in Some Rice Mutant Lines Using Stress Tolerance Indices

    Directory of Open Access Journals (Sweden)

    H Aminpanah

    2018-05-01

    Full Text Available Introduction Drought is a major problem that limits the adoption of high-yielding rice varieties in drought-prone rainfed rice environments. To improve crop productivity, it is necessary to understand the mechanism of plant responses to drought conditions with the ultimate goal of improving crop performance in the vast areas of the world where rainfall is limiting or unreliable. Safaei Chaeikar et al. (2008 reported that MP, GMP, HM and STI indices, which showed the highest correlation with grain yield under both optimal and stress conditions, can be used as the best indices to introduce drought-tolerant genotypes in rice breeding programs. They also were introduced Nemat, Sepidrood, IR64, IR50 and Bejar genotypes as tolerant varieties. The present study was conducted to determine how drought affects grain yield in rice mutant lines and also to test this hypothesis in order to identify the most suitable indices/genotypes. Materials and Methods A field trial was conducted at Iranian Rice Research Centers in North of Iran, Rasht (latitude 37◦28', longitude 49◦28'E and altitude 7m below the sea level, during the 2014-2015 growing season. The seeds were sown in a nursery on the 10 May and 25 day old seedlings were transplanted to the field. Two separately experiment was carried out under reproductive stage drought stress and controlled conditions based on randomized complete block design with three replications, in four-row plots of three m length. Transplanting was done using 1 seedling per hill; at hill spacing of 25 cm × 25 cm. 18 rice genotypes were consisted 14 M5 mutant lines and their four parental cultivars. Results and Discussion Analysis of variance indicated significant effects of drought stress, genotype and interaction effects of two factors on grain yield, plant height, flag leaf area, tiller number and grain fertility percentage. Drought stress at reproductive stage caused reduction in grain yield (59.47%, grain fertility

  2. Tauopathic changes in the striatum of A53T α-synuclein mutant mouse model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Jonathan Wills

    2011-03-01

    Full Text Available Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3β, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3β and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3β was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3β co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of

  3. Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

    International Nuclear Information System (INIS)

    Sato, K.; Hieda, N.

    1980-01-01

    The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Nicolas Lecland

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

    DEFF Research Database (Denmark)

    Roszko, Kelly L; Bi, Ruiye; Gorvin, Caroline M

    2017-01-01

    in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated...... human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11...

  7. Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1.

    Science.gov (United States)

    Guo, L-J; Luo, X-H; Xie, H; Zhou, H-D; Yuan, L-Q; Wang, M; Liao, E-Y

    2006-05-01

    We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

  8. Generation of gamma irradiation and EMS-induced mutant lines of the H7996 tomato (Solanum lycopersicum L.)

    International Nuclear Information System (INIS)

    Canama, Alma O.; Galvez, Hayde F.; Tongson, Eden Jane U.; Quilloy, Reynaldo B.; Hautea, Desiree M.

    2010-01-01

    Tomato (L.) is one of the most important vegetable crops grown worldwide for the fresh vegetable market and food processing industry. With the completion of the genome-sequencing projects in various crops, the major challenge will be determine the gene function. One approach is to generate and to analyze mutant phenotypes. The paper reports the generation of gamma-irradiated and ethy methane sulfonate (EMS)-treated mutant populations, identification and phenotypic characterization of dominant and visible mutations in tomato mutant lines. Mutant populations of tomato H7996 were created using physical (cobalt 60 gamma ray) and chemical EMS mutagens. Generally, based on high-throughput phenotypic characterization, mutations were observed on the plant habit, size, morphology, leaf and flower color and morphology and fruit characteristics. Specifically, the most common dominant and visible mutations noted in the M 1 generation were monopodial, compact, short internodes, multi-branch plant type, light yellow and ghost leaf coloration, tiny and long pedicel leaf morphology and small or short plant size. In the M2 generation, homogeneous and segregating M 2 families were selected to constitute the core set of visible tomato mutants. Initial bacterial wilt resistance (BWR) gene knockouts were also identified. The mutant lines will be used as a rich source of genetic materials for breeding and functional genomics of tomato. (author)

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  10. Field performance of thirty mutant lines of the rice (Oryza sativa L.) varieties ICTA-Virginia and Precoz-ICTA

    International Nuclear Information System (INIS)

    Montepeque, R.; Molina, L. G.; Lopez, J. J.; Pazos, W.; Ramirez, J.

    1993-01-01

    Fifteen mutant lines from the variety ICTA-Virginia and fifteen from the variety Precozicta were evaluated according to their agronomic characteristics under conditions of the Motagua river valley during 1992. The objective was to select genotypes showing resistance to disease caused by Pyricularia grisea. The analysis of variance did not show significative differences among ICTA-Virginia mutants. The highest yield was form MV-860, 8.17 TM/ha and the lowest 5.31 TM/ha for MV-411. Significant differences were found among mutant lines from Precozicta. The highest yields were 6.06, 5.80 and 5.52 TM/ha for MPI-1189, MPI-1664 and MPI-1346 respectively. Inoculation with Pyricularia was made spraying it over the crop. However, it was not possible the evaluation of the disease in the neck (neck blast) due to absence of the pathogen. 5 tabs.(Author)

  11. Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

    OpenAIRE

    Miyaji, E.N.; Johnson, R.T.; Downes, C.S.; Eveno, E.; Mezzina, M.; Sarasin, A.; Menck, C.F.M.

    2000-01-01

    Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, po...

  12. Development of technique on the induction and selection of in vitro mutant lines(Potato, Solanum tuberosum L.)

    International Nuclear Information System (INIS)

    Hong, Joo Bong; Lee, Young Il; Song, Hee Sup; Kim, Jae Sung; Byun, Myung Woo; Lee, Young Keun; Shin, In Chul; Lee, Sang Jae; Lee, Ki Woon; Lim, Yong Taek

    1992-08-01

    The radiosensitivity and salt resistance on the single cell and callus of potato, mass production method of plantlet and microtuber of potato by in vitro culture and microtuber formation from the stem irradiated with radiation were investigated to obtain a optimum condition for selection of mutant cell line. (Author)

  13. Chromosome-wise dissection of the genome of the extremely big mouse line DU6i

    NARCIS (Netherlands)

    M.R. Bevova (Marianna); Y.S. Aulchenko (Yurii); G. Aksu (Guzide); U. Renne (Ulla); K. Brockmann

    2006-01-01

    textabstractThe extreme high-body-weight-selected mouse line DU6i is a polygenic model for growth research, harboring many small-effect QTL. We dissected the genome of this line into 19 autosomes and the Y chromosome by the construction of a new panel of chromosome substitution strains (CSS). The

  14. Preferência de Bemisia tabaci biótipo B em linhagens mutantes de algodoeiro Bemisia tabaci biotype B preference in mutant cotton lines

    Directory of Open Access Journals (Sweden)

    Francisco das Chagas Vidal Neto

    2008-02-01

    Full Text Available Os efeitos de caracteres mutantes morfológicos do algodoeiro (Gossypium hirsutum L. r. latifolium Hutch.: folha okra, bráctea frego e planta vermelha, em relação à resistência à mosca-branca (Bemisia tabaci biótipo B Hemiptera: Aleyrodidae, foram avaliados em experimentos com ou sem chance de escolha. Os experimentos foram conduzidos em casa-de-vegetação, no delineamento de blocos ao acaso, em fatorial 23 + 1, com quatro repetições. O mutante com a característica planta vermelha foi menos atrativo e menos preferido para oviposição, em relação à planta verde, em ambos os ensaios, com ou sem escolha. Não houve preferência quanto à forma da folha e ao tipo de bráctea.The effects of cotton lines (Gossypium hirsutum L. r. latifolium Hutch. with mutants morphologic characteristics: okra leaf, frego bract and red plant in relation to host plant resistance to whitefly (Bemisia tabaci bioyipe B Hemiptera: Aleyrodidae, were evaluated in choice or no choice assays. The assays were carried out in the greenhouse conditions, according to a completely randomized block design, in a 23 + 1 in a factorial arrangement with four replications. The mutant with red plant characteristic was less attractive and less preferred for oviposition than the normal green plant does, in both, whit or without choice tests. It did not have preference in relation to the form of the leaf and bract type.

  15. Preclinical Studies of Signaling Pathways in a Mutant Mouse Model of Hormone-Refractory Prostate Cancer

    Science.gov (United States)

    2011-02-01

    intraepithelial neoplasia in the mouse prostate. Cancer Res 63: 8784–8790. Joshua AM, Vukovic B, Braude I, Hussein S, Zielenska M, Srigley J , Evans A, Squire JA...prostate tumors 4. Reportable Outcomes Kinkade, C.W., Castillo-Martin, M., Puzio-Kuter, A., Yan, J ., Foster, T.H., Gao, H., Sun,, Y., Ouyang, X...2) Uzgare, A. R. and Isaacs, J . T. (2004). Enhanced redundancy in Akt and mitogen-activated protein kinase-induced survival of malignant versus

  16. Detection of quantitative trait loci causing abnormal spermatogenesis and reduced testis weight in the small testis (Smt) mutant mouse.

    Science.gov (United States)

    Bolor, Hasbaira; Wakasugi, Noboru; Zhao, Wei Dong; Ishikawa, Akira

    2006-04-01

    The small testis (Smt) mutant mouse is characterized by a small testis of one third to one half the size of a normal testis, and its spermatogenesis is mostly arrested at early stages of meiosis, although a small number of spermatocytes at the late prophase of meiosis and a few spermatids can sometimes be seen. We performed quantitative trait locus (QTL) analysis of these spermatogenic traits and testis weight using 221 F2 males obtained from a cross between Smt and MOM (Mus musculus molossinus) mice. At the genome-wide 5% level, we detected two QTLs affecting meiosis on chromosomes 4 and 13, and two QTLs for paired testis weight as a percentage of body weight on chromosomes 4 and X. In addition, we found several QTLs for degenerated germ cells and multinuclear giant cells on chromosomes 4, 7 and 13. Interestingly, for cell degeneration, the QTL on chromosome 13 interacted epistatically with the QTL on chromosome 4. These results reveal polygenic participation in the abnormal spermatogenesis and small testis size in the Smt mutant.

  17. Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

    Science.gov (United States)

    Suijker, Johnny; Oosting, Jan; Koornneef, Annemarie; Struys, Eduard A; Salomons, Gajja S; Schaap, Frank G; Waaijer, Cathelijn J F; Wijers-Koster, Pauline M; Briaire-de Bruijn, Inge H; Haazen, Lizette; Riester, Scott M; Dudakovic, Amel; Danen, Erik; Cleton-Jansen, Anne-Marie; van Wijnen, Andre J; Bovée, Judith V M G

    2015-05-20

    Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

  18. Development of technique on the induction and selection of in vitro mutant lines (Potato, Solanum tuberosum L.)

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Jang Ryoel; Lee, Yeong Il; Song, Hee Seop; Kim, Jae Seong; Sin, In Cheol; Lee, Sang Jae; Lee, Ki Un; Lim, Yong Taek [Korea Atomic Energy Res. Inst., Taejon (Korea, Republic of)

    1993-09-01

    For the development of the technique on the plant tissue culture and application of nuclear technique in the in vitro mutation breeding, present research laid emphasis on the development of techniques of potato tissue culture, and on the induction and selection of radiation mutation. Another culture for haploid induction, optimum radiation dosage for cybrid formation of potato and mutation induction from in vitro cultured microtuber and plantlets were investigated for modelling the technique on the induction and selection of in vitro mutant lines. Inheritance stability of the selected mutants were also studied in field condition. In vitro system of micropropagation and selection of mutation was summarized.

  19. Development of technique on the induction and selection of in vitro mutant lines (Potato, Solanum tuberosum L.)

    International Nuclear Information System (INIS)

    Yoo, Jang Ryoel; Lee, Yeong Il; Song, Hee Seop; Kim, Jae Seong; Sin, In Cheol; Lee, Sang Jae; Lee, Ki Un; Lim, Yong Taek

    1993-09-01

    For the development of the technique on the plant tissue culture and application of nuclear technique in the in vitro mutation breeding, present research laid emphasis on the development of techniques of potato tissue culture, and on the induction and selection of radiation mutation. Another culture for haploid induction, optimum radiation dosage for cybrid formation of potato and mutation induction from in vitro cultured microtuber and plantlets were investigated for modelling the technique on the induction and selection of in vitro mutant lines. Inheritance stability of the selected mutants were also studied in field condition. In vitro system of micropropagation and selection of mutation was summarized

  20. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  1. Enhanced astrocytic nitric oxide production and neuronal modifications in the neocortex of a NOS2 mutant mouse.

    Directory of Open Access Journals (Sweden)

    Yossi Buskila

    Full Text Available BACKGROUND: It has been well accepted that glial cells in the central nervous system (CNS produce nitric oxide (NO through the induction of a nitric oxide synthase isoform (NOS2 only in response to various insults. Recently we described rapid astroglial, NOS2-dependent, NO production in the neocortex of healthy mice on a time scale relevant to neuronal activity. To explore a possible role for astroglial NOS2 in normal brain function we investigated a NOS2 knockout mouse (B6;129P2-Nos2(tm1Lau/J, Jackson Laboratory. Previous studies of this mouse strain revealed mainly altered immune responses, but no compensatory pathways and no CNS abnormalities have been reported. METHODOLOGY/PRINCIPAL FINDINGS: To our surprise, using NO imaging in brain slices in combination with biochemical methods we uncovered robust NO production by neocortical astrocytes of the NOS2 mutant. These findings indicate the existence of an alternative pathway that increases basal NOS activity. In addition, the astroglial mutation instigated modifications of neuronal attributes, shown by changes in the membrane properties of pyramidal neurons, and revealed in distinct behavioral abnormalities characterized by an increase in stress-related parameters. CONCLUSIONS/SIGNIFICANCE: The results strongly indicate the involvement of astrocytic-derived NO in modifying the activity of neuronal networks. In addition, the findings corroborate data linking NO signaling with stress-related behavior, and highlight the potential use of this genetic model for studies of stress-susceptibility. Lastly, our results beg re-examination of previous studies that used this mouse strain to examine the pathophysiology of brain insults, assuming lack of astrocytic nitrosative reaction.

  2. Pattern of retinal morphological and functional decay in a light-inducible, rhodopsin mutant mouse.

    Science.gov (United States)

    Gargini, Claudia; Novelli, Elena; Piano, Ilaria; Biagioni, Martina; Strettoi, Enrica

    2017-07-18

    Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of Rho Tvrm4 /Rho + rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.

  3. Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism.

    Science.gov (United States)

    Dhamne, Sameer C; Silverman, Jill L; Super, Chloe E; Lammers, Stephen H T; Hameed, Mustafa Q; Modi, Meera E; Copping, Nycole A; Pride, Michael C; Smith, Daniel G; Rotenberg, Alexander; Crawley, Jacqueline N; Sahin, Mustafa

    2017-01-01

    Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant ( Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each

  4. Developing Novel Automated Apparatus for Studying Battery of Social Behaviors in Mutant Mouse Models for Autism

    Science.gov (United States)

    2013-06-01

    coloured blobs ) and RFID data (open circles) are then independently processed offline to extract the X–Y coordinates of each identified mouse within the...such as in most rodents, fish and insects. Radio-frequency identified (RFID) tagging has been success- fully applied to track the position of uniquely...large animal groups (for example, insect colonies, fish schools)40. Methods Animals. Adult male and female mice from the C57BL/6Jx129sv and BTBR Toþ4

  5. Endogenous superoxide dismutase and catalase activities and radiation resistance in mouse cell lines

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Ostrand-Rosenberg, S.

    1988-01-01

    The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by γ-radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (Dsub(q)), or Dsub(o). None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent Ksub(i) of potassium cyanide for mouse CuZn-superoxide dismutase was determined (Ksub(i) = 6.5 μmol dm -3 ). (author)

  6. A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons

    DEFF Research Database (Denmark)

    Borgius, Lotta; Restrepo, C. Ernesto; Leao, Richardson N.

    2010-01-01

    Excitatory glutamatergic neurons are part of most of the neuronal circuits in the mammalian nervous system. We have used BAC-technology to generate a BAC-Vglut2::Cre mouse line where Cre expression is driven by the vesicular glutamate transporter 2 (Vglut2) promotor. This BAC-Vglut2::Cre mouse line...... showed specific expression of Cre in Vglut2 positive cells in the spinal cord with no ectopic expression in GABAergic or glycinergic neurons. This mouse line also showed specific Cre expression in Vglut2 positive structures in the brain such as thalamus, hypothalamus, superior colliculi, inferior...... colliculi and deep cerebellar nuclei together with nuclei in the midbrain and hindbrain. Cre-mediated recombination was restricted to Cre expressing cells in the spinal cord and brain and occurred as early as E 12.5. Known Vglut2 positive neurons showed normal electrophysiological properties in the BAC...

  7. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    International Nuclear Information System (INIS)

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development

  8. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    Energy Technology Data Exchange (ETDEWEB)

    Webb, Carol F., E-mail: carol-webb@omrf.org [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Ratliff, Michelle L., E-mail: michelle-ratliff@omrf.org [Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Powell, Rebecca, E-mail: rebeccapowell@gmail.com [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Wirsig-Wiechmann, Celeste R., E-mail: celeste-wirsig@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Lakiza, Olga, E-mail: olga-lakiza@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Obara, Tomoko, E-mail: tomoko-obara@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  9. Immunological characteristics and response to lipopolysaccharide of mouse lines selectively bred with natural and acquired immunities.

    Science.gov (United States)

    Narahara, Hiroki; Sakai, Eri; Katayama, Masafumi; Ohtomo, Yukiko; Yamamoto, Kanako; Takemoto, Miki; Aso, Hisashi; Ohwada, Shyuichi; Mohri, Yasuaki; Nishimori, Katsuhiko; Isogai, Emiko; Yamaguchi, Takahiro; Fukuda, Tomokazu

    2012-05-01

    Genetic improvement of resistance to infectious diseases is a challenging goal in animal breeding. Infection resistance involves multiple immunological characteristics, including natural and acquired immunity. In the present study, we developed an experimental model based on genetic selection, to improve immunological phenotypes. We selectively established three mouse lines based on phagocytic activity, antibody production and the combination of these two phenotypes. We analyzed the immunological characteristics of these lines using a lipopolysaccharide (LPS), which is one of the main components of Gram-negative bacteria. An intense immunological reaction was induced in each of the three mouse lines. Severe loss of body weight and liver damage were observed, and a high level of cytokine messenger RNA was detected in the liver tissue. The mouse line established using a combination of the two selection standards showed unique characteristics relative to the mouse lines selected on the basis of a single phenotype. Our results indicate that genetic selection and breeding is effective, even for immunological phenotypes with a relatively low heritability. Thus, it may be possible to improve resistance to infectious diseases by means of genetic selection. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

  10. Development of improved advanced mutant lines of cereal and native grains through radiation-induced mutagenesis in Peru

    International Nuclear Information System (INIS)

    Gomez, L.; Aldaba, G.; Yarango, D.; Argumedo, K.; Ibannez, N.; Falconi, J.

    2015-01-01

    In Peru it is very important to increase the food production in amount and quality, especially in the rural areas where a high poverty and malnutrition problems are usually founded. Mutation induction method is used to improve well adapted cultivars, thru the upgraded in one or two changed characteristics, retaining all its original attributes. Quinoa (Chenopodium quinoa), accession LM 89, was treated with gamma rays at the doses 150 and 250 Gray. In M 2 and following generations mutation in morphological traits were observed and 8 mutant lines were selected among them MQLM89-149 with higher yield equal to 4258.6 Kg/ha, surpassing the witness at 205.63% and MQLM89-42 with 14.7 of grain protein, superior to the parent material with 12.3%. Kiwicha (Amaranthus caudatus) CICA- UNASAC cultivar was irradiated with gamma ray (400 and 600 Gray). Mutations of morphological and physiological characteristics were identified and nine mutant lines with 27 to 50% better yield potential than the parent material were selected. In barley (Hordeum vulgare) mutant lines were developed from the cultivar UNALM 96, through the application of gamma rays at a dose of 200 and 300 Gray. Mutant lines were selected a M 8 generation with higher agronomic performance and nutritive quality adapted to the highland with grain yield within the range of 5100 - 8731 kg/ha, over the value of the parent material with of 4246 kg/ha and had improvement in the content of P-131 mg/g DW, Zn66 mg/g DW, Mn55 mg/g DW, Fe57 mg/g DW and Cu63 ug/g DW. (Author)

  11. Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant

    Science.gov (United States)

    Nag, Nabanita; Peterson, Katherine; Wyatt, Keith; Hess, Sonja; Ray, Sugata; Favor, Jack; Bogani, Debora; Lyon, Mary; Wistow, Graeme

    2007-01-01

    No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for γE-crystallin. Recombinant protein for the mutant γE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of γE. Quantitative RT-PCR showed that γE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of γE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity. PMID:17223009

  12. Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity.

    Directory of Open Access Journals (Sweden)

    Tao Lin

    Full Text Available The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

  13. Mouse DRG Cell Line with Properties of Nociceptors.

    Science.gov (United States)

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  14. Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

    Science.gov (United States)

    Carlisle, L; Steel, K; Forge, A

    1990-11-01

    Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

  15. Pentobarbital Sleep Time in Mouse Lines Selected for Resistance and Susceptibility to Fescue Toxicosis

    OpenAIRE

    Arthur, Kimberly Ann

    2002-01-01

    In previous work with mouse lines selected for resistance (R) and susceptibility (S) to fescue toxicosis, R mice had higher activities of Phase II liver enzymes glutathione S-transferase and uridine diphosphate glucuronosyl-transferase than S mice. Objectives of this study were: 1. to determine whether selection for toxicosis response had also caused divergence between lines in hepatic Phase I enzyme activity (as assessed by sleep time following sodium pentobarbital anesthesia), 2. to determi...

  16. Yield and Quality Traits of some Rice Mutant Lines as Affected by Different Nitrogen Levels

    International Nuclear Information System (INIS)

    Sobieh, S.El-S.S.

    2007-01-01

    Two field experiments were carried out during two growing seasons (2004 and 2005) at a farm located in Sahafa village, Sharkia Governorate, to evaluate newly rice mutants comparing with the local cultivar Sakha 104 for yield and quality characteristics as affected by nitrogen fertilizer levels. The obtained results showed that: 1- Rice grain yield and yield attributes were significantly increased with increasing N levels from 23 to 69 kg N fed '. 2- Both mutant MG 16 and MS 6 exhibited highly significant increases in mean values for yield attributes except for number of panicles/m2, as compared with the local cultivar Sakha 104. 3- Percentage of yield increases were 26.85 and 16.21 % for mutant MG16 and MS6 comparing with the local variety Sakha 104, respectively. Mutant MG 16 showed the highest mean values for plant height, panicle length, number of grains per panicle, panicle weight, 1000-grain weight, grain yield/fed, and straw yield/fed, as compared with the mutant MS 6 and Sakha 104. 4- Hulling and milling % were significantly increased as increasing of nitrogen levels from 23 to 69 kg N fed 1 , whereas head rice, gel consistency and amylose content were not significantly affect. 5- Significant differences were obtained between the three rice genotypes for hulling %, milling %, head rice %, amylose content and gel consistency

  17. Study of genetic behavior of some early maturing and high yielding mutant lines of soybean in different locations

    International Nuclear Information System (INIS)

    Mir Ali, N.; Moualla, M.

    2007-01-01

    this study aimed at checking the stability of some mutant lines from soybean varieties in different locations and to select the best performing lines in each location. These lines 15 were selected according to previous experiments as being early maturing and/or that yield higher than the control. The study lasted three years, the experiment plants were grown in 3 locations: Raqa, Idleb and Lattakia. The experiment was designed as RCBD with 3 replicates for each variety. Results showed significant difference between lines, Locations and year in both earliness and yield, A significant interaction was realized between (line X location) and (line X year) for earliness and yield. For earliness (line X year) was not significant. The reverse situation was realized for yield. Location X year of yield and earliness was significant. Earliness was correlated positively with all characters (except for 100-seed-weight). Yield was positively and significantly correlated with characters of all lines. Three lines with higher yield than the control (142.61%) and same maturity time were selected. (author)

  18. The streptomycin-treated mouse intestine selects Escherichia coli envZ missense mutants that interact with dense and diverse intestinal microbiota.

    Science.gov (United States)

    Leatham-Jensen, Mary P; Frimodt-Møller, Jakob; Adediran, Jimmy; Mokszycki, Matthew E; Banner, Megan E; Caughron, Joyce E; Krogfelt, Karen A; Conway, Tyrrell; Cohen, Paul S

    2012-05-01

    Previously, we reported that the streptomycin-treated mouse intestine selected nonmotile Escherichia coli MG1655 flhDC deletion mutants of E. coli MG1655 with improved colonizing ability that grow 15% faster in vitro in mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). Here, we report that the 10 to 20% remaining motile E. coli MG1655 are envZ missense mutants that are also better colonizers of the mouse intestine than E. coli MG1655. One of the flhDC mutants, E. coli MG1655 ΔflhD, and one of the envZ missense mutants, E. coli MG1655 mot-1, were studied further. E. coli MG1655 mot-1 is more resistant to bile salts and colicin V than E. coli MG1655 ΔflhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 ΔflhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 ΔflhD appear to colonize equally well in one intestinal niche, but E. coli MG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly by E. coli MG1655 ΔflhD. Evidence is also presented that E. coli MG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the "Restaurant" hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results.

  19. Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling

    NARCIS (Netherlands)

    Singh, Simar Pal; Pillai, Saravanan Y.; de Bruijn, Marjolein J. W.; Stadhouders, Ralph; Corneth, Odilia B. J.; van den Ham, Henk Jan; Muggen, Alice; van Ijcken, Wilfred; Slinger, Erik; Kuil, Annemieke; Spaargaren, Marcel; Kater, Arnon P.; Langerak, Anton W.; Hendriks, Rudi W.

    2017-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5(+) B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6,

  20. Basement membrane components secreted by mouse yolk sac carcinoma cell lines

    DEFF Research Database (Denmark)

    Damjanov, A; Wewer, U M; Tuma, B

    1990-01-01

    Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac...

  1. The toxicity of silver and silica nanoparticles in comparable human and mouse cell lines

    DEFF Research Database (Denmark)

    Foldbjerg, Rasmus; Beer, Christiane; Sutherland, Duncan S

    The toxicity of silica (SiO2) and PVP-coated silver (Ag) nanoparticles (NPs) was investigated in two pairs of human or mouse cell lines originating from lung epithelium (A549 and ASB-XIV) and macrophages (THP-1 and J744A.1). Both NPs were characterized in H2O and cell media and demonstrated to be...

  2. The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations

    DEFF Research Database (Denmark)

    Fuchs, Helmut; Sabrautzki, Sibylle; Przemeck, Gerhard K H

    2016-01-01

    The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and the...

  3. A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model.

    Science.gov (United States)

    Wang, Fang; Travins, Jeremy; Lin, Zhizhong; Si, Yaguang; Chen, Yue; Powe, Josh; Murray, Stuart; Zhu, Dongwei; Artin, Erin; Gross, Stefan; Santiago, Stephanie; Steadman, Mya; Kernytsky, Andrew; Straley, Kimberly; Lu, Chenming; Pop, Ana; Struys, Eduard A; Jansen, Erwin E W; Salomons, Gajja S; David, Muriel D; Quivoron, Cyril; Penard-Lacronique, Virginie; Regan, Karen S; Liu, Wei; Dang, Lenny; Yang, Hua; Silverman, Lee; Agresta, Samuel; Dorsch, Marion; Biller, Scott; Yen, Katharine; Cang, Yong; Su, Shin-San Michael; Jin, Shengfang

    2016-11-01

    D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

  4. Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines

    DEFF Research Database (Denmark)

    Christensen, C R; Klingelhöfer, Jörg; Tarabykina, S

    1998-01-01

    identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse...

  5. Pea mutant risnod27 as reference line for field assessment of impact of symbiotic nitrogen fixation

    Czech Academy of Sciences Publication Activity Database

    Biedermannová, E.; Novák, Karel; Vondrys, J.

    2002-01-01

    Roč. 25, č. 9 (2002), s. 2051-2066 ISSN 0190-4167 R&D Projects: GA ČR GA521/00/0937 Institutional research plan: CEZ:AV0Z5020903 Keywords : pea mutant * symbiotic nodules Subject RIV: EE - Microbiology, Virology Impact factor: 0.593, year: 2002

  6. Evaluation of some chemical and technological properties of induced erect chickpea mutant lines developed under drought stressed conditions

    International Nuclear Information System (INIS)

    Moustafa, R.A.K.; Ali, H.G.M.

    2009-01-01

    Seeds of the chickpea variety Flip 99-47 C were treated with gamma rays at doses of 0, 50 and 75 Gy and sown in the winter season of 2004/2005 to raise M1 generation under ordinary (normal) irrigation conditions. Bulked seeds from each treatment were planted in the subsequent growing seasons of 2005/2006 and 2006/2007 to advance M2 and M3 generations, respectively under either ordinary (normal) irrigation or drought stress condition. In the third generation, three erect mutant lines were derived from 75 Gy mutagenic treatment under drought stress compared to semi spreading growth habit of the initiated variety Flip 99-47 C. In the winter season of 2007/2008, M4 bulked seeds from the three erect lines as well as unirradiated seeds of the original variety grown under either ordinary (normal) irrigation (2152.5 m 3 /fad.) or drought (1159.2 m 3 /fad.) conditions were analyzed for the chemical composition and nutritional values. Obtained results indicated that there were slight decreases in protein and fat contents accompanied with marginal increases in both ash and carbohydrates in seed samples of the erect mutant developed under drought stress as compared to unirradiated seeds of the original variety grown under ordinary (normal) irrigation treatment. An opposite trend was noticed between seed samples derived from the erect lines compared to seeds of the parent variety developed under drought condition. Negligible changes in levels of the minerals (iron, magnesium, calcium and phosphorus) were detected between seeds of the erect lines and the original variety that grown under either ordinary (normal) irrigation or drought conditions. Cooking time (min) and hydration coefficient values did not much differ between the three tested seed samples. Marginal differences in essential and non-essential amino acids were detected between seeds of the erect mutants and those of the initial variety grown under ordinary (normal) irrigation or drought stressed conditions

  7. Radiation response of mouse lymphoid and myeloid cell lines. Pt. 1

    International Nuclear Information System (INIS)

    Radford, I.R.

    1994-01-01

    The sensitivity of 10 mouse lymphoid or myeloid cell lines to γ-ray- and DNA-associated 125 I-decay-induced clonogenic cell killing have been compared with their rate of loss of viability (membrane integrity) and with their putative cell type of origin. The increased sensitivity of haematopoietic cell lines to killing by DNA dsb may be related to their mode of death (apoptosis versus necrosis). Mode of cell death may thus be an important factor in determining the 'inherent radiosensitivity' of normal cells/tissues. Haematopoietic cell lines that undergo rapid interphase apoptotic death showed extreme sensitivity to DNA dsb. (author)

  8. Differential Expression of Claudin Family Proteins in Mouse Ovarian Serous Papillary Epithelial Adenoma in Aging FSH Receptor-Deficient Mutants

    Directory of Open Access Journals (Sweden)

    Jayaprakash Aravindakshan

    2006-12-01

    Full Text Available Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of folliclestimulating hormone receptor (FSH-R signaling and to explore why the atrophic and anovulatory ovaries of follitropin receptor knockout (FORKO mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we used mRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative reverse transcription-polymerase chain reaction, protein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 decreases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction proteins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma.

  9. Evaluation of symbiotic performance of some mutant lines of soybean inoculated with two bradyrhizobium japonicum strains using 15N technique

    International Nuclear Information System (INIS)

    Kurdali, F.; Mir-Ali, N.; Al-Nabulsi, I.

    2002-11-01

    A pot experiment was conducted to study the symbiotic performance of two soybean varieties and some of their mutants (that were obtained as a result of a previous mutation breeding program) with two bradyrhizobium japonicum strains (RG and FA3) using 15 N isotopic dilution method. Random amplified polymorphic DNA technique (RAPD) was used to study the genetic relationships among the soybean genotypes and to make sure that the two rhizobial strains are different. The 25 random primers used discriminated the different soybean genotypes and the dendrogram resultants from shared polymorphic fragments put each variety and its mutants in two separate clusters asserting that the mutants and their mother lines are different. Both strains of B. japonicum were able to form effective nodules on all soybean plants. However, number of nodules, dry matter yield and N-uptake from the available sources by soybeans were affected by both plant genotype and rhizobial strains. N 2 -fixation was affected to a large extent by different strain and plant genotype combinations. Percentage of fixed N 2 (N dfa) ranged between 35 and 49%; whereas, the actual amounts of fixed N 2 were between 105 and 210 mg N/pot. Amounts of N 2 -fixed by FA3 strain were higher than of RG in both soybean varieties, whereas, the latter strain showed higher performance in the mutant lines. The results showed that total plant N estimation may not be a sufficient indicator for high N 2 -fixation. the results also showed that it is very important to determine both the amount of nitrogen derived from N 2 -fixation and N derived from soil for evaluating the symbiotic performance ability. Moreover, the performance of symbiotic N 2 -fixation in soybean was shown to depend on both plant genotype and rhizobial strain and the amount of N 2 -fixation can be increased by combining the best plant genotypes and the most adapted strain. (author)

  10. Oxygen Association-Dissociation and Stability Analysis on Mouse Hemoglobins with Mutant α- and β-Globins

    Science.gov (United States)

    D'Surney, S. J.; Popp, R. A.

    1992-01-01

    Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an α-globin (α89, His to Leu) and a β-globin (β59, Lys to Ile). The variant α-globin, designated chain 5(m) in the Hba(g2) haplotype, had a high oxygen affinity and was stable. The variant β-globin, (β(s2)) of the Hbb(s2) haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) X Hba(a)/Hba(a);Hbb(s2)/Hbb(s2)) F(2) genotypes can be grouped into five classes of P(50) values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hba(a)/Hba(a);Hbb(s)/Hbb(s)) had a P(50) = 40 mm Hg and the hemoglobin of Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) F(2) mice had a P(50) = 25 mm Hg (human P(50) = 26 mm Hg). Peripheral blood from Hba(g2)/Hba(g2);Hbb(s)/Hbb(s), Hba(a)/Hba(a);Hbb(s2)/Hbb(s2) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice exhibited normal hematological values except for a slightly higher hematocrit for Hba(g2)/Hba(g2);Hbb(s)/Hbb(s) and Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hba(g2)/Hba(g2);Hbb(s2)/Hbb(s2) mice. PMID:1427042

  11. Efficient inhibition of murine breast cancer growth and metastasis by gene transferred mouse survivin Thr34→Ala mutant

    Directory of Open Access Journals (Sweden)

    Chen li-Juan

    2008-09-01

    Full Text Available Abstract Background Metastasis in breast cancer is a vital concern in treatment because most women with primary breast cancer have micrometastases to distant sites at diagnosis. As a member of the inhibitor of apoptosis protein (IAP family, survivin has been proposed as an attractive target for new anticancer interventions. In this study, we investigated the role of the plasmid encoding the phosphorylation-defective mouse survivin threonine 34→alanine mutant (Msurvivin T34A plasmid in suppressing both murine primary breast carcinomas and pulmonary metastases. Methods In vitro study, induction of apoptosis by Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol was examined by PI staining fluorescence microscopy and flow cytometric analysis. The anti-tumor and anti-metastases activity of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol was evaluated in female BALB/c mice bearing 4T1 s.c. tumors. Mice were treated twice weekly with i.v. administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol, PORF-9 null plasmid complexed with cationic liposome (DOTAP/Chol, 0.9% NaCl solution for 4 weeks. Tumor volume was observed. After sacrificed, tumor net weight was measured and Lung metastatic nodules of each group were counted. Assessment of apoptotic cells by TUNEL assay was conducted in tumor tissue. Microvessel density within tumor tissue was determined by CD31 immunohistochemistry. Alginate-encapsulated tumor cells test was conducted to evaluate the effect on angiogenesis. By experiment of cytotoxicity T lymphocytes, we test whether Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol can induce specific cell immune response. Results Administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol resulted in significant inhibition in the growth and metastases of 4T1 tumor model. These anti-tumor and anti-metastases responses were associated with

  12. Seed coat color, weight and eye pattern inheritance in gamma-rays induced cowpea M2-mutant line

    Directory of Open Access Journals (Sweden)

    Reda M. Gaafar

    2016-06-01

    Full Text Available Gamma radiation is a very effective tool for inducing genetic variation in characters of many plants. Black seeds of M2 mutant were obtained after exposure of an Egyptian cowpea cultivar (Kaha 1 to a low dose of gamma rays. Segregation of seed coat color, weight of 100 seeds and seed eye pattern of the black seeds of this mutant line were further examined in this study. Four colors were observed for seed coat in the M3 plants ranging from cream to reddish brown and three eye patterns were distinguished from each other. SDS–PAGE of the seed storage proteins showed 18 protein bands; five of these bands disappeared in the seeds of M3 plants compared to M2 and M0 controls while other 5 protein bands were specifically observed in seeds of M3 plants. PCR analysis using twelve ISSR primers showed 47 polymorphic and 8 unique amplicons. The eight unique amplicons were characteristic of the cream coat color and brown wide eye pattern (M03-G10 while the polymorphic bands were shared by 6 coat-color groups. A PCR fragment of 850 bp was amplified, using primer HB-12, in M3-G04 which showed high-100 seed weight. These results demonstrated the mutagenic effects of gamma rays on seed coat color, weight of 100 seeds and eye pattern of cowpea M3 mutant plants.

  13. Generation and characterization of PDGFRα-GFPCreERT2 knock-In mouse line.

    Science.gov (United States)

    Miwa, Hiroyuki; Era, Takumi

    2015-05-01

    Platelet-derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα-expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα-expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock-in mouse line generated here could be useful for studying PDGFRα-expressing cells and their descendants in vivo at various stages of development. © 2015 Wiley Periodicals, Inc.

  14. Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

    Directory of Open Access Journals (Sweden)

    Miyaji E.N.

    2000-01-01

    Full Text Available Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2 that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  16. Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line.

    Science.gov (United States)

    Kawaguchi, Daichi; Sahara, Setsuko; Zembrzycki, Andreas; O'Leary, Dennis D M

    2016-04-01

    Foxg1 expression is highly restricted to the telencephalon and other head structures in the early embryo. This expression pattern has been exploited to generate conditional knockout mice, based on a widely used Foxg1-Cre knock-in line (Foxg1(tm1(cre)Skm)), in which the Foxg1 coding region was replaced by the Cre gene. The utility of this line, however, is severely hampered for two reasons: (1) Foxg1-Cre mice display ectopic and unpredictable Cre activity, and (2) Foxg1 haploinsufficiency can produce neurodevelopmental phenotypes. To overcome these issues, we have generated a new Foxg1-IRES-Cre knock-in mouse line, in which an IRES-Cre cassette was inserted in the 3'UTR of Foxg1 locus, thus preserving the endogenous Foxg1 coding region and un-translated gene regulatory sequences in the 3'UTR, including recently discovered microRNA target sites. We further demonstrate that the new Foxg1-IRES-Cre line displays consistent Cre activity patterns that recapitulated the endogenous Foxg1 expression at embryonic and postnatal stages without causing defects in cortical development. We conclude that the new Foxg1-IRES-Cre mouse line is a unique and advanced tool for studying genes involved in the development of the telencephalon and other Foxg1-expressing regions starting from early embryonic stages. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

    Science.gov (United States)

    Bodel, P

    1978-05-01

    Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

  18. Radiation response of mouse lymphoid and myeloid cell lines. Pt. 3

    International Nuclear Information System (INIS)

    Radford, I.R.; Murphy, T.K.

    1994-01-01

    The authors have examined the timing of γ-irradiation-induced death in relation to cell cycle progression using a panel of mouse lymphoid or myeloid cell lines. Death was found to occur immediately after irradiation ('rapid interphase' death), or after arrest in G 2 phase ('delayed interphase' death), or following one or more mitoses ('mitotic/delayed mitotic' death). In part II of this series of papers the authors demonstrated the occurrence of radiation-induced apoptosis in all these cell lines. Several of the cell lines showed different timing of death dependent upon the radiation dose used. These differences in the timing of radiation-induced death are shown to be useful indicators of the relative radiosensitivity of haematopoietic cell lines. (author)

  19. Accumulation of dry matter and nitrogen in the developing seeds of high protein mutant lines of Triticum Aestivum (L.) produced by the IAEA

    International Nuclear Information System (INIS)

    Mir Ali, N.; Nabulsi, I.

    1993-03-01

    Accumulation patterns of dry matter and nitrogen in the developing seeds of nine mutant lines produced by the IAEA and their mother Triticum Aestivum (L.) line were studied. The experiments lasted 2 years under rain fed conditions. Significant differences were found among the lines in dry matter and nitrogen rates, and periods of accumulation, whereas no significant differences were found in the final seed weight of the lines. The highest rates of accumulation for dry matter and nitrogen were accompanied with the shortest period of accumulation in two late flowering mutant lines. However, these two lines were the lowest in their yield per plot. The other mutant lines achieved the high nitrogen percentage in their seeds through the relative reduction in dry matter accumulation rate compared to their mother line rather than through higher rate of nitrogen accumulation. This study revealed some of the potential reasons behind the higher percentage of protein in the seeds of the mutant lines under investigation. (author). 17 refs., 3 figs., 2 tabs

  20. Nuclear hormone receptor expression in mouse kidney and renal cell lines.

    Directory of Open Access Journals (Sweden)

    Daisuke Ogawa

    Full Text Available Nuclear hormone receptors (NHRs are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN, the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m, and cell lines of mesangial (MES13, podocyte (MPC, proximal tubular epithelial (mProx24 and collecting duct (mIMCD3 origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77, nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

  1. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    Energy Technology Data Exchange (ETDEWEB)

    Huimin, Zhou [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Li, Jia [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Shujing, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Hongmei, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Department of Medical Microbiology and Parasitology, School of Medicine, Liaodong College, Dandong 118000 (China); Haiying, Chu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Yichuan, Hu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jun, Cao [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jianing, Zhang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China)

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  2. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    International Nuclear Information System (INIS)

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing

    2006-01-01

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression

  3. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike

    2006-01-01

    Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines ...

  4. Radiation susceptibility of the mouse smalleye mutants, Del(2)Sey3Hpax6 and Del(2)Sey4Hpax6, which delete the chromosome 2 middle regions

    International Nuclear Information System (INIS)

    Nitta, Y.; Hoshi, M.; Yoshida, K.; Yamate, J.; Peters, J.; Cattanach, B.M.

    2003-01-01

    Full text: LOH at the chromosome 2 middle regions is common in the radiation-induced mouse acute myeloid leukemia (AML). To identify the suppressor or the modifier gene of AML at this region, the mouse deletion mutants, Del(2)Sey3H pax6 and Del(2)Sey3H pax6 could be the good models, as they deleted the chromosome 2 middle regions hemizygously. The allele of the partially deleted chromosome 2 was paternally generated and maintained hemizygously. The exact deleted regions of the two mutants were mapped by the PCR-based detection of polymorphism of the STS markers. The length of the deletions was 3.01Mb and 10.11MB for Del(2)Sey3H pax6 and Del(2)Sey3H pax6 , respectively. For the induction of tumors, a radiation, 3.0Gy of Co-60 and a chemical carcinogen, N-methyl-N-nitrosourea were applied to the mutants. Their tumorigenicity was compared with those of control as well as normal sibs by the Kaplan-Meier analysis. Both mutants were found to predispose to small intestinal tumors. Intestinal tumors developed spontaneously with the incidence of 30%. The radiation and the chemical accelerated the malignancy and increased the incidence of the intestinal tumors. Radiation shortened the latency of AML development in the Del(2)Sey3H pax6 mutant but not in the Del(2)Sey3H pax6 . Spontaneous AML has not been observed, nor any increase in the incidence of induced AMLs. The commonly deleted region of the two mutants, the 3.01Mb region, must be critical for the development of tumors and the high susceptibility to radiation. The role of Pax6 gene should be considered in the intestinal tumorigenesis, as the Pax6 gene plays an important role in the pancreas development during the embryogenesis. The Wt1, a tumor suppressor gene, which is deleted hemizygously in these mutants as well. The screening of homozygous deletion has been started using the induced as well as spontaneously developed tumors

  5. Antiproliferative and Apoptotic Effect of Dendrosomal Curcumin Nanoformulation in P53 Mutant and Wide-Type Cancer Cell Lines.

    Science.gov (United States)

    Montazeri, Maryam; Pilehvar-Soltanahmadi, Younes; Mohaghegh, Mina; Panahi, Alireza; Khodi, Samaneh; Zarghami, Nosratollah; Sadeghizadeh, Majid

    2017-01-01

    The aim of this paper is to investigate the effect of dendrosomal curcumin (DNC) on the expression of p53 in both p53 mutant cell lines SKBR3/SW480 and p53 wild-type MCF7/HCT116 in both RNA and protein levels. Curcumin, derived from Curcumin longa, is recently considered in cancer related researches for its cell growth inhibition properties. p53 is a common tumor-suppressor gene involved in cancers and its mutation not only inhibits tumor suppressor activity but also promotes oncogenic activity. Here, p53 mutant/Wild-type cells were employed to study the toxicity of DNC using MTT assay, Flow cytometry and Annexin-V, Real-time PCR and Western blot were used to analyze p53, BAX, Bcl-2, p21 and Noxa changes after treatment. During the time, DNC increased the SubG1 cells and decreased G1, S and G2/M cells, early apoptosis also indicated the inhibition of cell growth in early phase. Real-Time PCR assay showed an increased mRNA of BAX, Noxa and p21 during the time with decreased Bcl-2. The expression of p53 mutant decreased in SKBR3/SW480, and the expression of p53 wild-type increased in MCF7/HCT116. Consequently, p53 plays an important role in mediating the survival by DNC, which can prevent tumor cell growth by modulating the expression of genes involved in apoptosis and proliferation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis.

    Science.gov (United States)

    Peters, Brian M; Luna-Tapia, Arturo; Tournu, Hélène; Rybak, Jeffrey M; Rogers, P David; Palmer, Glen E

    2017-06-01

    We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21 Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro , it does not seem to affect azole susceptibility in vivo . Copyright © 2017 American Society for Microbiology.

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

    Directory of Open Access Journals (Sweden)

    Yun Xia

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

  8. Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line

    OpenAIRE

    Kumagai, Ayako; Fujita, Akira; Yokoyama, Tomoki; Nonobe, Yuki; Hasaba, Yasuhiro; Sasaki, Tsutomu; Itoh, Yumi; Koura, Minako; Suzuki, Osamu; Adachi, Shigeki; Ryo, Haruko; Kohara, Arihiro; Tripathi, Lokesh; Sanosaka, Masato; Fukushima, Toshiki

    2014-01-01

    Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer’s disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mu...

  9. Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

    International Nuclear Information System (INIS)

    Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

    1989-01-01

    Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

  10. Correlation between ultraviolet survival and DNA repair efficiency in mouse cell hybrids and their parent lines

    International Nuclear Information System (INIS)

    Limbosch, S.

    1982-01-01

    Three hybrid cell lines formed between mouse lymphoma (LS) and mouse fibroblasts (A9) have been tested for their capacity to perform unscheduled DNA synthesis; their recovery characteristics after uv irradiation have also been studied to determine if DNA repair is implicated in the high survival observed in one hybrid (clone 3). The results of these investigations indicate that hybrid clone 3 was distinguishable from the more uv sensitive parental and other hybrid cell lines by its higher uv-induced unscheduled DNA synthesis, its greater clonogenic survival in plateau phase, and its faster recovery when maintained in conditioned medium after irradiation. The simultaneous increase of these three properties in hybrid clone 3 suggest that, by three different approaches, we have evidenced the same molecular process, a process involved in the elimination of potentially lethal damage, most probably the excision repair pathway. This report also shows that the low efficiency in excision repair in the parent line A9 is probably not due to deletion but rather to repression of the relevant gene(s) and that somatic cell hybridization can result in a stimulation of a previously poorly expressed repair process

  11. Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5

    International Nuclear Information System (INIS)

    Yasui, L.S.; Fink, T.J.; Enrique, A.M.

    1994-01-01

    Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. (author)

  12. Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

    International Nuclear Information System (INIS)

    Bouffler, S.D.; Jha, B.; Johnson, R.T.

    1990-01-01

    DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate

  13. Disjunction or non-disjunction in F2 generation according to the cross between a wheat mutant and its original lines

    International Nuclear Information System (INIS)

    Touvin, H.

    1973-01-01

    An early homogeneous mutant line (B) was obtained in M 2 generation following the gamma-rays (15kR) treatment of the dry seeds of a fixed homogeneous line of soft wheat (A). The study of this mutant leads to the following observations: the earliness is stable in the mutant stock during successive generations and in different climatic conditions; the products of reciprocal crosses between the mutant B and the original line A, compared in micro-tests under greenhouse conditions gave following different results according to the cross; in the F 1 , the reciprocal hybrids (AxB, BxA) are earlier than the mutant. The F 1 offsprings (BxA) which received the earliness characteristic from the female parent, develop more rapidly than the reciprocal F 1 hybrid (AxB). Thus, this shows that there exist a maternal effect from the mutant side. In the F 2 , the descendants of the hybrid (AxB) segregated phenotypically in two classes, early and late, whereas the other hybrid (BxA) produces only early plants. The F 3 offsprings confirm the observations made in the F 2 generation. Although the F 3 generation of the hybrid (AxB) is composed of the early homogenous, the heterogeneous and the late homogeneous lines, but no segregation occurs in the cross (BxA). The segregation ratio in F 2 and in most of the backcrosses progenies indicates that the transmission of the earliness character is monogenic. From these results the existence of a major gene for earliness can be supposed, the expression of which appears to be under the control of the cytoplasm. The conclusion emphasizes the importance of the reciprocal crosses in the use of the mutants [fr

  14. Cox4i2, Ifit2, and Prdm11 Mutant Mice

    DEFF Research Database (Denmark)

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as imm...

  15. Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

    Science.gov (United States)

    del Río, A; Barrio, M C; Murillo, J; Maldonado, E; López-Gordillo, Y; Martínez-Sanz, E; Martínez, M L; Martínez-Álvarez, C

    2011-01-01

    The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed. Copyright © 2010 S. Karger AG, Basel.

  16. Comparative Analyses of Transcriptional Profiles in Mouse Organs Using a Pneumonic Plague Model after Infection with Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant

    Directory of Open Access Journals (Sweden)

    Cristi L. Galindo

    2009-01-01

    Full Text Available We employed Murine GeneChips to delineate the global transcriptional profiles of the livers, lungs, and spleens in a mouse pneumonic plague infection model with wild-type (WT Y. pestis CO92 and its Braun lipoprotein (Δlpp mutant with reduced virulence. These organs showed differential transcriptional responses to infection with WT Y. pestis, but the overall host functional processes affected were similar across all three tissues. Gene expression alterations were found in inflammation, cytokine signaling, and apoptotic cell death-associated genes. Comparison of WT and Δlpp mutant-infected mice indicated significant overlap in lipopolysaccharide- (LPS- associated gene expression, but the absence of Lpp perturbed host cell signaling at critical regulatory junctions resulting in altered immune response and possibly host cell apoptosis. We generated a putative signaling pathway including major inflammatory components that could account for the synergistic action of LPS and Lpp and provided the mechanistic basis of attenuation caused by deletion of the lpp gene from Y. pestis in a mouse model of pneumonic plague.

  17. Functional Coding Variation in Recombinant Inbred Mouse Lines Reveals Novel Serotonin Transporter-Associated Phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Carneiro, Ana [Vanderbilt University; Airey, David [University of Tennessee Health Science Center, Memphis; Thompson, Brent [Vanderbilt University; Zhu, C [Vanderbilt University; Rinchik, Eugene M [ORNL; Lu, Lu [University of Tennessee Health Science Center, Memphis; Chesler, Elissa J [ORNL; Erikson, Keith [University of North Carolina; Blakely, Randy [Vanderbilt University

    2009-01-01

    The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, SLC6A4) figures prominently in the etiology or treatment of many prevalent neurobehavioral disorders including anxiety, alcoholism, depression, autism and obsessive-compulsive disorder (OCD). Here we utilize naturally occurring polymorphisms in recombinant inbred (RI) lines to identify novel phenotypes associated with altered SERT function. The widely used mouse strain C57BL/6J, harbors a SERT haplotype defined by two nonsynonymous coding variants (Gly39 and Lys152 (GK)). At these positions, many other mouse lines, including DBA/2J, encode Glu39 and Arg152 (ER haplotype), assignments found also in hSERT. Synaptosomal 5-HT transport studies revealed reduced uptake associated with the GK variant. Heterologous expression studies confirmed a reduced SERT turnover rate for the GK variant. Experimental and in silico approaches using RI lines (C57Bl/6J X DBA/2J=BXD) identifies multiple anatomical, biochemical and behavioral phenotypes specifically impacted by GK/ER variation. Among our findings are multiple traits associated with anxiety and alcohol consumption, as well as of the control of dopamine (DA) signaling. Further bioinformatic analysis of BXD phenotypes, combined with biochemical evaluation of SERT knockout mice, nominates SERT-dependent 5-HT signaling as a major determinant of midbrain iron homeostasis that, in turn, dictates ironregulated DA phenotypes. Our studies provide a novel example of the power of coordinated in vitro, in vivo and in silico approaches using murine RI lines to elucidate and quantify the system-level impact of gene variation.

  18. Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line.

    Science.gov (United States)

    Carpenter, Adam J; Porter, Andrew C G

    2004-12-01

    DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

  19. Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

    OpenAIRE

    Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

    2016-01-01

    Background Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunizat...

  20. Alterations in grooming activity and syntax in heterozygous SERT and BDNF knockout mice: the utility of behavior-recognition tools to characterize mutant mouse phenotypes.

    Science.gov (United States)

    Kyzar, Evan J; Pham, Mimi; Roth, Andrew; Cachat, Jonathan; Green, Jeremy; Gaikwad, Siddharth; Kalueff, Allan V

    2012-12-01

    Serotonin transporter (SERT) and brain-derived neurotrophic factor (BDNF) are key modulators of molecular signaling, cognition and behavior. Although SERT and BDNF mutant mouse phenotypes have been extensively characterized, little is known about their self-grooming behavior. Grooming represents an important behavioral domain sensitive to environmental stimuli and is increasingly used as a model for repetitive behavioral syndromes, such as autism and attention deficit/hyperactivity disorder. The present study used heterozygous ((+/-)) SERT and BDNF male mutant mice on a C57BL/6J background and assessed their spontaneous self-grooming behavior applying both manual and automated techniques. Overall, SERT(+/-) mice displayed a general increase in grooming behavior, as indicated by more grooming bouts and more transitions between specific grooming stages. SERT(+/-) mice also aborted more grooming bouts, but showed generally unaltered activity levels in the observation chamber. In contrast, BDNF(+/-) mice displayed a global reduction in grooming activity, with fewer bouts and transitions between specific grooming stages, altered grooming syntax, as well as hypolocomotion and increased turning behavior. Finally, grooming data collected by manual and automated methods (HomeCageScan) significantly correlated in our experiments, confirming the utility of automated high-throughput quantification of grooming behaviors in various genetic mouse models with increased or decreased grooming phenotypes. Taken together, these findings indicate that mouse self-grooming behavior is a reliable behavioral biomarker of genetic deficits in SERT and BDNF pathways, and can be reliably measured using automated behavior-recognition technology. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Genome characterization of the selected long- and short-sleep mouse lines.

    Science.gov (United States)

    Dowell, Robin; Odell, Aaron; Richmond, Phillip; Malmer, Daniel; Halper-Stromberg, Eitan; Bennett, Beth; Larson, Colin; Leach, Sonia; Radcliffe, Richard A

    2016-12-01

    The Inbred Long- and Short-Sleep (ILS, ISS) mouse lines were selected for differences in acute ethanol sensitivity using the loss of righting response (LORR) as the selection trait. The lines show an over tenfold difference in LORR and, along with a recombinant inbred panel derived from them (the LXS), have been widely used to dissect the genetic underpinnings of acute ethanol sensitivity. Here we have sequenced the genomes of the ILS and ISS to investigate the DNA variants that contribute to their sensitivity difference. We identified ~2.7 million high-confidence SNPs and small indels and ~7000 structural variants between the lines; variants were found to occur in 6382 annotated genes. Using a hidden Markov model, we were able to reconstruct the genome-wide ancestry patterns of the eight inbred progenitor strains from which the ILS and ISS were derived, and found that quantitative trait loci that have been mapped for LORR were slightly enriched for DNA variants. Finally, by mapping and quantifying RNA-seq reads from the ILS and ISS to their strain-specific genomes rather than to the reference genome, we found a substantial improvement in a differential expression analysis between the lines. This work will help in identifying and characterizing the DNA sequence variants that contribute to the difference in ethanol sensitivity between the ILS and ISS and will also aid in accurate quantification of RNA-seq data generated from the LXS RIs.

  2. Comparison of Two Mouse Ameloblast-like Cell Lines for Enamel-specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Juni eSarkar

    2014-07-01

    Full Text Available Ameloblasts are ectoderm-derived cells that produce an extracellular enamel matrix that mineralizes to form enamel. The development and use of immortalized cell lines, with a stable phenotype, is an important contribution to biological studies as it allows for the investigation of molecular activities without the continuous need for animals. In this study we compare the expression profiles of enamel-specific genes in two mouse derived ameloblast-like cell lines: LS8 and ALC cells. Quantitative PCR analysis indicates that, relative to each other, LS8 cells express greater mRNA levels for genes that define secretory-stage activities (Amelx, Ambn, Enam and Mmp20, while ALC express greater mRNA levels for genes that define maturation-stage activities (Odam and Klk4. Western blot analyses show that Amelx, Ambn and Odam proteins are detectable in ALC, but not LS8 cells. Unstimulated ALC cells form calcified nodules, while LS8 cells do not. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.

  3. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

    Science.gov (United States)

    Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

    2004-12-01

    The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

  4. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Behavioural effects of high fat diet in a mutant mouse model for the schizophrenia risk gene neuregulin 1

    DEFF Research Database (Denmark)

    Holm-hansen, S.; Low, J. K.; Zieba, J.

    2016-01-01

    on the behavioural phenotype of test mice and attenuated particular cognitive deficits of Nrg1 mutant females. This topic requires further investigations thereby also considering other dietary factors of relevance for schizophrenia as well as interactive effects of diet with medication and sex....

  7. Plant regeneration of bananas Ambon kuning and Barangan mutant lines were carried out by using female organ and shoot-tip as explants source

    International Nuclear Information System (INIS)

    Dewi, Azri K; Ishak

    1998-01-01

    Plant regeneration of bananas Ambon Kuning and Barangan mutant lines were carried out by using female organ and shoot-tip as explants source. Female organ was taken from heart of banana stem, while shoot-tip taken from sucker in banana plantation at Pasar Jumat, Jakarta. Those explants were cultured on MS medium containing 3 mg/l BAP, 0.5 mg/l IAA and supplemented by 100 tyrosin and 80 mg/l adenin hemisulphate. Observation showed that 180 and 42 buds were obtained from JBR 02 mutant lines respectively, while 84 and 79 buds for JAK 01 and JAK 02 respectively. The highest shoot formation was 1.013 shoots were obtained from BRC variety and lowest one was JBR 01 mutant line. statistical data analysis indicated that shoot formation between BRC variety and another mutant lines were significant difference using LSD test at level 0.05. Plantlet formation derived from female organ as well as shoot-tip showed that BRC variety produced number of plantlets per bottle was higher that another one. (author)

  8. Spontaneous and radiation-induced leukemogenesis of the mouse small eye mutant, Pax6Sey3H

    International Nuclear Information System (INIS)

    Nitta, Yumiko; Satoh, Kenichi; Yoshida, Kazuko; Senba, Kei; Nakagata, Naomi; Peters, J.; Cattanach, B.M.

    2004-01-01

    Allelic loss on the chromosome 2 is associated with radiation-induced murine acute myeloid leukemia. However, the gene, which contributes mainly to the leukemogenesis has not yet been identified. Expecting any predisposition to acute myeloid leukemia, we performed a radiation leukemogenesis experiment with Pax6 SeY3H , one of the small eye mutants carrying a congenital hemizygosity of the chromosome 2 middle region. A deletion mapping of Pax6 SeY3H with 50 sequence-tagged site (STS) markers indicated that the deleted segment extended between the 106.00 and 111.47 Mb site from the centromere with a length of 5.47 Mb. In the deleted segment, 6 known and 17 novel genes were located. Pax6 SeY3H mutants that crossed back into C3H/He did not develop myeloid leukemia spontaneously, but they did when exposed to gamma-rays. The final incidence of myeloid leukemia in mutants (25.8%) was as high as that in normal sibs (21.4%). Survival curves of leukemia-bearing mutants shifted toward the left (p=0.043 by the Log rank test). F1 hybrids of Pax6 SeY3H with JF1 were less susceptible to radiation than Pax6 SeY3H onto C3H/He in regard to survival (p=0.003 and p<0.00001 for mutants and normal sibs, respectively, by a test of the difference between two proportions). Congenital deletion of the 5.47 Mb segment at the middle region on chromosome 2 alone did not trigger myeloid stem cells to expand clonally in vivo; however, the deletion shortcut the latency of radiation-induced myeloid leukemia. (author)

  9. Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1987-01-01

    Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.

  10. Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

    Directory of Open Access Journals (Sweden)

    Chen Tsung-Chieh

    2012-05-01

    in pstpip2 causes autoinflammatory disease in an N-ethyl-N-nitrosourea mutagenesis mouse model. Thus, our pstpip2 mutant mice provide a new model for investigating the potential mechanisms of inflammatory pain.

  11. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    Science.gov (United States)

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  12. Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

    Science.gov (United States)

    Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

    2017-01-01

    Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

  13. Absence of Nrf2 or its selective overexpression in neurons and muscle does not affect survival in ALS-linked mutant hSOD1 mouse models.

    Directory of Open Access Journals (Sweden)

    Marcelo R Vargas

    Full Text Available The nuclear factor erythroid 2-related factor 2 (Nrf2 governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1 cause familial forms of amyotrophic lateral sclerosis (ALS, a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.

  14. Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line

    Directory of Open Access Journals (Sweden)

    Ayako Kumagai

    2014-12-01

    Full Text Available Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer’s disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds.

  15. Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line

    Science.gov (United States)

    Kumagai, Ayako; Fujita, Akira; Yokoyama, Tomoki; Nonobe, Yuki; Hasaba, Yasuhiro; Sasaki, Tsutomu; Itoh, Yumi; Koura, Minako; Suzuki, Osamu; Adachi, Shigeki; Ryo, Haruko; Kohara, Arihiro; Tripathi, Lokesh P.; Sanosaka, Masato; Fukushima, Toshiki; Takahashi, Hiroyuki; Kitagawa, Kazuo; Nagaoka, Yasuo; Kawahara, Hidehisa; Mizuguchi, Kenji; Nomura, Taisei; Matsuda, Junichiro; Tabata, Toshihide; Takemori, Hiroshi

    2014-01-01

    Memantine is a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer’s disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds. PMID:25513882

  16. Small-molecule aggregation inhibitors reduce excess amyloid in a trisomy 16 mouse cortical cell line

    Directory of Open Access Journals (Sweden)

    ANDRÉA C PAULA LIMA

    2008-01-01

    Full Text Available We have previously characterized a number of small molecule organic compounds that prevent the aggregation of the β-amyloid peptide and its neurotoxicity in hippocampal neuronal cultures. We have now evaluated the effects of such compounds on amyloid precursor protein (APP accumulation in the CTb immortalized cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down's syndrome. Compared to a non-trisomic cortical cell line (CNh, CTb cells overexpress APP and exhibit slightly elevated resting intracellular Ca2+ levéis ([Ca2+]¡. Here, we show that the compounds 2,4-dinitrophenol, 3-nitrophenol and 4-anisidine decreased intracellular accumulation of APP in CTb cells. Those compounds were non-toxic to the cells, and slightly increased the basal [Ca2+]¡. Results indícate that the compounds tested can be leads for the development of drugs to decrease intracellular vesicular accumulation of APP in trisomic cells.

  17. Chromosome-wise dissection of the genome of the extremely big mouse line DU6i.

    Science.gov (United States)

    Bevova, Marianna R; Aulchenko, Yurii S; Aksu, Soner; Renne, Ulla; Brockmann, Gudrun A

    2006-01-01

    The extreme high-body-weight-selected mouse line DU6i is a polygenic model for growth research, harboring many small-effect QTL. We dissected the genome of this line into 19 autosomes and the Y chromosome by the construction of a new panel of chromosome substitution strains (CSS). The DU6i chromosomes were transferred to a DBA/2 mice genetic background by marker-assisted recurrent backcrossing. Mitochondria and the X chromosome were of DBA/2 origin in the backcross. During the construction of these novel strains, >4000 animals were generated, phenotyped, and genotyped. Using these data, we studied the genetic control of variation in body weight and weight gain at 21, 42, and 63 days. The unique data set facilitated the analysis of chromosomal interaction with sex and parent-of-origin effects. All analyzed chromosomes affected body weight and weight gain either directly or in interaction with sex or parent of origin. The effects were age specific, with some chromosomes showing opposite effects at different stages of development.

  18. Cell suspension method to improve green spot in in-vitro culture of jarak pagar (Jatropha curcas L ) mutant lines

    International Nuclear Information System (INIS)

    Ita Dwimahyani

    2007-01-01

    Jatropha curcas has a high potential as an alternative energy source, since it can produce natural oil which could be processed into fuel replacing fossil energy. Increasing demand of biodiesel has resulted in increasing demand for high quality of Jatropha germplasm. Cell suspension method is expected to assure the production of a homogeneous germplasm of Jatropha. A laboratory experiment was conducted to evaluate the effectiveness cell suspension method in of Jatropha curcas cotyledon. The explant used in this experiment was Jatropha curcas seed mutant line (JH-38) which has superiority in plant height, early maturity and unseasonable fruiting. Two kinds of in-vitro medium were used for callus induction, i.e. medium A (MS + 2,4-D 2.0 mg/l + BAP 0.5 mg/l + malt extract 0.1 g + agar 8.0 g/l) and medium B (MS + 2,4-D 3.0 mg/l + BAP 0,5 mg/l + malt extract 0,1 g + agar 8.0 g/l). The same medium composition without agar was used for cell generating, and medium ECS (MS + glutamine 0.5 g + casein hydrolysate 0.5 g + IAA 0.5 mg/l + BAP 3.0 mg/l + agar 8.0 g/l for cell growth. Results of the experiment showed that the optimum growth of calli was obtained by explant JH-38/3 in medium A. The growth level of embryonic cell ranged from 0 to 130 %. The optimum percentage green spot is shown by JH-38/1 explant in medium A. (author)

  19. Morphological and functional responses of a metal-tolerant sunflower mutant line to a copper-contaminated soil series.

    Science.gov (United States)

    Kolbas, Aliaksandr; Kolbas, Natallia; Marchand, Lilian; Herzig, Rolf; Mench, Michel

    2018-04-02

    The potential use of a metal-tolerant sunflower mutant line for biomonitoring Cu phytoavailability, Cu-induced soil phytotoxicity, and Cu phytoextraction was assessed on a Cu-contaminated soil series (13-1020 mg Cu kg -1 ) obtained by fading a sandy topsoil from a wood preservation site with a similar uncontaminated soil. Morphological and functional plant responses as well as shoot, leaf, and root ionomes were measured after a 1-month pot experiment. Hypocotyl length, shoot and root dry weight (DW) yields, and leaf area gradually decreased as soil Cu exposure rose. Their dose-response curves (DRC) plotted against indicators of Cu exposure were generally well fitted by sigmoidal curves. The half-maximal effective concentration (EC 50 ) of morphological parameters ranged between 203 and 333 mg Cu kg -1 soil, corresponding to 290-430 μg Cu L -1 in the soil pore water, and 20 ± 5 mg Cu kg -1 DW in the shoots. The EC 10 for shoot Cu concentration (13-15 mg Cu kg -1 DW) coincided to 166 mg Cu kg -1 soil. Total chlorophyll content and total antioxidant capacity (TAC) were early biomarkers (EC 10 : 23 and 51 mg Cu kg -1 soil). Their DRC displayed a biphasic response. Photosynthetic pigment contents, e.g., carotenoids, correlated with TAC. Ionome was changed in Cu-stressed roots, shoots, and leaves. Shoot Cu removal peaked roughly at 280 μg Cu L -1 in the soil pore water.

  20. Correlation between activation of PPAR¿ and resistin downregulation in a mouse adipocyte cell line by a series of thiazolidinediones.

    NARCIS (Netherlands)

    Sotiriou, A.; Blaauw, R.H.; Meijer, C.; Gijsbers, L.H.; Burg, van der B.; Vervoort, J.; Rietjens, I.M.C.M.

    2013-01-01

    The present study shows significant correlations between the EC50 for PPAR¿ activation in a reporter gene cell line and resistin downregulation in mouse adipocytes, and between the IC50 for resistin downregulation and the already published minimum effective dose for antihyperglycemic activity in a

  1. The effects of tributyltin oxide and deoxynivalenol on the transcriptome of the mouse thymoma cell line EL-4

    NARCIS (Netherlands)

    Schmeits, P.J.M.; Kol, S.; Loveren, van H.; Peijnenburg, A.A.C.M.; Hendriksen, P.J.M.

    2014-01-01

    The main goal of this study was to assess the potential of the mouse thymoma EL-4 cell line in screening for chemical induced immunotoxicity. Therefore, EL-4 cells were exposed to two well-known immunotoxicants, organotin compound tributyltin oxide (TBTO, 0.5 and 1 µM for 3 or 6 h) and the mycotoxin

  2. mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

    Directory of Open Access Journals (Sweden)

    Jennifer L Anderson

    2017-11-01

    Full Text Available As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

  3. Loss and stabilization of resistance to aminopterin in mouse cell lines

    International Nuclear Information System (INIS)

    Safronov, V.V.; Kapitsa, O.S.; Sapegina, M.B.; Gorodetskii, S.I.

    1986-01-01

    Using step-wise selection, lines of mouse L-cells were obtained (clones B-82, TK-), resistance to aminopterin (AP) of which exceeds resistance of parental cells by 10 3 -5 x 10 4 times. Increased resistance is the result of amplification of the gene for dihydrofolate reductase (DHFR), which was established according to increase in enzyme activity by 15-120 times and by cytogenetic methods. Development and disappearance of resistance to AP was studied and karyological analysis of lines obtained was conducted. Two types of karylogical changes were revealed: the presence of double microchromosomes (DM) and of a marker chromosome having homogeneously staining regions (HSR). The localization of the DHFR and HSR genes was demonstrated using in situ hybridization. At early stages of the development of resistance and for a long time, an extrachromosomal localization of amplified genes in the structure of DM, which determine unstable resistance to toxin, is fundamental. The possibility was demonstrated of the long-term existence of cells in which DM and HSR are present simultaneously. Change in number of copies of the DHFR gene in lines of such cells occurs through change in the number of DM, while size and localization of HSR are constant in different conditions of cell culturing. The presence of HSR determines stable resistance to AP. Data were obtained in support of an intermediate relative stabilization of resistance, which is caused by temporary insertion of copies of the DHFR gene into other sections of chromosomes, in addition to HSR dispersed variously through the genome

  4. Absence of linkage of apparently single gene mediated ADHD with the human syntenic region of the mouse mutant coloboma

    Energy Technology Data Exchange (ETDEWEB)

    Hess, E.J.; Rogan, P.K.; Domoto, M. [Pennsylvania State Univ. College of Medicine, Hershey, PA (United States)] [and others

    1995-12-18

    Attention deficit disorder (ADHD) is a complex biobehavioral phenotype which affects up to 8% of the general population and often impairs social, academic, and job performance. Its origins are heterogeneous, but a significant genetic component is suggested by family and twin studies. The murine strain, coloboma, displays a spontaneously hyperactive phenotype that is responsive to dextroamphetamine and has been proposed as a genetic model for ADHD. Coloboma is a semi-dominant mutation that is caused by a hemizygous deletion of the SNAP-25 and other genes on mouse chromosome 2q. To test the possibility that the human homolog of the mouse coloboma gene(s) could be responsible for ADHD, we have carried out linkage studies with polymorphic markers in the region syntenic to coloboma (20p11-p12). Five families in which the pattern of inheritance of ADHD appears to be autosomal dominant were studied. Segregation analysis of the traits studied suggested that the best fitting model was a sex-influenced, single gene, Mendelian pattern. Several genetic models were evaluated based on estimates of penetrance, phenocopy rate, and allele frequency derived from our patient population and those of other investigators. No significant linkage was detected between the disease locus and markers spanning this chromosome 20 interval. 39 refs., 2 figs., 1 tab.

  5. Phosphorus Partitioning of Soybean Lines Containing Different Mutant Alleles of Two Soybean Seed-Specific Adenosine Triphosphate-Binding Cassette Phytic Acid Transporter Paralogs

    Directory of Open Access Journals (Sweden)

    Jason D. Gillman

    2013-03-01

    Full Text Available Seed phytate is a repository of P and minerals in soybean [ (L. Merr.] seeds that limits P and mineral bioavailability for monogastric animals (e.g., humans, swine [], and poultry [especially chicken, ] due to insufficient digestive tract phytase activity. We previously identified epistatic recessive mutations affecting two paralogous adenosine triphosphate-binding cassette phytic acid transporter genes (one a nonsense mutation in and the other a missense mutation in as the molecular genetic basis in the ethyl methanesulfonate (EMS-induced mutant low phytate soybean line M153. An additional mutant low phytate line, M766, contained one single nucleotide polymorphism within the ninth intron of the locus as well as a nonsense mutation in . The objectives of this research were to clarify the genetics underlying the low phytate phenotype in line M766 and to determine P partitioning in new combinations of mutant alleles from M766 and M153. Inheritance of nonsense alleles affecting both ( genes (one from M153 and one from M766 led to the production of viable seeds that contained transgressive reductions in total seed phytate and significantly higher levels of inorganic phosphate than has been reported for nontransgenic soybean material and will allow efficient molecular selection of soybeans with even greater reductions of phytate for improved quality soybean meal.

  6. Evaluation of Nitrogen Uptake and Growth Performance of Advanced Mutant Lines MR219-4 and MR219-9 Grown Under Aerobic Conditions

    International Nuclear Information System (INIS)

    Shyful Azizi Abdul Rahman; Abdul Rahim Harun; Rusli Ibrahim; Khairuddin Abdul Rahim

    2014-01-01

    Developing a good crop production management package; drought resistance variety, effective water and nutrient management in rice production practices is crucial for global climate change adaptation. A research project under IAEA RAS5065 (Supporting Climate-Proofing Rice Production Systems (CRiPS) Based on Nuclear Applications) was conducted from 2012 to 2013, in collaboration with MARDI. Two advanced mutant lines, MR219-4 and MR219-9 were used in this research project to evaluate growth, yield potential and fertilizer uptake under different water input condition (flooded and aerobic). The advanced mutant line MR219-9 showed comparable growth, yield and nitrogen uptake under both flooded and aerobic conditions. The yield and yield components are not significantly different from the parent variety (MR219) but total N uptake was lower than MR219 regardless of water regime. The field trial showed that MR219-9 has a better total N content which is comparable to the aerobic rice variety (MRIA 1) and this indicates that this advance mutant line MR219-9 is a potential aerobic rice variety. (author)

  7. Age-related variability of some characters of karyotype instability in the mouse line CC57W/Mv

    International Nuclear Information System (INIS)

    Glazko, T.T.; Safonova, N.A.; Kovaleva, O.A.; Stolina, M.P.; Solomko, A.P.; Malyuta, S.S.; Glazko, V.I.; AN Ukrainskoj SSR, Kiev

    1995-01-01

    The investigations of relations between cytogenetical variability in cells of bone marrow of the mouse line CC57W/Mv and factors of age and radioactivity pollution (the specific vivarium in the 30-km Chernobyl zone) were carried out. The karyotype instability on some characters were similarly between young mice in the Chernobyl zone and old mice under control conditions. The old Chernobyl mice differentiated from old control ones by a low frequency of some cytogenetic anomalies and higher values of the mitotic index. The contribution of the intensity of cell division into observed variabilities of cytogenetic character between different mouse groups was discussed

  8. Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours.

    Science.gov (United States)

    Chen, Lindi; Esfandiari, Arman; Reaves, William; Vu, Annette; Hogarty, Michael D; Lunec, John; Tweddle, Deborah A

    2018-03-01

    Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.

  9. Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

    Science.gov (United States)

    Al-Soudy, Al-Sayed; Nakanishi, Tsuyoshi; Mizuno, Seiya; Hasegawa, Yoshikazu; Shawki, Hossam H; Katoh, Megumi C; Basha, Walaa A; Ibrahim, Abdelaziz E; El-Shemy, Hany A; Iseki, Hiroyoshi; Yoshiki, Atsushi; Hiromori, Youhei; Nagase, Hisamitsu; Takahashi, Satoru; Oishi, Hisashi; Sugiyama, Fumihiro

    2016-07-01

    Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Evolving of mutant lines resistant to lodging, blast, and high yield in rice by induce mutation using gamma ray (physical mutagen)

    International Nuclear Information System (INIS)

    Majd, F.; Rahimi, M.; Rezazadeh, M.

    2003-01-01

    Induction of mutation for the purpose of producing variations in the gene pool has been used in recent years. In this experiment the locally adapted rice C V Moosa-Tarom was used as a high quality, tall and very lodging susceptible mutation material. The main purpose of this project was to evolve lodging resistant mutants of high yielding. The elite seeds of Moosa-Tarom variety after moisture regulation were exposed to 100, 200 and 300 Gy from Cobalt 60 source at the Nuclear Research Center. The irradiated seeds were sown in the field along with a comparable number of unirradiated seeds taken as control. All the first panicles of M1 plants were individually harvested and classified according to the dose rate as M2 material . Among M2 plant populations 203 plants that appeared from the agronomic point of view, along with a number of on unirradiated seeds, were selected and moved to the next generations. During subsequent screening for three generations (M 3-M 5) and due to lodging resistant, height and efficient factors of yield potential some mutant lines were harvested. From these lines in a preliminary and advanced randomized complete design agronomic traits, 13 promising lines were selected. From the experiment, line 43-3 were confirmed, which is characterized by lodging resistant and high yield. This line showed relative superiority and introduced to Rice Research Institute

  11. X-radiation-induced transformation in a C3H mouse embryo-derived cell line

    International Nuclear Information System (INIS)

    Terzaghi, M.; Little, J.B.

    1976-01-01

    Reproducible x-ray-induced oncogenic transformation has been demonstrated in an established cell line of mouse embryo fibroblasts. Cells derived from transformed foci formed malignant tumors when injected into syngeneic hosts. An exponential increase in the number of transformants per viable cell occurred with doses of up to 400 rads of x-radiation. The transformation frequency in exponentially growing cultures remained constant at 2.3 x 10 -3 following doses of 400 to 1500 rads. There was little change in survival following x-ray doses up to 300 rads. Doses greater than 300 rads were associated with an exponential decline in survival; the D 0 for the survival curve was 175 rads. Transformation frequency varied with changes in the number of viable cells seeded per dish. There was about a 10-fold decline in the transformation frequency when the number of cells was increased from 400 to 1000 viable cells/100-mm Petri dish. Below this density range there was little change in transformation frequency. The presence of lethally preirradiated cells was not associated with an enhancement of transformation in irradiated cells or with the induction of transformation in unirradiated cell cultures. Amphotericin B (Fungizone) inhibited the appearance of transformants when added to the culture medium within 2 to 3 weeks after initiation of the experiment

  12. A new Cre driver mouse line, Tcf21/Pod1-Cre, targets metanephric mesenchyme.

    Directory of Open Access Journals (Sweden)

    Yoshiro Maezawa

    Full Text Available Conditional gene targeting in mice has provided great insight into the role of gene function in kidney development and disease. Although a number of Cre-driver mouse strains already exist for the kidney, development of additional strains with unique expression patterns is needed. Here we report the generation and validation of a Tcf21/Pod1-Cre driver strain that expresses Cre recombinase throughout the condensing and stromal mesenchyme of developing kidneys and in their derivatives including epithelial components of the nephron and interstitial cells. To test the efficiency of this line, we crossed it to mice transgenic for either loss or gain of function β-catenin conditional alleles. Mice with deletion of β-catenin from Tcf21-expressing cells are born with hypoplastic kidneys, hydroureters and hydronephrosis. By contrast, Tcf21-Cre driven gain of function for β-catenin in mice results in fused midline kidneys and hypoplastic kidneys. Finally, we report the first renal mesenchymal deletion of Patched1 (Ptch1, the receptor for sonic hedgehog (Shh, which results in renal cysts demonstrating a functional role of Shh signaling pathway in renal cystogensis. In summary, we report the generation and validation of a new Cre driver strain that provides robust excision in metanephric mesenchyme.

  13. [Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

    Science.gov (United States)

    Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

    2010-02-01

    This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

  14. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    Directory of Open Access Journals (Sweden)

    Andrew Parker

    2015-12-01

    Full Text Available Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182 in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  15. Evaluation and characterization of advanced rice mutant line of rice (Oryza sativa), MR219-4 and MR219-9 under drought condition

    International Nuclear Information System (INIS)

    Abdul Rahim Harun; Zarith Shafika Kamarudin; Abdullah, M.Z.; Anna, L.P.K.; Sobri Hussain; Rusli Ibrahim; Khairuddin abdul Rahim

    2012-01-01

    Two advance rice mutant lines, MR219-4 and MR219-9 derived from mutagenesis of Oryza sativa cv. MR219 with gamma radiation at 300 Gy were evaluated in simulated drought condition in the greenhouse at Malaysian Nuclear Agency. The mutants were evaluated simultaneously with ARN1, a drought resistant variety and MR211 a susceptible cultivar as a check. Randomized complete block design with three replicates was used in the experiment. The evaluation and selection were done based on leaf rolling and leaf drying as well as other agronomic traits, such as, number of tillers per plant, plant height, flag leaf area, grain weight per plant, grain yield per plant, 100-grain weight, harvest index, panicle length and plant biomass. The mutants MR219-4 showed moderate tolerance and MR219-9 showed tolerance to drought respectively as compare to the check variety (ARN1, MR211) and control MR219. Leaf rolling, leaf drying, days to flowering and days to maturity are valuable secondary traits that may provide additional information for selection because of associating with the plant survival under water stress. Further research on expression of drought-tolerant lines under different drought conditions is essential in order to identify particular traits that are associated with drought tolerance and high yield potential. Similarly the importance of secondary traits, relative to other putative traits for drought tolerance, needs to be tested in various environments. (author)

  16. Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research

    NARCIS (Netherlands)

    Tetteh, Paul W.; Kretzschmar, Kai; Begthel, Harry; Van Den Born, Maaike; Korving, Jeroen; Morsink, Folkert; Farin, Henner; Van Es, Johan H.; Offerhaus, G. Johan A; Clevers, Hans

    2016-01-01

    Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic

  17. The Utilization of Premix Flour with Sorghum Mutant Lines Zh-30 Based as Material For Dough Making And Dry Noodle Industry

    International Nuclear Information System (INIS)

    Dwi Djoko Slamet Santosa

    2009-01-01

    Sorghum mutant line Zh-30 is a breeding line developed at the Center for the Application of Isotope and Radiation Technology, BATAN by using mutation techniques. Gamma irradiation with the dose of 300 Gy was used to induced plant genetic variability. Through selection processes on several generations, the mutant line Zh-30 was identified to have better agronomic characteristics, better grain quality and higher yield than the original variety. Research on flour quality of this mutant line was done to identify its potential use in dry noodle. Subsequent experiments, i.e. the effect of kansui (alkaline salt Na 2 CO 3 and K 2 CO 3 ) on rheological properties of dough, the effect of egg addition on rheological properties of dough and cooked noodles. Observations were done on dough which were premix flour I, II and III with 10.2 %, 14.5 % and 17.4 % protein content respectively. The influence of each alkaline salt and their mixture on dough rheology i.e., dough consistency and resistant to extension and extensibility. The kansui Concentration applied were 0, 0.5, 1.0 and 1.5 %. Obviously premix flour I + 0.5 % kansui gave optimal consistency, resistance and extensibility of the dough. The addition of five ml egg to premix I dough + 0.5 % kansui gave optimal results. The increase of egg mellowed the dough, and increase noodle texture and reduce stickiness. Addition of five ml egg already gave significant increase of elasticity, with the highest elasticity was reached by addition of 35 ml egg, although no difference was found for 5 - 35 ml.. (author)

  18. The antioxidative effect of bread crust in a mouse macrophage reporter cell line.

    Science.gov (United States)

    Pötzsch, Sandy; Dalgalarrondo, Michele; Bakan, Benedicte; Marion, Didier; Somoza, Veronika; Stangl, Gabriele; Silber, Rolf-Edgar; Simm, Andreas; Navarrete Santos, Alexander

    2014-10-01

    Free radicals and oxidative stress are important factors in the biology of aging and responsible for the development of age-related diseases. One way to reduce the formation of free radicals is to boost the antioxidative system by nutrition. Heat treatment of food promote the Maillard reaction which is responsible for their characteristic color and taste. During the Maillard reaction reducing sugars react with proteins in a non-enzymatic way leading to the formation of advanced glycation end products (AGEs). As an AGE-rich source our group used bread crust (BCE) to investigate the effect of AGEs on the antioxidant defense. It is well known that the NF-kB pathway is activated by treatment of cells with AGEs. Therefore for stimulation with the BCE we used the macrophage reporter cell line RAW/NF-kB/SEAPorter™. Amino acid analysis and LC-MS/MS by Orbitrap Velo was used to determine the bioactive compounds in the soluble BCE. The radical scavenging effect was conducted by the DPPH-assay. BCE induced the NF-kB pathway in RAW/NF-kB/SEAPorter™ cells and also showed a concentration-dependent antioxidative capacity by the DPPH-assay. With the LC/MS and amino acid analyses, we identified the presence of gliadin in BCE confirmed by using specific gliadin antibodies. By immunoprecipitation (IP) with an antibody against γ-gliadin and western blot probing against the AGE carboxymethyllysine (CML) the presence of AGE-gliadin in BCE was confirmed. Stimulation of the RAW/NF-kB/SEAPorter™ cells with the γ-gliadin depleted fractions did not activate the NF-kB pathway. CML-modified gliadin in the BCE is a bioactive compound of the bread crust which is responsible for the antioxidative capacity and for the induction of the NF-kB pathway in mouse macrophages. Copyright © 2014. Published by Elsevier Inc.

  19. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

    Science.gov (United States)

    Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

  20. High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting

    Directory of Open Access Journals (Sweden)

    Amanda M. Ackermann

    2017-03-01

    Full Text Available Objective: α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER “knock-in” mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. Methods: We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3′ UTR of the Glucagon (Gcg locus in mouse embryonic stem cells (ESCs. Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+ enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Results: Tamoxifen injection of Gcg-CreERT2;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively, as well as in first-wave fetal α-cells (36% and adult enteroendocrine L-cells (33%. Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal. Conclusions: We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice

  1. Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain

    Directory of Open Access Journals (Sweden)

    Hiroshi Kitani

    2014-01-01

    Full Text Available We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7–10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5 was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4–5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

  2. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis.

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

    Full Text Available A lysosomal storage disease (LSD results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.

  3. Absence-like and tonic seizures in aspartoacylase/attractin double-mutant mice.

    Science.gov (United States)

    Gohma, Hiroshi; Kuramoto, Takashi; Matalon, Reuben; Surendran, Sankar; Tyring, Stephen; Kitada, Kazuhiro; Sasa, Masashi; Serikawa, Tadao

    2007-04-01

    The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn(mg-3J) mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.

  4. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    Science.gov (United States)

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. © 2015. Published by The Company of

  5. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

    Science.gov (United States)

    Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

    2012-07-01

    Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma

  6. Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

    International Nuclear Information System (INIS)

    Steinberg, T.H.; Silverstein, S.C.

    1987-01-01

    Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86 Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86 Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86 Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86 Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86 Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86 Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-

  7. Nitrogen Use Efficiency and Carbon Isotope Discrimination Study on NMR151 and NMR152 Mutant Lines Rice at Field Under Different Nitrogen Rates and Water Potentials

    International Nuclear Information System (INIS)

    Ahmad Nazrul Abdul Wahid; Shyful Azizi Abdul Rahman; Abdul Rahim Harun; Latiffah Nordin; Abdul Razak Ruslan; Hazlina Abdullah; Khairuddin Abdul Rahim

    2016-01-01

    This study was conducted to evaluate the nitrogen use efficiency and "1"3C isotope discrimination of rice mutant lines viz. NMR151 and NMR152. Both cultivars are developed under rice radiation mutagenesis programme for adaptability to aerobic conditions. In the present study, NMR151 and NMR152 were grown under conditions of varying water potentials and nitrogen levels in a field. Two water potentials and three nitrogen rates in a completely randomized design with three replications were carried out. The rice mutants were grown for 110 days under two water potentials, (i) Field capacity from 0 to 110 DAS [FC], and (ii) Field capacity from 0 to 40 DAS and 30 % dry of field capacity from 41 to 110 DAS [SS] and three nitrogen rates, (i) 0 kg N/ ha (0N), (ii) 60 kg N/ ha (60N), and (iii) 120 kg N/ ha (120N). "1"5N isotopic tracer technique was used in this study, whereby the "1"5N labeled urea fertilizer 5.20 % atom excess (a.e) was utilized as a tracer for nitrogen use efficiency (NUE) study. "1"5N isotope presence in the samples was determined using emission spectrometry and percentage of total nitrogen was determined by the Kjeldahl method. "1"5N a.e values of the samples were used in the determination of the NUE. The value of "1"3C isotope discrimination (Δ"1"3C) in the sample was determined using isotope ratio mass spectrometry (IRMS). The "1"3C isotope discrimination technique was used as a tool to identify drought resistance rice cultivars with improves water use efficiency. The growth and agronomy data, viz. plant height, number of tillers, grain yield, straw yield, and 1000 grain weight also were recorded. Results from this study showed nitrogen rates imparted significant effects on yield (grain and straw) plant height, number of tillers and 1000 grain weight. Water potentials had significant effects only on 1000 grain weight and Δ"1"3C. The NUE for both mutant lines rice showed no significant different between treatments. Both Rice mutant lines rice NMR151

  8. Microclones derived from the mouse chromosome 7 D-E bands map within the proximal region of the c14CoS deletion in albino mutant mice

    International Nuclear Information System (INIS)

    Toenjes, R.R.W.; Weith, A.; Rinchik, E.M.; Winking, H.; Carnwath, J.W.; Kaliner, B.; Paul, D.

    1991-01-01

    A group of radiation-induced perinatal-lethal deletions that include the albino (c) locus on mouse chromosome 7 causes failure of expression of various hepatocyte-specific genes when homozygous. The transcription of such genes could be controlled in trans by a regulatory gene(s) located within the proximal region of the C14CoS deletion. To identify this potential regulatory gene, a microclone library was established from microdissected D and E bands of chromosome 7. Three nonoverlapping microclones (E305, E336B, and E453B) hybridizing with wildtype but not with C14CoS/C14CoS DNA were isolated. E336B represents a single-copy DNA fragment, whereas E305 and E453B hybridized with 3 and 10 EcoRI DNA restriction fragments, respectively. All fragments map exclusively within the deletion. The microclones hybridized to DNA of viable C6H/C14CoS deletion heterozygotes but not to DNA of homozygotes for the lethal mutation c10R75M, which belongs to the same complementation group as c14CoS. DNA of viable homozygous mutant C62DSD, which carries a deletion breakpoint proximal to that of c6H, hybridized only with E453B. This microclone identified 6 EcoRI restriction fragments in C62DSD/C62DSD DNA. The results demonstrate that of the isolated microclones, E453B identifies a locus (D7RT453B) that maps closest to the hsdr-1 (hepatocyte-specific developmental regulation) locus, which maps between the proximal breakpoints of deletions c10R75M and c62DSD

  9. Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

    Science.gov (United States)

    Masuya, Hiroshi; Shimizu, Kunihiko; Sezutsu, Hideki; Sakuraba, Yoshiyuki; Nagano, Junko; Shimizu, Aya; Fujimoto, Naomi; Kawai, Akiko; Miura, Ikuo; Kaneda, Hideki; Kobayashi, Kimio; Ishijima, Junko; Maeda, Takahide; Gondo, Yoichi; Noda, Tetsuo; Wakana, Shigeharu; Shiroishi, Toshihiko

    2005-03-01

    Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

  10. The pht4;1-3 mutant line contains a loss of function allele in the Fatty Acid Desaturase 7 gene caused by a remnant inactivated selection marker-a cautionary tale.

    Science.gov (United States)

    Nilsson, Anders K; Andersson, Mats X

    2017-01-01

    A striking and unexpected biochemical phenotype was found in an insertion mutant line in the model plant Arabidopsis thaliana . One of two investigated insertion mutant lines in the gene encoding the phosphate transporter PHT4;1 demonstrated a prominent loss of trienoic fatty acids, whereas the other insertion line was indistinguishable from wild type in this aspect. We demonstrate that the loss of trienoic fatty acids was due to a remnant inactive negative selection marker gene in this particular transposon tagged line, pht4;1-3 . This constitutes a cautionary tale that warns of the importance to confirm the loss of this type of selection markers and the importance of verifying the relationship between a phenotype and genotype by more than one independent mutant line or alternatively genetic complementation.

  11. Gamma-ray induction of a mutant soybean [Glycine max (L.) Merrill] line lacking all seed lipoxygenases

    International Nuclear Information System (INIS)

    Hajika, Makita; Suda, Ikuo; Sakai, Shinji; Takahashi, Masakazu

    1997-01-01

    Induction of a soybean line lacking all isozymes of seed lipoxygenase was attempted using γ-radiation and of 1,813 seeds in M 3 generation, only one was identified as a seed lacking all the isozymes by SDS-PAGE. This line did not present any physiological abnormality over 10 generations or more (M 4 -M 14 ) and no significant influence of the enzyme on the agricultural traits was observed during the performance test in fields. In the resistance test against insect pests, significant differences were not found among the varieties and the lines tested. These results suggest that deletion of all lipoxygenase isozymes would not affect the soybean production in practice. The lipoxygenase activity was not detected in the leaves as well as the seeds of this line, suggesting that this enzyme are not indispensable for the soybean growth. The validity of this line in food processing fields was examined through determining the levels of hexanal production and DETBA. This line was found able to improve the taste of soybean cookies and use in combination with other materials as flour, egg, etc. because the line has no lipoxygenase activity. (M.N.)

  12. The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

    Directory of Open Access Journals (Sweden)

    Paudyal Anju

    2010-08-01

    Full Text Available Abstract Background The planar cell polarity (PCP signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. Results We report the identification of a new N-ethyl-N-nitrosourea (ENU-induced mutant with craniorachischisis, which we have named chuzhoi (chz. We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. Conclusions The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation

  13. Effect of pentoxifylline on P-glycoprotein mediated vincristine resistance of L1210 mouse leukemic cell line

    International Nuclear Information System (INIS)

    Breier, A.; Uhrik, B.; Barancik, M.; Stefankova, Z.; Tribulova, N.

    1994-01-01

    Effect of pentoxifylline (PTX) on vincristine (VCR) resistance of multidrug resistant L1210/VCR mouse leukemic cell line was studied. Reversal effect of PTX (in concentration 50-150 mg dm -3 ) on vincristine resistance, i.e. potentiation of vincristine cytotoxicity on L1210/VCR cells by PTX was found. PTX alone in the above concentration did not exert any significant effect on sensitive or resistant cell lines in the absence of vincristine. Resistance of L1210/VCR cell line was found previously to be accompanied with overexpression of drug transporting P-glycoprotein. Indeed, lower level of 3 H-vincristine accumulation by resistant L1210/VCR cell line in comparison with sensitive L1210 cell line was observed. Accumulation of 3 H-vincristine by L1210/VCR cell line was significantly increased in the presence of PTX. PTX in the same condition did not exert any considerable effect on accumulation of 3 H-vincristine by nonresistant L1210 cells. Observable morphological damage was observed in 1210/VCR cells cultivated in medium containing vincristine (0.2 mg dm -3 ) and pentoxifylline (100 mg dm -3 ) in comparison with the non-damaged cells in the presence of vincristine or pentoxifylline alone. The results obtained indicate that pentoxifylline may be considered as a reversal agent in multidrug resistance. (author)

  14. Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

    Directory of Open Access Journals (Sweden)

    M.A. Sukoyan

    2002-05-01

    Full Text Available Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

  15. Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5

    International Nuclear Information System (INIS)

    Yasui, L.S.

    1992-01-01

    Survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125 I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125 I decays. D o values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. Difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125 IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137 Cs gamma radiation survival values (D o and D 10 ) were higher in xrs-5 cells compared with CHO cells, although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125 I decay. (Author)

  16. Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

    Science.gov (United States)

    Tetteh, Paul W; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; Morsink, Folkert; Farin, Henner; van Es, Johan H; Offerhaus, G Johan A; Clevers, Hans

    2016-10-18

    Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1 CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1 CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1 CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

  17. Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background

    OpenAIRE

    Carstea, Ana Claudia; Pirity, Melinda K; Dinnyes, Andras

    2009-01-01

    In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression (“knock-out”), or to introduce mutant or modified alleles at the locus of interest (“knock-in”). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence ger...

  18. Isolation and characterization of a new cell line from spontaneous mouse mammary tumour, MBL-6, for in vivo cancer studies

    Directory of Open Access Journals (Sweden)

    Ladan Langroudi

    2017-12-01

    Full Text Available In search for treatments against breast cancer, cell lines are one of the basic resources, particularly as in vitro models. Additionally, animal models of cancer are used as the successive step in therapeutics research. In this regard, human breast cancer cell lines provide fundamental models in vitro. However, in vivo studies require immunodeficient mice, which lack the influence of other in vivo factors such as the native microenvironment and the immune system. There are few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer. In this study, a new mouse breast cancer cell line, MBL-6, was successfully established and characterized from tissues of a spontaneous mammary tumor. The cell line had epithelial morphology, formed adherent monolayer, maintained continuously in vitro and was able to form new tumors when injected subcutaneously in syngeneic mice. The growth pattern and metastasis evaluations revealed a considerable in situ duration before invading distant organs. Real time polymerase chain reaction (PCR analysis showed the expression of ER-, PR- and Her-2 receptors. The chromosome analysis showed numerous chromosomal abnormalities. Aggressive tumorigenecity in tumorigenesis test and the IC50 to cyclophosphamide (CTX, celecoxib (CLX and cisplatin (CPN was also evaluated. The numerous tests performed on the new MBL-6 cell line suggest that it is in good quality and may be used in animal models of breast cancer studies.

  19. Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

    Science.gov (United States)

    Jaiswal, A K; Nebert, D W; Eisen, H W

    1985-08-01

    The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

  20. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira

    2007-01-01

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53ΔC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53ΔC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain

  1. The perfect host: a mouse host embryo facilitating more efficient germ line transmission of genetically modified embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Robert A Taft

    Full Text Available There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs. We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium. ESC germline transmission was observed in 9/11 (82% lines using PH blastocysts, compared to 6/11 (55% when conventional host blastocysts were used. Furthermore, less than 35% (83/240 of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137 of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the

  2. Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

    Energy Technology Data Exchange (ETDEWEB)

    Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

    1988-03-25

    The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human x-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, the authors introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.

  3. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  4. Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

    Science.gov (United States)

    De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

    2017-06-01

    Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') 2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can

  5. A knock-in mouse line conditionally expressing the tumor suppressor WTX/AMER1.

    Science.gov (United States)

    Boutet, Agnès; Comai, Glenda; Charlet, Aurélie; Jian Motamedi, Fariba; Dhib, Haroun; Bandiera, Roberto; Schedl, Andreas

    2017-11-01

    WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele. © 2017 Wiley Periodicals, Inc.

  6. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    International Nuclear Information System (INIS)

    Medina, D.; Oborn, C.J.; Li, M.L.; Bissell, M.J.

    1987-01-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of β-casein mRNA in the presence or absence of prolactin. The inducibility of β-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types

  7. Altered Baseline and Nicotine-Mediated Behavioral and Cholinergic Profiles in ChAT-Cre Mouse Lines.

    Science.gov (United States)

    Chen, Edison; Lallai, Valeria; Sherafat, Yasmine; Grimes, Nickolas P; Pushkin, Anna N; Fowler, J P; Fowler, Christie D

    2018-02-28

    The recent development of transgenic rodent lines expressing cre recombinase in a cell-specific manner, along with advances in engineered viral vectors, has permitted in-depth investigations into circuit function. However, emerging evidence has begun to suggest that genetic modifications may introduce unexpected caveats. In the current studies, we sought to extensively characterize male and female mice from both the ChAT (BAC) -Cre mouse line, created with the bacterial artificial chromosome (BAC) method, and ChAT (IRES) -Cre mouse line, generated with the internal ribosome entry site (IRES) method. ChAT (BAC) -Cre transgenic and wild-type mice did not differ in general locomotor behavior, anxiety measures, drug-induced cataplexy, nicotine-mediated hypolocomotion, or operant food training. However, ChAT (BAC) -Cre transgenic mice did exhibit significant deficits in intravenous nicotine self-administration, which paralleled an increase in vesicular acetylcholine transporter and choline acetyltransferase (ChAT) hippocampal expression. For the ChAT (IRES) -Cre line, transgenic mice exhibited deficits in baseline locomotor, nicotine-mediated hypolocomotion, and operant food training compared with wild-type and hemizygous littermates. No differences among ChAT (IRES) -Cre wild-type, hemizygous, and transgenic littermates were found in anxiety measures, drug-induced cataplexy, and nicotine self-administration. Given that increased cre expression was present in the ChAT (IRES) -Cre transgenic mice, as well as a decrease in ChAT expression in the hippocampus, altered neuronal function may underlie behavioral phenotypes. In contrast, ChAT (IRES) -Cre hemizygous mice were more similar to wild-type mice in both protein expression and the majority of behavioral assessments. As such, interpretation of data derived from ChAT-Cre rodents must consider potential limitations dependent on the line and/or genotype used in research investigations. SIGNIFICANCE STATEMENT Altered

  8. Estimation of genetic variability level in inbred CF1 mouse lines ...

    Indian Academy of Sciences (India)

    To estimate the genetic variability levels maintained by inbred lines selected for body weight and to compare them with a nonselected population from which the lines were derived, we calculated the per cent polymorphic loci (P) and marker diversity (MD) index from data on 43 putative loci of inter simple sequence repeats ...

  9. The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Frentzel Stefan

    2010-06-01

    Full Text Available Abstract Background Huntington's disease (HD is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD. In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD. Results Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels. Conclusions Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

  10. Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Hashiguchi, Kohtaro; Ozaki, Masumi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Kuraoka, Isao [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, Kumamoto (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.

  11. Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

    International Nuclear Information System (INIS)

    Mill, Christopher P.; Gettinger, Kathleen L.; Riese, David J.

    2011-01-01

    Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.

  12. Germ-line mutations at a mouse ESTR (Pc-3) locus and human microsatellite loci

    International Nuclear Information System (INIS)

    Ryo, Haruko; Nakajima, Hiroo; Nomura, Taisei

    2006-01-01

    We examined the use of the mouse Pc-3 ESTR (expanded simple tandem repeat) locus and 72 human microsatellite loci as potentially sensitive biomarkers for mutagenic exposures to germ cells in mice and humans respectively. In the mouse work, we treated male mice with TCDD (2, 3, 7, 8-tetrachlo-rodibenzo-p-dioxin; a chemical known to induce congenital anomalies in humans and mice) and, analysed the F 1 fetuses for Pc-3 mutations. Although the incidence of anomalies was higher in the TCDD group, there were no induced mutations. However, respiratory distress syndrome (RDS) was observed in 3 of 7 fetuses born to male mice which were treated with TCDD and which showed abnormal length of Pc-3 allele. In the human studies, the children of Chernobyl liquidators were examined for mutations at a total of 72 (31 autosomal, 1 X-linked and 40 Y-linked) microsatellite loci. This study was prompted by earlier findings of increases in microsatellite mutations in barn swallows and wheat in the highly contaminated areas after the Chernobyl accident. We examined 64 liquidator families (70 children) and 66 control families (70 children). However, no increases in mutation rates were found. The estimated mean dose to the liquidators was about 39 mSv and this might be one possible reason why no increases of mutations could be found. (author)

  13. A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

    International Nuclear Information System (INIS)

    Suzuki, N.

    1984-01-01

    When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl- 3 H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity. (Auth.)

  14. Assessment of estradiol-induced gene regulation and proliferation in an immortalized mouse immature Sertoli cell line.

    Science.gov (United States)

    Kumar, Narender; Srivastava, Swati; Burek, Malgorzata; Förster, Carola Y; Roy, Partha

    2016-03-01

    The number of Sertoli cells during proliferative phase determines the fate of the germ cells in male reproductive system. A well-characterized cell line may help in better understanding of Sertoli cell biology. Hence, the present study assessed estradiol signaling in a mouse immature Sertoli cell line (MSC-1) as an alternative model in place of primary culture of Sertoli cells. In this study, we used MSC-1 cell line, derived from 10-day old mice. The cell cycle parameters were assessed, and the expression and regulation of Sertoli cell-specific secretory genes (ABP; androgen-binding protein) and tight junction genes (claudin-5, occludin, and vimentin) in response to estradiol was studied. The results obtained suggested the presence of both estrogen receptors (ERα and ERβ) in MSC-1 cells. In vitro scratch assay and cell-cycle analysis suggested the proliferative effects of estradiol in both time- and dose-dependent manner. The gene expression profiles of ABP, claudin-5, and occludin showed biphasic regulation at low and high doses of estradiol. Analysis of signaling pathways suggested the activation of extracellular signal-regulated kinase (ERK) pathway with significantly increased pERK/ERK ratio (p<0.05). The results also suggested down regulation in the expression of mir-17 family members (mir-17, mir-20b, and mir-106a) (p<0.05). Considering the limited number of Sertoli cell lines and long-term survival inability of primary culture of Sertoli cells, MSC-1 cells could be a potential cell line for understanding the mechanisms of various cellular events in Sertoli cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance.

    Science.gov (United States)

    Monderer, David; Luseau, Alexandrine; Bellec, Amélie; David, Emmanuelle; Ponsolle, Stéphanie; Saiagh, Soraya; Bercegeay, Sylvain; Piloquet, Philippe; Denis, Marc G; Lodé, Laurence; Rédini, Françoise; Biger, Marine; Heymann, Dominique; Heymann, Marie-Françoise; Le Bot, Ronan; Gouin, François; Blanchard, Frédéric

    2013-10-01

    Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance

  16. Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

    Science.gov (United States)

    Tamada, Hiromi; Kiyama, Hiroshi

    2015-01-01

    Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

  17. SkyMouse: A smart interface for astronomical on-line resources and services

    OpenAIRE

    CUI, Chen-Zhou; SUN, Hua-Ping; ZHAO, Yong-Heng; LUO, Yu; QI, Da-Zhi

    2007-01-01

    With the development of network and the World Wide Web (WWW), the Internet has been growing and changing dramatically. More and more on-line database systems and different kinds of services are available for astronomy research. How to help users find their way through the jungle of information services becomes an important challenge. Although astronomers have been aware of the importance of interoperability and introduced the concept of Virtual Observatory as a uniform environment for future ...

  18. Chromosome alterations in the X-ray-induced transformants of cultured mouse cell line

    International Nuclear Information System (INIS)

    Kodama, Seiji; Komatsu, Kenshi; Okumura, Yutaka; Sasaki, M.S.

    1989-01-01

    Mouse m5S cells were subjected to soft X-ray irradiation. Twenty-four transformants were separated as indicators of focus formation. Two clones, cl.4103 and cl.6310, were chosen for the analysis of chromosome alterations in transformants. A parent strain, m5S/1M, served as the control. Anchorage independence (AG) was not detected in the control strain, irrespective of culture conditions and population doubling number (PDN). In the case of transformants, the frequency of AG was increased with increasing PDN for cl.4103, and was constant for cl.6310, irrespective of PDN. Karyotype of m5S/1M was 42, X, -Y, +der (6) t(6;13), t(8;8), +8, +15. In addition, -13, der(10) and -der(6)t(6;13), der(5), +mar occurred as karyotype alterations for cl.4103 and cl. 6310, respectively. The present experiment indicated that chromosome alterations secondary to primary alterations occur in a high frequency in the transforming process of X-ray irradiated cells, and that the secondary chromosome alterations result in selective proliferation of transformed clones. (Namekawa, K)

  19. Mouse Model Resources for Vision Research

    Directory of Open Access Journals (Sweden)

    Jungyeon Won

    2011-01-01

    Full Text Available The need for mouse models, with their well-developed genetics and similarity to human physiology and anatomy, is clear and their central role in furthering our understanding of human disease is readily apparent in the literature. Mice carrying mutations that alter developmental pathways or cellular function provide model systems for analyzing defects in comparable human disorders and for testing therapeutic strategies. Mutant mice also provide reproducible, experimental systems for elucidating pathways of normal development and function. Two programs, the Eye Mutant Resource and the Translational Vision Research Models, focused on providing such models to the vision research community are described herein. Over 100 mutant lines from the Eye Mutant Resource and 60 mutant lines from the Translational Vision Research Models have been developed. The ocular diseases of the mutant lines include a wide range of phenotypes, including cataracts, retinal dysplasia and degeneration, and abnormal blood vessel formation. The mutations in disease genes have been mapped and in some cases identified by direct sequencing. Here, we report 3 novel alleles of Crxtvrm65, Rp1tvrm64, and Rpe65tvrm148 as successful examples of the TVRM program, that closely resemble previously reported knockout models.

  20. A Comparison Between House Mouse Lines Selected for Attack Latency or Nest-Building : Evidence for a Genetic Basis of Alternative Behavioral Strategies

    NARCIS (Netherlands)

    Sluyter, Frans; Bult, Abel; Lynch, Carol B.; Oortmerssen, Geert A. van; Koolhaas, Jaap M.

    House mouse lines bidirectionally selected for either nest-building behavior or attack latency were tested for both attack latency and nest-building behavior under identical conditions. Male mice selected for high nest-building behavior had shorter attack latencies, i.e., were more aggressive, than

  1. Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

    Science.gov (United States)

    Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

    2012-11-01

    Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

  2. [Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

    Science.gov (United States)

    Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

    2010-10-01

    This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

  3. 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

    Science.gov (United States)

    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

  4. Intrinsic properties of lumbar motor neurones in the adult G127insTGGG superoxide dismutase-1 mutant mouse in vivo: evidence for increased persistent inward currents

    DEFF Research Database (Denmark)

    Meehan, Claire Francesca; Moldovan, Mihai; Marklund, Stefan L.

    2010-01-01

    Aim: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a preferential loss of motoneurones. Previous publications using in vitro neonatal preparations suggest an increased excitability of motoneurones in various superoxide dismutase-1 (SOD1) mutant mice...... of an increased PIC and less spike frequency adaptation which may contribute to excitotoxity of these neurones as the disease progresses....

  5. Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

    Science.gov (United States)

    Tamada, Hiromi; Kiyama, Hiroshi

    2015-01-01

    Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

  6. RPLC-lon-trap-FTMS method for lipid profiling of plasma: Method validation And application to p53 mutant mouse model

    NARCIS (Netherlands)

    Hu, C.; Dommelen, J. van; Heijden, R. van der; Spijksma, G.; Reijmers, T.H.; Wang, M.; Slee, E.; Lu, X.; Xu, G.; Greef, J. van der; Hankemeier, T.

    2008-01-01

    A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometry method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C8 column and a binary gradient, more than 160 lipids belonging to

  7. Genes and Alcohol Consumption: Studies with Mutant Mice

    Science.gov (United States)

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  8. Cell line established starting with a mouse mammary tumor. Effect of the addition of hormones

    Energy Technology Data Exchange (ETDEWEB)

    Mouriquand, J

    1973-12-31

    From 7th international conference on mammary tumors; SaintPierre-de- Chartreuse, France (12 Jun 1972). The PS-MT cell line was defined (18th passage); isolated from a pool of mammary tumors of the PS strain of mice, it remained dormant for 6 months and then grew out very slowly. Subcultures were possible only after 19 months. The morphology is epithelial. After storage in liquid nitrogen in a medium containing 5% DMSO, the viability was approximately 80%. It was not possible to disclose the presence of mycoplasmas. With the standard insulincontaining medium, a few C-type particles were observed by electron-microscopic examination. The addition of hydrocortisone or prolactin, or both hormones together, increases slightly the production of C-type particles. If the secretory stimulating activity of hydrocortisone is maintained for one week before the addition of prolactin for another week, a large amount of A and B particles are found, mixed with C-type particles. They are present in large number in the pellets obtained from the tissue culture media. Five mammary tumors within 6 months were obtained in BALB/c females. Thus, the production of A- and B-type particles is hormone-dependent and requires the same sequence of hormones as the production of casein by mammary glands in organ cultures. (auth)

  9. Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

    Science.gov (United States)

    Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

    2015-10-29

    Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier

  10. Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line.

    Science.gov (United States)

    Liu, Chuan; Duan, Weixia; Xu, Shangcheng; Chen, Chunhai; He, Mindi; Zhang, Lei; Yu, Zhengping; Zhou, Zhou

    2013-03-27

    Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

  11. Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

    Science.gov (United States)

    Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

    2014-04-01

    The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

  12. Transgenic Mouse Lines Subdivide External Segment of the Globus Pallidus (GPe) Neurons and Reveal Distinct GPe Output Pathways

    Science.gov (United States)

    Mastro, Kevin J.; Bouchard, Rachel S.; Holt, Hiromi A. K.

    2014-01-01

    Cell-type diversity in the brain enables the assembly of complex neural circuits, whose organization and patterns of activity give rise to brain function. However, the identification of distinct neuronal populations within a given brain region is often complicated by a lack of objective criteria to distinguish one neuronal population from another. In the external segment of the globus pallidus (GPe), neuronal populations have been defined using molecular, anatomical, and electrophysiological criteria, but these classification schemes are often not generalizable across preparations and lack consistency even within the same preparation. Here, we present a novel use of existing transgenic mouse lines, Lim homeobox 6 (Lhx6)–Cre and parvalbumin (PV)–Cre, to define genetically distinct cell populations in the GPe that differ molecularly, anatomically, and electrophysiologically. Lhx6–GPe neurons, which do not express PV, are concentrated in the medial portion of the GPe. They have lower spontaneous firing rates, narrower dynamic ranges, and make stronger projections to the striatum and substantia nigra pars compacta compared with PV–GPe neurons. In contrast, PV–GPe neurons are more concentrated in the lateral portions of the GPe. They have narrower action potentials, deeper afterhyperpolarizations, and make stronger projections to the subthalamic nucleus and parafascicular nucleus of the thalamus. These electrophysiological and anatomical differences suggest that Lhx6–GPe and PV–GPe neurons participate in different circuits with the potential to contribute to different aspects of motor function and dysfunction in disease. PMID:24501350

  13. Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

    Science.gov (United States)

    Kern, R M; Yang, Z; Kim, P S; Grody, W W; Iyer, R K; Cederbaum, S D

    2007-01-01

    Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

  14. Estimation of genetic variability, mutagenic effectiveness and efficiency in M2 flower mutant lines of Capsicum annuum L. treated with caffeine and their analysis through RAPD

    Directory of Open Access Journals (Sweden)

    Rumana Aslam

    2017-07-01

    Full Text Available In the present investigation healthy and certified seeds of Capsicum annuum were treated with five concentrations of caffeine i.e. 0.10%, 0.25%, 0.50%, 0.75% and 1.0%. Germination percentage, plants survival and pollen fertility were decreased with the increase of caffeine concentrations. Similarly root length and shoot length were decreased as the concentrations increased in M1 generation. Different mutants were isolated in M1 generation. In M2 generation, various flower mutants with changes in number of sepals, petals, anther size colour i.e. Trimerous, tetramerous, pentamerous with fused petals, hexamerous etc were segregated. Heptamerous and anther change was not observed in lower concentration viz. 0.1%. All these mutants showed significant changes in morphological characters and good breeding values at lower and intermediate concentrations. Mutagenic effectiveness and efficiency was observed on the basis of M2 flower mutant frequency. It was generally decreased with the increase of mutagen concentrations. Cytological aberrations in mutants showed the decreasing trend at meiotic final stages. These mutants were further analysed through RAPD method and on the basis of appearance of polymorphic DNA bands, they distinguished these flower mutants genotypically. Among 93 bands 44 bands were polymorphic which showed great genetic variation produced by caffeine. As an outcome of that the above caffeine concentrations are good for the induction of genetic variability in Capsicum genotype.

  15. The mouse small eye mutant, Del(2)Sey3H, which deletes the putative tumor suppressor region of the radiation-induced acute myeloid leukemia is susceptible to radiation

    International Nuclear Information System (INIS)

    Nitta, Yumiko; Yoshida, Kazuko; Tanaka, Kimio; Peters, Jo; Cattanach, Bruce M.

    2003-01-01

    Radiation-induced murine acute myeloid leukemia (AML) is characterized by the chromosome 2 deletions. Standing on the hypothesis that an AML suppressor gene would locate on the chromosome 2, a deletion-wide screen was performed on radiation-induced AMLs by the fluorescence in situ hybridization (FISH) method. The hemizugous deletion of the D2Mit15, a marker DNA at the 49.0cM region from the centromere, associated with the AMLs in 97 out of the 105 cases (92.4%). As the deletion region was close to the region of human WAGR syndrome (MIM194072), the mouse small eye mutants could be the animal model for radiation-induced AMLs. The mutant, Del(2)Sey3H (Sey3H) was found to delete around the 49.0cM region by the allelic loss mapping. The Sey3H showed high susceptibility to radiation to develop tumors including the myeloid leukemia with shorter latency. These finding support the existence of a putative tumor suppressor gene responsible for the radiation-leukemogenesis near the D2Mit15 region. (author)

  16. A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal huntingtin’s polyglutamine stretch on CAG140 mouse model pathogenesis

    Directory of Open Access Journals (Sweden)

    Zheng Shuqiu

    2012-08-01

    Full Text Available Abstract Background Huntington’s disease (HD is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ stretch within Huntingtin (htt, the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR in modulating HD mouse model pathogenesis is currently unknown. Results We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh encoding N-terminal hemaglutinin (HA or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt. Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

  17. Towards mouse models of perseveration: a heritable component in extinction of operant behavior in fourteen standard and recombinant inbred mouse lines.

    NARCIS (Netherlands)

    Malkki, H.A.I.; Donga, L.A.B.; de Groot, S.E.; Brussaard, A.B.; Borst, J.G.G.; Elgersma, J.W.; Galjart, N.; van der Horst, G.T.; Levelt, C.N.; Pennartz, C.M.A.; Smit, A.B.; Spruijt, B.M.; Verhage, M.; de Zeeuw, C.I.; Battaglia, F.P.

    2011-01-01

    Extinction of instrumental responses is an essential skill for adaptive behavior such as foraging. So far, only few studies have focused on extinction following appetitive conditioning in mice. We studied extinction of appetitive operant lever-press behavior in six standard inbred mouse strains

  18. Productive mutants of niger

    International Nuclear Information System (INIS)

    Misra, R.C.

    2001-01-01

    Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

  19. ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function.

    Science.gov (United States)

    Sharma, Aarti; Lyashchenko, Alexander K; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z; Shneider, Neil A

    2016-02-04

    Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

  1. Localization of the homolog of a mouse craniofacial mutant to human chromosome 18q11 and evaluation of linkage to human CLP and CPO

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-06-15

    The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locus ax. To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci. 23 refs., 4 figs., 2 tabs.

  2. Lessons from the use of genetically modified Drosophila melanogaster in ecological studies: Hsf mutant lines show highly trait-specific performance in field and laboratory thermal assays

    DEFF Research Database (Denmark)

    Sørensen, Jesper Givskov; Loeschcke, Volker; Kristensen, Torsten Nygård

    2009-01-01

    . 2.  We have tested the importance of inducible heat shock proteins (Hsps) under different thermal conditions using two heat shock factor (Hsf) mutant lines (either able (Hsf+) or unable (Hsf0) to mount a heat stress response) and an outbred laboratory adapted wild-type line of Drosophila......1.  Laboratory studies on genetically modified strains may reveal important information on mechanisms involved in coping with thermal stress. However, to address the evolutionary significance of specific genes or physiological mechanisms, ecologically relevant field tests should also be performed...

  3. New hybrid rice cultivar 'Zhefuliangyou 12' with improved grain quality produced by leaf color marker-labeled male sterile line and mutant with enhanced tillers and improved grain quality

    International Nuclear Information System (INIS)

    Mei Shufang; Zhao Hua; Wang Yongqiang; Shu Xiaoli; Wu Dianxing

    2013-01-01

    In order to breed high yielding and good grain quality new variety with controllable seed purity, hybrid rice 'Zhefuliangyou 12' was produced by green-revertible albino leaf color marker-labeled two-line male sterile line 'NHR111S' and mutant 'ZF-2' with enhanced tillers and improved grain quality, which was characterized by improved grain quality, rice blast resistance and lodging resistance. Breeding protocol, characteristics, and high yielding cultivation techniques of 'Zhefuliangyou 12' were briefly introduced in the current paper. (authors)

  4. Baseline and Trend of Lymphocyte-to-Monocyte Ratio as Prognostic Factors in Epidermal Growth Factor Receptor Mutant Non-Small Cell Lung Cancer Patients Treated with First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

    Directory of Open Access Journals (Sweden)

    Yu-Mu Chen

    Full Text Available Patients with early-stage lung cancer who have a high baseline lymphocyte-to-monocyte ratio (LMR have a favorable prognosis. However, the prognostic significance of LMR in patients with advanced-stage EGFR-mutant non-small cell lung cancer (NSCLC receiving first-line epidermal growth factor receptor (EGFR-tyrosine kinase inhibitors (TKIs has not been established. We conducted a retrospective analysis to investigate the influence of LMR on clinical outcomes including progression-free survival (PFS and overall survival (OS in EGFR-mutant patients with NSCLC.Of 1310 lung cancer patients diagnosed between January 2011 and October 2013, 253 patients receiving first-line EGFR-TKIs for EGFR-mutant NSCLC were included. The cut-off values for baseline and the 1-month-to-baseline ratio of LMR (MBR, determined by using receiver operating characteristic curves, were 3.29 and 0.63, respectively. Patients were divided into 3 prognostic groups: high LMR and MBR, high LMR or MBR, and low LMR and MBR.The mean patient age was 65.2 years, and 41% were men. The median PFS and OS were 10.3 and 22.0 months, respectively. The PFS in patients with high LMR and MBR, high LMR or MBR, and low LMR and MBR were 15.4, 7.1, and 2.0 months, respectively (p < 0.001, whereas the OS were 32.6, 13.7, and 5.1 months, respectively (p < 0.001.A combination of baseline and trend of LMR can be used to identify patients with a high mortality risk in EGFR-mutant NSCLC patients receiving first-line EGFR-TKIs.

  5. Repair of exogenous DNA double-strand breaks promotes chromosome synapsis in SPO11-mutant mouse meiocytes, and is altered in the absence of HORMAD1.

    Science.gov (United States)

    Carofiglio, Fabrizia; Sleddens-Linkels, Esther; Wassenaar, Evelyne; Inagaki, Akiko; van Cappellen, Wiggert A; Grootegoed, J Anton; Toth, Attila; Baarends, Willy M

    2018-03-01

    Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11 YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11 YF/YF mice, 140-160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11 YF/YF compared to Spo11 +/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11 YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Effect of iso-osmotic salt and water stress in relation to adjustment on mutant sugarcane (Saccharum officinarum L.) plant lines

    International Nuclear Information System (INIS)

    Ahuja, Akash V.; Kalwade, Sachin B.; Nikam, Ashok A.; Devarumath, R.M.; Chauvan, Viraj S.; Kanse, Sangram S.

    2014-01-01

    Gamma radiation induced mutagenesis followed by in vitro selection was employed for salt tolerance in popular sugarcane (Saccharum officinarum L.) cv.CoM0265. Assimilated regenerated mutant plantlets were planted on control as well as salt affected soil. Mutants which showed relatively good response with respect to its quantitative and qualitative parameters were selected for priming experiment. Nine mutants and its respective control and parent control which are known to vary in salt tolerance under field conditions were studied. In order to discriminate between the ionic and osmotic components of salt stress, mutant plants were treated with NaCl salt (100 mM) or polyethylene glycol-PEG 8000 solutions (20%) for 10 days. Both NaCI and PEG treatment significantly reduced leaf width, number of green leaves and chlorophyll stability index. Osmotic adjustment indicated that the NaCI and PEG stress lead to accumulation of osmolytes, however sugar level changes non significantly. The ion concentration was drastically affected upon NaCI treatment, whereas PEG stress accumulated relatively less amount of Na + ions in comparison to NaCl. However, there was an increase in K + concentration upon PEG treatment, whereas NaCI stress accumulated less K + concentration with respect to PEG and control. The NaCI and PEG treated mutant plants showed increased activities of superoxide dismutase (SOD) and Catalase (CAT) in comparison to its control and parent control. Among the mutant selected gamma rays irradiation in corporation with enhanced tolerance to abiotic stress is one of the important goals for the biotechnological improvement of crop plants. Enhanced salinity tolerance may prove beneficial to improve the competitiveness of the popular sugarcane cultivars and their commercial cultivation in saline areas. (author)

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Decreased reactivation of a herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) mutant using the in vivo mouse UV-B model of induced reactivation

    Science.gov (United States)

    BenMohamed, Lbachir; Osorio, Nelson; Srivastava, Ruchi; Khan, Arif A.; Simpson, Jennifer L.; Wechsler, Steven L.

    2015-01-01

    Blinding ocular herpetic disease in humans is due to herpes simplex virus type 1 (HSV-1) reactivations from latency, rather than to primary acute infection. The cellular and molecular mechanisms that control the HSV-1 latency-reactivation cycle remain to be fully elucidated. The aim of this study was to determine if reactivation of the HSV-1 latency associated transcript (LAT) deletion mutant (dLAT2903) was impaired in this model, as it is in the rabbit model of induced and spontaneous reactivation and in the explant TG induced reactivation model in mice. The eyes of mice latently infected with wild type HSV-1 strain McKrae (LAT(+) virus) or dLAT2903 (LAT(−) virus) were irradiated with UV-B and reactivation was determined. We found that compared to LAT(−) virus, LAT(+) virus reactivated at a higher rate as determined by shedding of virus in tears on days 3 to 7 after UV-B treatment. Thus, the UV-B induced reactivation model of HSV-1 appears to be a useful small animal model for studying the mechanisms involved in how LAT enhances the HSV-1 reactivation phenotype. The utility of the model for investigating the immune evasion mechanisms regulating the HSV-1 latency/reactivation cycle and for testing the protective efficacy of candidate therapeutic vaccines and drugs are discussed. PMID:26002839

  10. Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

    Science.gov (United States)

    Migliarini, Sara; Pacini, Giulia; Pasqualetti, Massimo

    2015-01-01

    Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2 flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2 null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2 null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2 null/null mice. Finally, we set

  11. Gaucher disease: transcriptome analyses using microarray or mRNA sequencing in a Gba1 mutant mouse model treated with velaglucerase alfa or imiglucerase.

    Directory of Open Access Journals (Sweden)

    Nupur Dasgupta

    Full Text Available Gaucher disease type 1, an inherited lysosomal storage disorder, is caused by mutations in GBA1 leading to defective glucocerebrosidase (GCase function and consequent excess accumulation of glucosylceramide/glucosylsphingosine in visceral organs. Enzyme replacement therapy (ERT with the biosimilars, imiglucerase (imig or velaglucerase alfa (vela improves/reverses the visceral disease. Comparative transcriptomic effects (microarray and mRNA-Seq of no ERT and ERT (imig or vela were done with liver, lung, and spleen from mice having Gba1 mutant alleles, termed D409V/null. Disease-related molecular effects, dynamic ranges, and sensitivities were compared between mRNA-Seq and microarrays and their respective analytic tools, i.e. Mixed Model ANOVA (microarray, and DESeq and edgeR (mRNA-Seq. While similar gene expression patterns were observed with both platforms, mRNA-Seq identified more differentially expressed genes (DEGs (∼3-fold than the microarrays. Among the three analytic tools, DESeq identified the maximum number of DEGs for all tissues and treatments. DESeq and edgeR comparisons revealed differences in DEGs identified. In 9V/null liver, spleen and lung, post-therapy transcriptomes approximated WT, were partially reverted, and had little change, respectively, and were concordant with the corresponding histological and biochemical findings. DEG overlaps were only 8-20% between mRNA-Seq and microarray, but the biological pathways were similar. Cell growth and proliferation, cell cycle, heme metabolism, and mitochondrial dysfunction were most altered with the Gaucher disease process. Imig and vela differentially affected specific disease pathways. Differential molecular responses were observed in direct transcriptome comparisons from imig- and vela-treated tissues. These results provide cross-validation for the mRNA-Seq and microarray platforms, and show differences between the molecular effects of two highly structurally similar ERT

  12. AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington’s disease

    International Nuclear Information System (INIS)

    Zuleta, Amparo; Vidal, Rene L.; Armentano, Donna; Parsons, Geoffrey; Hetz, Claudio

    2012-01-01

    Highlights: ► The contribution of ER stress to HD has not been directly addressed. ► Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. ► We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington’s disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptation to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588 Q95 -mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588 Q95 -mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.

  13. LINES

    Directory of Open Access Journals (Sweden)

    Minas Bakalchev

    2015-10-01

    Full Text Available The perception of elements in a system often creates their interdependence, interconditionality, and suppression. The lines from a basic geometrical element have become the model of a reductive world based on isolation according to certain criteria such as function, structure, and social organization. Their traces are experienced in the contemporary world as fragments or ruins of a system of domination of an assumed hierarchical unity. How can one release oneself from such dependence or determinism? How can the lines become less “systematic” and forms more autonomous, and less reductive? How is a form released from modernistic determinism on the new controversial ground? How can these elements or forms of representation become forms of action in the present complex world? In this paper, the meaning of lines through the ideas of Le Corbusier, Leonidov, Picasso, and Hitchcock is presented. Spatial research was made through a series of examples arising from the projects of the architectural studio “Residential Transformations”, which was a backbone for mapping the possibilities ranging from playfulness to exactness, as tactics of transformation in the different contexts of the contemporary world.

  14. PGC-1 silencing compounds the perturbation of mitochondrial function caused by mutant SOD1 in skeletal muscle of ALS mouse model

    Directory of Open Access Journals (Sweden)

    Yan eQi

    2015-10-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a lethal neurodegenerative disease causing death of motor neurons. This study investigated the roles of energy metabolism in the pathogenesis of ALS in the SOD1(G93A transgenic mouse model. Control and SOD1(G93A mice were administered with shcontrol or shPGC-1α in combination with PBS or TZD for 8 weeks. Gene expression was analyzed by quantitative real-time PCR and western blot. ROS and fibrosis were assessed with a colorimetric kit and Sirius staining respectively. Inflammatory cytokines were measured using ELISA kits. The levels of tissue ROS and serum inflammatory cytokines were significantly higher in SOD1(G93A mice compared to control mice, and knocking down PGC-1α drastically increased cytokine levels in both control and SOD1(G93A mice. Muscle fibrosis was much severer in SOD1(G93A mice, and worsened by silencing PGC-1α and attenuate d by TZD. The expression levels of PGC-1α, SOD1, UCP2, and cytochrome C were substantially reduced by shPGC-1α and increased by TZD in muscle of both control and SOD1(G93A mice whereas the level of NF-B was significantly elevated in SOD1(G93A mice, which was further increased by PGC-1α silencing. These data indicated that disruption of energy homeostasis would exacerbate the pathological changes caused by SOD1 mutations to promote the pathogenesis of ALS.

  15. Genome wide analysis of inbred mouse lines identifies a locus containing Ppar-gamma as contributing to enhanced malaria survival.

    Directory of Open Access Journals (Sweden)

    Selina E R Bopp

    2010-05-01

    Full Text Available The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-gamma in malaria infection.

  16. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40 0 C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G 1 -phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35 0 C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs

  17. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    Energy Technology Data Exchange (ETDEWEB)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  18. An X-linked Myh11-CreERT2 mouse line resulting from Y to X chromosome-translocation of the Cre allele.

    Science.gov (United States)

    Liao, Mingmei; Zhou, Junmei; Wang, Fen; Ali, Yasmin H; Chan, Kelvin L; Zou, Fei; Offermanns, Stefan; Jiang, Zhisheng; Jiang, Zhihua

    2017-09-01

    The Myh11-CreER T2 mouse line (Cre + ) has gained increasing application because of its high lineage specificity relative to other Cre drivers targeting smooth muscle cells (SMCs). This Cre allele, however, was initially inserted into the Y chromosome (X/Y Cre+ ), which excluded its application in female mice. Our group established a Cre + colony from male ancestors. Surprisingly, genotype screening identified female carriers that stably transmitted the Cre allele to the following generations. Crossbreeding experiments revealed a pattern of X-linked inheritance for the transgene (k > 1000), indicating that these female carries acquired the Cre allele through a mechanism of Y to X chromosome translocation. Further characterization demonstrated that in hemizygous X/X Cre+ mice Cre activity was restricted to a subset arterial SMCs, with Cre expression in arteries decreased by 50% compared to X/Y Cre+ mice. This mosaicism, however, diminished in homozygous X Cre+ /X Cre+ mice. In a model of aortic aneurysm induced by a SMC-specific Tgfbr1 deletion, the homozygous X Cre+ /X Cre+ Cre driver unmasked the aortic phenotype that is otherwise subclinical when driven by the hemizygous X/X Cre+ Cre line. In conclusion, the Cre allele carried by this female mouse line is located on the X chromosome and subjected to X-inactivation. The homozygous X Cre+ /X Cre+ mice produce uniform Cre activity in arterial SMCs. © 2017 Wiley Periodicals, Inc.

  19. Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

    Science.gov (United States)

    Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

    2009-01-01

    Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

  20. Mutation spectrum in FE1-MUTA(TM) Mouse lung epithelial cells exposed to nanoparticulate carbon black

    DEFF Research Database (Denmark)

    Jacobsen, Nicklas Raun; White, Paul A; Gingerich, John

    2011-01-01

    It has been shown previously that carbon black (CB), Printex 90 exposure induces cII and lacZ mutants in the FE1-Muta(TM) Mouse lung epithelial cell line and causes oxidatively damaged DNA and the production of reactive oxygen species (ROS). The purpose of this study was to determine the mutation...

  1. Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Savina Apolloni

    2014-09-01

    Full Text Available In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS: the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG, a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising

  2. miR-345 in metastatic colorectal cancer: a non-invasive biomarker for clinical outcome in non-KRAS mutant patients treated with 3rd line cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Schou, Jakob V; Rossi, Simona; Jensen, Benny V

    2014-01-01

    for overall survival (OS) in patients with metastatic colorectal cancer (mCRC) treated with cetuximab and irinotecan. METHODS: From 138 patients with mCRC in 3rd line therapy with cetuximab and irinotecan in a prospective phase II study, 738 pretreatment miRNAs were isolated and profiled from whole blood...... to treatment with cetuximab and irinotecan. CONCLUSION: We identified miR-345 in whole blood as a potential biomarker for clinical outcome. MiR-345 was a single prognostic biomarker for both OS and PFS in all patients and also in the non-KRAS mutant population....

  3. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  4. Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens

    International Nuclear Information System (INIS)

    Spring, B.; Pawelec, G.; Ziegler, A.

    1986-01-01

    To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray-induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA=A2, and TU161 for HLA-B13 molecules, respectively. Bo- th MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells. (author)

  5. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-01-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, was compared to the wild-type cell, CHO-SC1, in single- and split-radiation-dose schemes. When the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal X-ray damage. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Distinct perturbations in the cell-cycle progression were noted following heat alone or heat with radiation. A delay in the progression of synchronized G 1 -phase and S-phase cells was demonstrated autoradiographically after inhibition of protein synthesis. In addition, treated S-phase cells showed a transient increase in the percent labelled cells after the cells were returned to their normal growth temperature of 35 0 C. This observation was suggestive of an unusual pattern of DNA synthesis during the recovery period. Split-dose experiments were done using incubation with cycloheximide to chemically inhibit protein synthesis. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the repair of sublethal damage

  6. Generation of Pet1210-Cre Transgenic Mouse Line Reveals Non-Serotonergic Expression Domains of Pet1 Both in CNS and Periphery

    Science.gov (United States)

    Pelosi, Barbara; Migliarini, Sara; Pacini, Giulia; Pratelli, Marta; Pasqualetti, Massimo

    2014-01-01

    Neurons producing serotonin (5-hydroxytryptamine, 5-HT) constitute one of the most widely distributed neuronal networks in the mammalian central nervous system (CNS) and exhibit a profuse innervation throughout the CNS already at early stages of development. Serotonergic neuron specification is controlled by a combination of secreted molecules and transcription factors such as Shh, Fgf4/8, Nkx2.2, Lmx1b and Pet1. In the mouse, Pet1 mRNA expression appears between 10 and 11 days post coitum (dpc) in serotonergic post-mitotic precursors and persists in serotonergic neurons up to adulthood, where it promotes the expression of genes defining the mature serotonergic phenotype such as tryptophan hydroxylase 2 (Tph2) and serotonin transporter (SERT). Hence, the generation of genetic tools based on Pet1 specific expression represents a valuable approach to study the development and function of the serotonergic system. Here, we report the generation of a Pet1210-Cre transgenic mouse line in which the Cre recombinase is expressed under the control of a 210 kb fragment from the Pet1 genetic locus to ensure a reliable and faithful control of somatic recombination in Pet1 cell lineage. Besides Cre-mediated recombination accurately occurred in the serotonergic system as expected and according to previous studies, Pet1210-Cre transgenic mouse line allowed us to identify novel, so far uncharacterized, Pet1 expression domains. Indeed, we showed that in the raphe Pet1 is expressed also in a non-serotonergic neuronal population intermingled with Tph2-expressing cells and mostly localized in the B8 and B9 nuclei. Moreover, we detected Cre-mediated recombination also in the developing pancreas and in the ureteric bud derivatives of the kidney, where it reflected a specific Pet1 expression. Thus, Pet1210-Cre transgenic mouse line faithfully drives Cre-mediated recombination in all Pet1 expression domains representing a valuable tool to genetically manipulate serotonergic and non

  7. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv.

    Directory of Open Access Journals (Sweden)

    Carla Bianca Luena Victorio

    Full Text Available Since its identification in 1969, Enterovirus 71 (EV71 has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71 is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv, which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1 and viral RNA-dependent RNA polymerase (3D. Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  8. Protective Effects of Mouse Bone Marrow Mesenchymal Stem Cell Soup on Staurosporine Induced Cell Death in PC12 and U87 Cell Lines

    Directory of Open Access Journals (Sweden)

    Hossein Zhaleh

    2016-11-01

    Full Text Available Mouse bone marrow mesenchymal stem cells (mBMSCs soup is promising tool for the treatment of neurodegenerative diseases. mBMSCs soup is easily obtained and is capable of transplantation without rejection. We investigated the effects of mBMSC soup on staurosporine-induced cell death in PC12 and U87 cells lines. The percentage of cell viability, cell death, NO concentration, total neurite length (TNL and fraction of cell differentiation (f% were assessed. Viability assay showed that mBM soup (24 and 48h in time dependent were increased cell viability (p<0.05 and also cell death assay showed that cell death in time dependent were decreased, respectively (p<0.05. TNL and fraction of cell differentiation significantly were increased compared with treatment1 (p<0.05. Our data showed that mBM Soup protects cells, increases cell viability, suppresses cell death and improvement the neurite elongation. We concluded that Mouse bone marrow mesenchymal stem cell soup plays an important protective role in staurosporine-induced cell death in PC12 and U87 cell lines.

  9. Generation of a tenascin-C-CreER2 knockin mouse line for conditional DNA recombination in renal medullary interstitial cells.

    Directory of Open Access Journals (Sweden)

    Wenjuan He

    Full Text Available Renal medullary interstitial cells (RMIC are specialized fibroblast-like cells that exert important functions in maintaining body fluid homeostasis and systemic blood pressure. Here, we generated a RMIC specific tenascin-C promoter driven inducible CreER2 knockin mouse line with an EGFP reporter. Similar as endogenous tenascin-C expression, the reporter EGFP expression in the tenascin-C-CreER2(+/- mice was observed in the inner medulla of the kidney, and co-localized with COX2 but not with AQP2 or AQP1, suggesting selective expression in RMICs. After recombination (tenascin-C-CreER2(+/-/ROSA26-lacZ(+/- mice + tamoxifen, β-gal activity was restricted to the cells in the inner medulla of the kidney, and didn't co-localize with AQP2, consistent with selective Cre recombinase activity in RMICs. Cre activity was not obvious in other major organs or without tamoxifen treatment. This inducible RMIC specific Cre mouse line should therefore provide a novel tool to manipulate genes of interest in RMICs.

  10. Deoxynivalenol (DON) is toxic to human colonic, lung and monocytic cell lines, but does not increase the IgE response in a mouse model for allergy

    International Nuclear Information System (INIS)

    Instanes, Christine; Hetland, Geir

    2004-01-01

    We examined whether the common crop mycotoxin deoxynivalenol (DON) from Fusarium species is toxic to human colonic (Caco-2), lung (A549) and monocytic (U937) cell lines. Moreover, since DON reportedly induces increased levels of Th2 cytokines and total IgE, and we have observed that mould extracts adjuvated allergy development in mice, possible adjuvant effect of DON on allergy was studied in a mouse model. For all the cells, exposure to DON for 24 h reduced cellular protein synthesis, proliferation and survival rate dose-dependently. In addition, production of IL-8 in the U937 cell line increased up to eight-fold at levels of DON just lower than the most toxic one, suggesting that IL-8 can be used as an additional index for cytotoxicity in mononuclear phagocytes. However, DON did not increase levels of allergen-specific IgE or IgG1 in the mouse model for allergy. These results suggest that DON, when inhaled or ingested, may have toxic effect on human alveolar macrophages and epithelial cells in lungs and colon, but does not increase the allergic response to allergens

  11. Opposite effects of alcohol in regulating stress-induced changes in body weight between the two mouse lines with enhanced or low opioid system activity.

    Science.gov (United States)

    Sacharczuk, Mariusz; Sadowski, Bogdan; Jaszczak, Kazimierz; Lipkowski, Andrzej W; Swiergiel, Artur H

    2010-04-19

    Considering the involvement of the opioid system in alcoholism, depression and metabolism - known risk factors in human obesity, we studied the effects of chronic mild stress (CMS) and alcohol intake on body weight in two mouse lines selected for high (HA-high analgesia) or low (LA-low analgesia) swim stress-induced analgesia. In comparison to LA mice, HA mice exhibit an upregulation of opioid receptor system function, different depression-like behavior and reduced energy expenditure in stress. LA animals showed enhanced basal and CMS-induced alcohol drinking versus HA. Now we report different effects of alcohol under no stress (control) and CMS conditions on food intake and body weight between the lines. CMS in animals with no access to alcohol increased body weight in both HA and LA mice, with no effect of CMS on food intake in either line and without differences between the lines. In LA mice alcohol reduced body weight under both conditions although significantly more under the control than CMS conditions. In contrast, in HA mice alcohol increased body weight more under the CMS than under control conditions. The results suggest that opioid system may modulate effects of alcohol on stress -induced changes in body weight. (c) 2010 Elsevier Inc. All rights reserved.

  12. The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.

    Science.gov (United States)

    Gays, F; Unnikrishnan, M; Shrestha, S; Fraser, K P; Brown, A R; Tristram, C M; Chrzanowska-Lightowlers, Z M; Brooks, C G

    2000-05-15

    As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.

  13. Characterization of a rabbit germ-line VH gene that is a candidate donor for VH gene conversion in mutant Alicia rabbits.

    Science.gov (United States)

    Chen, H T; Alexander, C B; Mage, R G

    1995-06-15

    Normal rabbits preferentially rearrange the 3'-most VH gene, VH1, to encode Igs with VHa allotypes, which constitute the majority of rabbit serum Igs. A gene conversion-like mechanism is employed to diversify the primary Ab repertoire. In mutant Alicia rabbits that derived from a rabbit with VHa2 allotype, the VH1 gene was deleted. Our previous studies showed that the first functional gene (VH4) or VH4-like genes were rearranged in 2- to 8-wk-old homozygous Alicia. The VH1a2-like sequences that were found in splenic mRNA from 6-wk and older Alicia rabbits still had some residues that were typical of VH4. The appearances of sequences resembling that of VH1a2 may have been caused by gene conversions that altered the sequences of the rearranged VH or there may have been rearrangement of upstream VH1a2-like genes later in development. To investigate this further, we constructed a cosmid library and isolated a VH1a2-like gene, VH12-1-6, with a sequence almost identical to VH1a2. This gene had a deleted base in the heptamer of its recombination signal sequence. However, even if this defect diminished or eliminated its ability to rearrange, the a2-like gene could have acted as a donor for gene-conversion-like alteration of rearranged VH genes. Sequence comparisons suggested that this gene or a gene like it could have acted as a donor for gene conversion in mutant Alicia and in normal rabbits.

  14. The Cytolethal Distending Toxin Subunit CdtB of Helicobacter hepaticus Promotes Senescence and Endoreplication in Xenograft Mouse Models of Hepatic and Intestinal Cell Lines

    Directory of Open Access Journals (Sweden)

    Christelle Péré-Védrenne

    2017-06-01

    Full Text Available Cytolethal distending toxins (CDTs are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB. For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3 was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

  15. R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

    Science.gov (United States)

    Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

    2016-10-01

    The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

  16. Expression of Caytaxin protein in Cayman Ataxia mouse models correlates with phenotype severity.

    Directory of Open Access Journals (Sweden)

    Kristine M Sikora

    Full Text Available Caytaxin is a highly-conserved protein, which is encoded by the Atcay/ATCAY gene. Mutations in Atcay/ATCAY have been identified as causative of cerebellar disorders such as the rare hereditary disease Cayman ataxia in humans, generalized dystonia in the dystonic (dt rat, and marked motor defects in three ataxic mouse lines. While several lines of evidence suggest that Caytaxin plays a critical role in maintaining nervous system processes, the physiological function of Caytaxin has not been fully characterized. In the study presented here, we generated novel specific monoclonal antibodies against full-length Caytaxin to examine endogenous Caytaxin expression in wild type and Atcay mutant mouse lines. Caytaxin protein is absent from brain tissues in the two severely ataxic Atcay(jit (jittery and Atcay(swd (sidewinder mutant lines, and markedly decreased in the mildly ataxic/dystonic Atcay(ji-hes (hesitant line, indicating a correlation between Caytaxin expression and disease severity. As the expression of wild type human Caytaxin in mutant sidewinder and jittery mice rescues the ataxic phenotype, Caytaxin's physiological function appears to be conserved between the human and mouse orthologs. Across multiple species and in several neuronal cell lines Caytaxin is expressed as several protein isoforms, the two largest of which are caused by the usage of conserved methionine translation start sites. The work described in this manuscript presents an initial characterization of the Caytaxin protein and its expression in wild type and several mutant mouse models. Utilizing these animal models of human Cayman Ataxia will now allow an in-depth analysis to elucidate Caytaxin's role in maintaining normal neuronal function.

  17. Basal and stress-induced differences in HPA axis, 5-HT responsiveness, and hippocampal cell proliferation in two mouse lines

    NARCIS (Netherlands)

    Veenema, AH; Koolhaas, JM; De Kloet, ER; Pacak, K; Aguilera, B; Saban, E; Kvetnansky, R

    2004-01-01

    To characterize individual differences in neuroendocrine and neurochemical correlates of stress coping, two lines of wild house mice were studied. These mice are genetically selected for high and low aggression and show distinctly different behavioral strategies toward environmental stimuli. Long

  18. An extra early mutant of pigeonpea

    International Nuclear Information System (INIS)

    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

  19. Regulation of radiation-induced protein kinase Cδ activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines

    International Nuclear Information System (INIS)

    Nakajima, Tetsuo; Yukawa, Osami; Tsuji, Hideo; Ohyama, Harumi; Wang, Bing; Tatsumi, Kouichi; Hayata, Isamu; Hama-Inaba, Hiroko

    2006-01-01

    Protein kinase Cδ (PKCδ) has an important role in radiation-induced apoptosis. The expression and function of PKCδ in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCδ-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCδ activation correlated with the degradation of PKCδ, indicating that PKCδ activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCδ activation was lower than that in radiosensitive 3SBH5. Cytosol PKCδ levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCδ levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCδ activation compared to that of 3SBH5. On the other hand, Atm -/- mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm -/- cells, had decreased PKCδ levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCδ degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCδ activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells

  20. Various ocular MR imaging in a mouse implanted with a new cell line of retinoblastoma and the correlation with the pathology: preliminary study

    International Nuclear Information System (INIS)

    Kim, Dong Hun; Kim, Il Joong; Yang, Jae Han; Byun, Joo Nam; Lee, Bong Jae; Kim, Jeong Hun; Yu, Young Suk

    2007-01-01

    We wanted to show various MR and correlated pathologic images of retinoblastoma in nude mouse with a new human retinoblastoma cell line (SNUOT-Rb1), which was inoculated into the intravitreous cavity. The established cell line was inoculated into the intravitreous cavity of 36 eyeballs of 18 mice and the transplanted retinoblastoma was examined for 3 months. The T1-weighted (T1WI), T2-weighted (T2WI), and contrast enhanced (Gd-DTPA) T1-weighted images were obtained with using a small loop coil. After scanning, the mice's eyeballs were extracted and the hematoxylin and eosin stained specimens were examined with a microscope. We compared the MR imagings with pathologic findings and evaluated the character of the tumors. The innoculated cells in the eyeballs of the mice grew into retinoblastoma (23/36, 64%). The eyeballs with retinoblastoma protruded externally and showed focal hemorrhage. Most tumors showed iso-signal intensity on TIWI (13/23, 57%), high signal intensity on T2WI (17/23, 74%), and good enhancement (21/23, 91%) with contrast. Almost all of the tumors (n = 21) were located in the retina and three extraretinal tumors were confirmed by pathology. Involvement of the optic nerve was suspected on MRI and this was confirmed by pathology in 6 cases and 5 cases, respectively. We could demonstrate various MR imagings of transplanted retinoblastoma by using the new tumor cell line in vivo

  1. Various ocular MR imaging in a mouse implanted with a new cell line of retinoblastoma and the correlation with the pathology: preliminary study

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Hun; Kim, Il Joong; Yang, Jae Han; Byun, Joo Nam; Lee, Bong Jae [Chosun University, College of Medicine, Gwangju (Korea, Republic of); Kim, Jeong Hun; Yu, Young Suk [Seoul National University, College of Medicine, Seoul (Korea, Republic of)

    2007-05-15

    We wanted to show various MR and correlated pathologic images of retinoblastoma in nude mouse with a new human retinoblastoma cell line (SNUOT-Rb1), which was inoculated into the intravitreous cavity. The established cell line was inoculated into the intravitreous cavity of 36 eyeballs of 18 mice and the transplanted retinoblastoma was examined for 3 months. The T1-weighted (T1WI), T2-weighted (T2WI), and contrast enhanced (Gd-DTPA) T1-weighted images were obtained with using a small loop coil. After scanning, the mice's eyeballs were extracted and the hematoxylin and eosin stained specimens were examined with a microscope. We compared the MR imagings with pathologic findings and evaluated the character of the tumors. The innoculated cells in the eyeballs of the mice grew into retinoblastoma (23/36, 64%). The eyeballs with retinoblastoma protruded externally and showed focal hemorrhage. Most tumors showed iso-signal intensity on TIWI (13/23, 57%), high signal intensity on T2WI (17/23, 74%), and good enhancement (21/23, 91%) with contrast. Almost all of the tumors (n = 21) were located in the retina and three extraretinal tumors were confirmed by pathology. Involvement of the optic nerve was suspected on MRI and this was confirmed by pathology in 6 cases and 5 cases, respectively. We could demonstrate various MR imagings of transplanted retinoblastoma by using the new tumor cell line in vivo.

  2. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Directory of Open Access Journals (Sweden)

    Anne Frentzen

    2011-04-01

    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  3. Comparative proteome analysis of three mouse lung adenocarcinoma CMT cell lines with different metastatic potential by two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Zhang, Kelan; Wrzesinski, Krzysztof; Stephen, J Fey; Larsen, Peter Mose; Zhang, Xumin; Roepstorff, Peter

    2008-12-01

    Metastasis is a lethal attribute of a cancer and presents a continuing therapeutic challenge. Metastasis is a highly complex process and more knowledge about the mechanisms behind metastasis is highly desirable. Isogenic CMT cell lines were selected from a spontaneous mouse lung adenocarcinoma and characterized in vivo to have different metastatic potential. In this study, the comprehensive protein expression profiles of three of these CMT cell lines at passage 5, 15 and 35 were analyzed by 2-DE separation followed by MS identification. As a result, 82 and 40 unique proteins were found to be significantly up- or down-regulated between cell lines with different metastatic potential at passages 5 and 15, respectively. These proteins were identified by MS and most of them have previously been reported to be related to cancer development and/or metastasis. Bioinformatics analysis indicated that several of the proteins were involved in proteasome, cell-cycle and cell-communication pathways. Among them, some keratins, 14-3-3 proteins and 26S proteasome proteins were identified and their aberrant expression may be directly or indirectly involved in cancer development and metastasis. In conclusion, our comprehensive 2-DE-based proteomics studies revealed some candidate proteins, protein families and signaling pathways, which might be important in cancer development and metastasis.

  4. Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Takuya Sato

    2015-07-01

    Full Text Available Mouse spermatogonial stem cells (SSCs can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

  5. Isolation of a novel chronic lymphocytic leukemic (CLL) cell line and development of an in vivo mouse model of CLL.

    Science.gov (United States)

    Kellner, Joshua; Wierda, William; Shpall, Elizabeth; Keating, Michael; McNiece, Ian

    2016-01-01

    Leukemic cell lines have become important tools for studies of disease providing a monoclonal cell population that can be extensively expanded in vitro while preserving leukemic cellular characteristics. However, studies of chronic lymphocytic leukemia (CLL) have been impeded in part by the lack of continuous human cell lines. CLL cells have a high spontaneous apoptosis rate in vitro and exhibit minimal proliferation in xenograft models. Therefore, there is a need for development of primary CLL cell lines and we describe the isolation of such a line from the bone marrow of a CLL patient (17p deletion and TP53 mutation) which has been in long term culture for more than 12 months with continuous proliferation. The CLL cell line (termed MDA-BM5) which was generated in vitro with continuous co-culture on autologous stromal cells is CD19+CD5+ and shows an identical pattern of somatic hypermutation as determined in the patient's bone marrow (BM), confirming the origin of the cells from the original CLL clone. MDA-BM5 cells were readily transplantable in NOD/SCID gamma null mice (NSG) with disease developing in the BM, liver and spleen. BM cells from quaternary serial transplantation in NSG mice demonstrated the presence of CD19+CD5+ cells with Ig restricted to lambda which is consistent with the original patient cells. These studies describe a new CLL cell line from a patient with del(17p) that provides a unique model for in vitro and in vivo studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29

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    Goran Christoph Söhl

    2013-06-01

    Full Text Available The potential gap junction forming mouse connexin29 (Cx29 protein is concomitantly expressed with connexin32 (Cx32 in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47 in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harbouring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29 mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

  7. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  8. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

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    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  9. RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function.

    Science.gov (United States)

    Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

    2011-02-01

    Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

  10. Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

    Science.gov (United States)

    Ben-Daniel, Bat-Hen; Bar-Zvi, Dudy; Tsror Lahkim, Leah

    2012-02-01

    Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  11. Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis

    NARCIS (Netherlands)

    Meng, X.; de rooij, D. G.; Westerdahl, K.; Saarma, M.; Sariola, H.

    2001-01-01

    We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have

  12. Establishment of A Malignant Pleural Effusion Mouse Model with Lewis Lung 
Carcinoma Cell Lines Expressing Enhanced Green Fluorescent Protein

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    Xingqun MA

    2012-06-01

    Full Text Available Background and objective Malignant pleural effusion (MPE is a poor prognosis factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC cell lines expressing enhanced green fluorescent protein (EGFP. Methods The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of mice that were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT imaging periodically to analyze the formation rate of pleural effusion. The survival rate and tumor metastasis were also observed. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore pleural effusion at the same time, we calculated the average volume. The correlation of pleural effusion with the integrated optical density (IOD were analyzed. Results Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastasis sites were mediastinal, and the hilar lymph nodes were contralateral pleural as well as pericardial. The metastasis rates were 87%, 73% and 20%, respectively. The CT scan revealed that the formation rates of pleural effusion on days 7, 14 and 21 were 13%, 46% and 53%, respectively. The average volume of pleural effusion increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The mean survival time of nude mice was 28.8 days. The volume of pleural effusion and IOD were significantly correlated (r=0.91, P<0.000,1. Conclusion A mouse model of lung cancer malignant pleural effusion was successfully established by injecting LLC lines expressing EGFP into the pleural cavity under a microscope. The model can enable dynamic observations of the

  13. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

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    Ana de la Cueva

    Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

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

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    Katherine Maringer

    2014-08-01

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

  15. Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

    Science.gov (United States)

    Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

    1992-08-01

    Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

  16. In Vivo Tau Imaging for a Diagnostic Platform of Tauopathy Using the rTg4510 Mouse Line

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    Naruhiko Sahara

    2017-12-01

    Full Text Available Association of tau deposition with neurodegeneration in Alzheimer’s disease (AD and related tau-positive neurological disorders collectively referred to as tauopathies indicates contribution of tau aggregates to neurotoxicity. The discovery of tau gene mutations in FTDP-17-tau kindreds has provided unequivocal evidence that tau abnormalities alone can induce neurodegenerative disorders. Therefore, visualization of tau accumulation would offer a reliable, objective index to aid in the diagnosis of tauopathy and to assess the disease progression. Positron emission tomography (PET imaging of tau lesions is currently available using several tau PET ligands. Because most tau PET ligands have the property of an extrinsic fluorescent dye, these ligands are considered to be useful for both PET and fluorescence imaging. In addition, small-animal magnetic resonance imaging (MRI is available for both structural and functional imaging. Using these advanced imaging techniques, in vivo studies on a mouse model of tauopathy will provide significant insight into the translational research of neurodegenerative diseases. In this review, we will discuss the utilities of PET, MRI, and fluorescence imaging for evaluating the disease progression of tauopathy.

  17. Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

    Energy Technology Data Exchange (ETDEWEB)

    Colmenares, C.; Lopez, C.

    1986-05-01

    Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, the authors used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). They also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the /sup 51/Cr-release assay in the presence of anti-interferon serum. The data show that HSV-1 infection of NK/NC targets induces increased cytotoxity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.

  18. Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity

    International Nuclear Information System (INIS)

    Kruszewski, M.; Iwanenko, T.

    1998-01-01

    The role of nuclear proteins in protection of DNA against ionizing radiation and their contribution to the radiation sensitivity was examined by an alkaline version of comet assay in two L5178Y (LY) mouse lymphoma cell lines differing in sensitivity t o ionizing radiation. LY-S cells are twice more sensitive to ionizing radiation than LY-R cells (D 0 values of survival curves are 0.5 Gy and 1 Gy, respectively). Sequential removal of nuclear proteins by extraction with NaCl of different concentrations increased the X-ray induced DNA damage in LY-R nucleoids. In contrast, in the radiation sensitive LY-S cell line, depletion of nuclear proteins practically did not affect DNA damage. Although there is no doubt that the main cause of LY-S cells' sensitivity to ionizing radiation is a defect in the repair of double-strand breaks, our data support the concept that nuclear matrix organization may contribute to the cellular susceptibility to DNA damaging agents. (author)

  19. Studies of radioinduced mutations in sorghum grain: 1. Comparison of phenotypic variability obtained through hybridation and mutagenesis of F2 and M2 populations; 2. Agronomical and physiotechnical characterization of mutants lines in the original collection and in the advanced lines of the Chapingo Postgraduate college

    International Nuclear Information System (INIS)

    Parra Negrete, L.A.

    1986-01-01

    Genetic inprovement of cultivated plants consists essentially of three phases (1) generation of genetic variability (2) selection of genotypes and (3) evaluation of selected genotypes. Hybridization and spontaneous or induced mutations are, responsible for the generation of and increase in genetic vegetative variability. Accordingly, such methods are used alternatively in local programs for plant improvement either for introducing improved genotypes or as sources of germplasma. This thesis is based on two experiments of mutations induced by cobalt 60 ionizing radiation while using distinct materials and methods presented in two parts, the first section analyze the mutation variability and the second the evaluation of mutant lines. (author)

  20. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

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    Ryan Aideen E

    2006-02-01

    Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

  1. Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines

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    Alonso Daniel F

    2009-12-01

    Full Text Available Abstract Background Carbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH, an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues. Methods We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth. Results In the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone. Conclusion Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.

  2. Comprehensive behavioral analysis of ENU-induced Disc1-Q31L and -L100P mutant mice

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    Shoji Hirotaka

    2012-02-01

    Full Text Available Abstract Background Disrupted-in-Schizophrenia 1 (DISC1 is considered to be a candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder, and major depression. A recent study reported that N-ethyl-N-nitrosourea (ENU-induced mutations in exon 2 of the mouse Disc1 gene, which resulted in the amino acid exchange of Q31L and L100P, caused an increase in depression-like behavior in 31 L mutant mice and schizophrenia-like behavior in 100P mutant mice; thus, these are potential animal models of psychiatric disorders. However, remaining heterozygous mutations that possibly occur in flanking genes other than Disc1 itself might induce behavioral abnormalities in the mutant mice. Here, to confirm the effects of Disc1-Q31L and Disc1-L100P mutations on behavioral phenotypes and to investigate the behaviors of the mutant mice in more detail, the mutant lines were backcrossed to C57BL/6JJcl through an additional two generations and the behaviors were analyzed using a comprehensive behavioral test battery. Results Contrary to expectations, 31 L mutant mice showed no significant behavioral differences when compared with wild-type control mice in any of the behavioral tests, including the Porsolt forced swim and tail suspension tests, commonly used tests for depression-like behavior. Also, 100P mutant mice exhibited no differences in almost all of the behavioral tests, including the prepulse inhibition test for measuring sensorimotor gating, which is known to be impaired in schizophrenia patients; however, 100P mutant mice showed higher locomotor activity compared with wild-type control mice in the light/dark transition test. Conclusions Although these results are partially consistent with the previous study in that there was hyperactivity in 100P mutant mice, the vast majority of the results are inconsistent with those of the previous study; this discrepancy may be explained by differences in the genetic background of the

  3. Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

    Science.gov (United States)

    Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

    1993-03-01

    Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

  4. Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

    Science.gov (United States)

    Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

    2017-10-01

    In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of β-endorphin in a mouse pituitary cell line

    International Nuclear Information System (INIS)

    Fagarasan, M.O.; Axelrod, J.; Bishop, J.F.; Rinaudo, M.S.

    1990-01-01

    Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates β-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced β-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced β-endorphin secretion. In contrast, IL-1-induced β-endorphin release was independent of PKC. The authors observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of β-endorphin

  6. Mobile phone radiation induces mode-dependent DNA damage in a mouse spermatocyte-derived cell line: a protective role of melatonin.

    Science.gov (United States)

    Liu, Chuan; Gao, Peng; Xu, Shang-Cheng; Wang, Yuan; Chen, Chun-Hai; He, Min-Di; Yu, Zheng-Ping; Zhang, Lei; Zhou, Zhou

    2013-11-01

    To evaluate whether exposure to mobile phone radiation (MPR) can induce DNA damage in male germ cells. A mouse spermatocyte-derived GC-2 cell line was exposed to a commercial mobile phone handset once every 20 min in standby, listen, dialed or dialing modes for 24 h. DNA damage was determined using an alkaline comet assay. The levels of DNA damage were significantly increased following exposure to MPR in the listen, dialed and dialing modes. Moreover, there were significantly higher increases in the dialed and dialing modes than in the listen mode. Interestingly, these results were consistent with the radiation intensities of these modes. However, the DNA damage effects of MPR in the dialing mode were efficiently attenuated by melatonin pretreatment. These results regarding mode-dependent DNA damage have important implications for the safety of inappropriate mobile phone use by males of reproductive age and also suggest a simple preventive measure: Keeping mobile phones as far away from our body as possible, not only during conversations but during 'dialed' and 'dialing' operation modes. Since the 'dialed' mode is actually part of the standby mode, mobile phones should be kept at a safe distance from our body even during standby operation. Furthermore, the protective role of melatonin suggests that it may be a promising pharmacological candidate for preventing mobile phone use-related reproductive impairments.

  7. Preliminary comparison of grating-based and in-line phase contrast X-ray imaging with synchrotron radiation for mouse kidney at TOMCAT

    International Nuclear Information System (INIS)

    Sun, J; Liu, P; Xu, L X; Irvine, S; Pinzer, B; Stampanoni, M

    2013-01-01

    Phase contrast imaging has been demonstrated to be advantageous in revealing detailed structures inside biological specimens without contrast agents. Grating-based differential phase contrast (DPC) and in-line phase contrast (ILPC) X-ray imaging are the two modalities frequently used at the beamline of TOmographic Microscopy and Coherent rAdiology experimenTs (TOMCAT) at the Swiss Light Source (SLS). In this paper, we preliminarily compared the abilities of two types of phase contrast imaging in distinguishing micro structures in mouse kidneys. The 3D reconstructions showed that the microstructures in kidney, such as micro vessels and renal tubules, were displayed clearly with both imaging modalities. The two techniques may be viewed as complementary. For larger features with very small density variations DPC is the desirable method. In cases where dose and time limits may prohibit the multiple steps required for DPC, and when the focus is on finer features, the ILPC method may be considered as a more viable alternative. Moreover, high resolution ILPC images are comparable with histological results.

  8. Quantitative evaluation of a single-distance phase-retrieval method applied on in-line phase-contrast images of a mouse lung

    International Nuclear Information System (INIS)

    Mohammadi, Sara; Larsson, Emanuel; Alves, Frauke; Dal Monego, Simeone; Biffi, Stefania; Garrovo, Chiara; Lorenzon, Andrea; Tromba, Giuliana; Dullin, Christian

    2014-01-01

    Quantitative analysis concerning the application of a single-distance phase-retrieval algorithm on in-line phase-contrast images of a mouse lung at different sample-to-detector distances is presented. Propagation-based X-ray phase-contrast computed tomography (PBI) has already proven its potential in a great variety of soft-tissue-related applications including lung imaging. However, the strong edge enhancement, caused by the phase effects, often hampers image segmentation and therefore the quantitative analysis of data sets. Here, the benefits of applying single-distance phase retrieval prior to the three-dimensional reconstruction (PhR) are discussed and quantified compared with three-dimensional reconstructions of conventional PBI data sets in terms of contrast-to-noise ratio (CNR) and preservation of image features. The PhR data sets show more than a tenfold higher CNR and only minor blurring of the edges when compared with PBI in a predominately absorption-based set-up. Accordingly, phase retrieval increases the sensitivity and provides more functionality in computed tomography imaging

  9. Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

    Science.gov (United States)

    Hama, Taketsugu; Nakanishi, Koichi; Sato, Masashi; Mukaiyama, Hironobu; Togawa, Hiroko; Shima, Yuko; Miyajima, Masayasu; Nozu, Kandai; Nagao, Shizuko; Takahashi, Hisahide; Sako, Mayumi; Iijima, Kazumoto; Yoshikawa, Norishige; Suzuki, Hiroyuki

    2017-12-01

    Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/ cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention. Copyright © 2017 the American Physiological Society.

  10. In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

    Science.gov (United States)

    Soverini, Simona; De Benedittis, Caterina; Castagnetti, Fausto; Gugliotta, Gabriele; Mancini, Manuela; Bavaro, Luana; Machova Polakova, Katerina; Linhartova, Jana; Iurlo, Alessandra; Russo, Domenico; Pane, Fabrizio; Saglio, Giuseppe; Rosti, Gianantonio; Cavo, Michele; Baccarani, Michele; Martinelli, Giovanni

    2016-08-02

    Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal

  11. Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

    International Nuclear Information System (INIS)

    Hashimoto, Osamu; Shinkawa, Masako; Torimura, Takuji; Nakamura, Toru; Selvendiran, Karuppaiyah; Sakamoto, Masaharu; Koga, Hironori; Ueno, Takato; Sata, Michio

    2006-01-01

    Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription

  12. Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

    Science.gov (United States)

    Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

    2016-05-25

    Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of

  13. Cartap-induced cytotoxicity in mouse C2C12 myoblast cell line and the roles of calcium ion and oxidative stress on the toxic effects.

    Science.gov (United States)

    Liao, Jiunn-Wang; Kang, Jaw-Jou; Jeng, Chian-Ren; Chang, Shao-Kuang; Kuo, Ming-Jang; Wang, Shun-Cheng; Liu, Michael R S; Pang, Victor Fei

    2006-02-15

    Our previous study has demonstrated that instead of neuromuscular blockage cartap, an organonitrogen insecticide, could cause a marked irreversible Ca2+-dependent contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of cartap and the possible roles of calcium ion and oxidative stress on cartap-induced muscle cell injury using the mouse myoblast cell line, C2C12. Cartap exerted a dose- and time-dependent cytotoxic effect in C2C12 cells measured by MTT colorimetric assay and trypan blue dye exclusion. The extracellular activities of both creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated in the cartap-treated groups at or greater than 100 microM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3, LDH-3, and LDH-4. Following the addition of 0.5-2.5mM EGTA, a Ca2+ chelator, or 30-100 microM verapamil, an L-type Ca2+ channel blocker, the cartap-induced reduction in MTT metabolic rate of C2C12 cells was significantly restored in a dose-dependent manner in both EGTA and verapamil-treated cells. Furthermore, EGTA could significantly reduce the cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally, cartap significantly increased the level of endogenous reactive oxygen species (ROS) in C2C12 cells in a dose- and time-dependent manner. The cartap-induced ROS generation could be significantly inhibited by antioxidants, including Vitamins C and E, catalase, and superoxide dismutase, with catalase the most effective. EGTA could significantly inhibit cartap-induced ROS generation in a dose-dependent manner. The results suggested that cartap could induce ROS generation in C2C12 cells via a Ca2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C2C12 cells. It is speculated that both Ca2+ and Ca2+-induced ROS may also play the central role on the myogenic contracture and myofiber injury

  14. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    Energy Technology Data Exchange (ETDEWEB)

    Levova, Katerina; Moserova, Michaela [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic); Nebert, Daniel W. [Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati (United States); Phillips, David H. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Frei, Eva [Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg (Germany); Schmeiser, Heinz H. [Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg (Germany); Arlt, Volker M. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Stiborova, Marie, E-mail: stiborov@natur.cuni.cz [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  15. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    International Nuclear Information System (INIS)

    Levova, Katerina; Moserova, Michaela; Nebert, Daniel W.; Phillips, David H.; Frei, Eva; Schmeiser, Heinz H.; Arlt, Volker M.; Stiborova, Marie

    2012-01-01

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  16. Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

    2014-06-04

    Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

  17. Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

    Science.gov (United States)

    Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

    2015-01-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

  18. Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

    Directory of Open Access Journals (Sweden)

    James F Powers

    Full Text Available There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1 inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and

  19. Conditional inactivation of p53 in mouse ovarian surface epithelium does not alter MIS driven Smad2-dominant negative epithelium-lined inclusion cysts or teratomas.

    Directory of Open Access Journals (Sweden)

    Suzanne M Quartuccio

    Full Text Available Epithelial ovarian cancer is the most lethal gynecological malignancy among US women. The etiology of this disease, although poorly understood, may involve the ovarian surface epithelium or the epithelium of the fallopian tube fimbriae as the progenitor cell. Disruptions in the transforming growth factor beta (TGFβ pathway and p53 are frequently found in chemotherapy-resistant serous ovarian tumors. Transgenic mice expressing a dominant negative form of Smad2 (Smad2DN, a downstream transcription factor of the TGFβ signaling pathway, targeted to tissues of the reproductive tract were created on a FVB background. These mice developed epithelium-lined inclusion cysts, a potential precursor lesion to ovarian cancer, which morphologically resembled oviductal epithelium but exhibited protein expression more closely resembling the ovarian surface epithelium. An additional genetic "hit" of p53 deletion was predicted to result in ovarian tumors. Tissue specific deletion of p53 in the ovaries and oviducts alone was attempted through intrabursal or intraoviductal injection of Cre-recombinase expressing adenovirus (AdCreGFP into p53 (flox/flox mice. Ovarian bursal cysts were detected in some mice 6 months after intrabursal injection. No pathological abnormalities were detected in mice with intraoviductal injections, which may be related to decreased infectivity of the oviductal epithelium with adenovirus as compared to the ovarian surface epithelium. Bitransgenic mice, expressing both the Smad2DN transgene and p53 (flox/flox, were then exposed to AdCreGFP in the bursa and oviductal lumen. These mice did not develop any additional phenotypes. Exposure to AdCreGFP is not an effective methodology for conditional deletion of floxed genes in oviductal epithelium and tissue specific promoters should be employed in future mouse models of the disease. In addition, a novel phenotype was observed in mice with high expression of the Smad2DN transgene as validated

  20. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

    Directory of Open Access Journals (Sweden)

    Mark P DeAndrade

    Full Text Available Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS, a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

  1. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

    Science.gov (United States)

    DeAndrade, Mark P; Zhang, Li; Doroodchi, Atbin; Yokoi, Fumiaki; Cheetham, Chad C; Chen, Huan-Xin; Roper, Steven N; Sweatt, J David; Li, Yuqing

    2012-01-01

    Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

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

    Science.gov (United States)

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

    2015-01-01

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

  3. High yielding mutants of blackgram variety 'PH-25'

    International Nuclear Information System (INIS)

    Misra, R.C.; Mohapatra, B.D.; Panda, B.S.

    2001-01-01

    Seeds of blackgram (Vigna mungo L.) variety 'PH-5' were treated with chemical mutagens ethyl methanesulfonate (EMS), nitrosoguanidine (NG), maleic hydrazide (MH) and sodium azide (NaN 3 ), each at 3 different concentrations. Thirty six mutant lines developed from mutagenic treatments along with parent varieties were tested in M 4 generation. The mutants showed wide variation in most of the traits and multivariante D 2 analysis showed genetic divergence among themselves. Twenty of the thirty mutants showed genetic divergence from parent. Ten selected high yielding mutants were tested in M 5 . Yield and other productive traits of five high yielding mutants in M 4 and M 5 are presented

  4. Development of high yielding mutants in lentil

    International Nuclear Information System (INIS)

    Rajput, M.A.; Sarwar, G.; Siddiqui, K.A.

    2001-01-01

    Full text: Lentil (Lens culinaris Medik.) locally known as Masoor, is the second most important rabi pulse crop, after chickpea, in Pakistan. It is cultivated on an area of over 63,400 ha, which constitutes about 4.83% of the total area under pulses. The annual production of the crop is 28,200 tones with an average yield of 445 kg/ha. Yield at the national level is very low, about one-half of the world's yield, which is mainly due to non-availability of high yield potential genotypes. Keeping in view the importance of mutants in developing a large number of new varieties, an induced mutations programme was initiated at AEARC, Tandojam during 1987-88, to develop high yielding varieties in lentil. For this, seeds of two lentil varieties, 'Masoor-85' and 'ICARDA-8' had been irradiated with gamma-rays ranging from 100-600 Gy in NIAB, Faisalabad during 1990. Selections were made in M2 on the basis of earliness, plant height, branches/plant and 100 grain weight. After confirming these mutants in M3 they were promoted in station yield trials and studied continuously for three consecutive years (1993- 1995). Overall results revealed that these mutants have consistent improvement of earliness in flowering and maturity. Plant height also increased in all mutant lines except AEL 23/40/91 where reduction in this attribute was observed as compared to parent variety. Mutant lines AEL 49/20/91 and AEL 13/30/91 showed improvement in 100 grain weight. The improvement of some agronomic characters enhanced the yield of mutant lines in comparison to parent varieties (Masoor-85 and ICARDA-8). The diversity in yield over the respective parents was computed from 6.94 to 60.12%. From these encouraging results it is hoped that mutant lines like AEL 12/30/91 and AEL 49/20/91 may serve as potential lentil genotypes in future. (author)

  5. c-erbA and v-erbA modulate growth and gene expression of a mouse glial precursor cell line.

    Science.gov (United States)

    Iglesias, T; Llanos, S; López-Barahona, M; Pérez-Aranda, A; Rodríguez-Peña, A; Bernal, J; Höhne, A; Seliger, B; Muñoz, A

    1994-07-01

    The c-erbA alpha protooncogene coding for the thyroid hormone (T3) receptor (TR alpha 1) and the viral, mutated v-erbA oncogene were expressed in an immortal mouse glial cell line (B3.1) using retroviral vectors. c-erbA alpha expression led to a decrease in cell proliferation in high and low serum conditions, both in the presence and in the absence of T3. In serum-free medium, c-erbA-expressing cells (B3.1 + TR alpha 1) were completely arrested, whereas cells expressing v-erbA (B3.1 + v-erbA) showed a higher DNA synthesis rate than normal B3.1 cells. Although proliferation of all three cell types was stimulated by platelet-derived growth factor and basic fibroblast growth factor, differences were also observed in the response to these agents. B3.1 + TR alpha 1 cells were more sensitive to platelet-derived growth factor than B3.1 and B3.1 + v-erbA cells. In contrast, B3.1 cells responded to basic fibroblast growth factor better than B3.1 + TR alpha 1 or B3.1 + v-erbA cells. Insulin-like growth factor I potentiated the action of platelet-derived growth factor and basic fibroblast growth factor. Again, different responses to treatment with insulin-like growth factor I alone were observed; B3.1 + TR alpha 1 cells did not respond to it, whereas B3.1 + v-erbA cells showed a dramatic stimulation by this agent. Interestingly, in the presence of T3, the blockade in B3.1 + TR alpha 1 cell proliferation was accompanied by the down-regulation of the typical astrocytic genes, glial fibrillary acidic protein and vimentin. These hormone effects were not found in v-erbA-expressing cells. In addition, v-erbA inhibited the basal expression of the cyclic nucleotide phosphodiesterase gene, an oligodendrocytic marker.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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

  7. 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.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    Lisa K. Mullany

    2015-10-01

    Full Text Available Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC. As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17; copy number variation (R175H; chromosome 9; and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31% of HGSCs exhibit loss of heterozygosity, a significant number (24% maintain a wild-type (WT allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing “proof of principle” that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.

  10. 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

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  13. Zinc stimulates glucose oxidation and glycemic control by modulating the insulin signaling pathway in human and mouse skeletal muscle cell lines.

    Science.gov (United States)

    Norouzi, Shaghayegh; Adulcikas, John; Sohal, Sukhwinder Singh; Myers, Stephen

    2018-01-01

    Zinc is a metal ion that is an essential cell signaling molecule. Highlighting this, zinc is an insulin mimetic, activating cellular pathways that regulate cellular homeostasis and physiological responses. Previous studies have linked dysfunctional zinc signaling with several disease states including cancer, obesity, cardiovascular disease and type 2 diabetes. The present study evaluated the insulin-like effects of zinc on cell signaling molecules including tyrosine, PRSA40, Akt, ERK1/2, SHP-2, GSK-3β and p38, and glucose oxidation in human and mouse skeletal muscle cells. Insulin and zinc independently led to the phosphorylation of these proteins over a 60-minute time course in both mouse and human skeletal muscle cells. Similarly, utilizing a protein array we identified that zinc could active the phosphorylation of p38, ERK1/2 and GSK-3B in human and ERK1/2 and GSK-3B in mouse skeletal muscle cells. Glucose oxidation assays were performed on skeletal muscle cells treated with insulin, zinc, or a combination of both and resulted in a significant induction of glucose consumption in mouse (pzinc alone. Insulin, as expected, increased glucose oxidation in mouse (pzinc and insulin did not augment glucose consumption in these cells. Zinc acts as an insulin mimetic, activating key molecules implicated in cell signaling to maintain glucose homeostasis in mouse and human skeletal muscle cells. Zinc is an important metal ion implicated in several biological processes. The role of zinc as an insulin memetic in activating key signaling molecules involved in glucose homeostasis could provide opportunities to utilize this ion therapeutically in treating disorders associated with dysfunctional zinc signaling.

  14. Mutant genes in pea breeding

    International Nuclear Information System (INIS)

    Swiecicki, W.K.

    1990-01-01

    Full text: Mutations of genes Dpo (dehiscing pods) and A (anthocyanin synthesis) played a role in pea domestication. A number of other genes were important in cultivar development for 3 types of usage (dry seeds, green vegetable types, fodder), e.g. fn, fna, le, p, v, fas and af. New genes (induced and spontaneous), are important for present ideotypes and are registered by the Pisum Genetics Association (PGA). Comparison of a pea variety ideotype with the variation available in gene banks shows that breeders need 'new' features. In mutation induction experiments, genotype, mutagen and method of treatment (e.g. combined or fractionated doses) are varied for broadening the mutation spectrum and selecting more genes of agronomic value. New genes are genetically analysed. In Poland, some mutant varieties with the gene afila were registered, controlling lodging by a shorter stem and a higher number of internodes. Really non-lodging pea varieties could strongly increase seed yield. But the probability of detecting a major gene for lodging resistance is low. Therefore, mutant genes with smaller influence on plant architecture are sought, to combine their effect by crossing. Promising seem to be the genes rogue, reductus and arthritic as well as a number of mutant genes not yet genetically identified. The gene det for terminal inflorescence - similarly to Vicia faba - changes plant development. Utilisation of assimilates and ripening should be better. Improvement of harvest index should give higher seed yield. A number of genes controlling disease resistance are well known (eg. Fw, Fnw, En, mo and sbm). Important in mass screening of resistance are closely linked gene markers. Pea gene banks collect respective lines, but mutants induced in highly productive cultivars would be better. Inducing gene markers sometimes seems to be easier than transfer by crossing. Mutation induction in pea breeding is probably more important because a high number of monogenic features are

  15. Selection of mutants of capsicum annuum induced by gamma ray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

    1998-06-01

    For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

  16. Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

    Science.gov (United States)

    de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed; White, Jacqui; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl Mj; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve Dm

    2015-09-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

  17. Increased susceptibility to cortical spreading depression in the mouse model of familial hemiplegic migraine type 2.

    Directory of Open Access Journals (Sweden)

    Loredana Leo

    2011-06-01

    Full Text Available Familial hemiplegic migraine type 2 (FHM2 is an autosomal dominant form of migraine with aura that is caused by mutations of the α2-subunit of the Na,K-ATPase, an isoform almost exclusively expressed in astrocytes in the adult brain. We generated the first FHM2 knock-in mouse model carrying the human W887R mutation in the Atp1a2 orthologous gene. Homozygous Atp1a2(R887/R887 mutants died just after birth, while heterozygous Atp1a2(+/R887 mice showed no apparent clinical phenotype. The mutant α2 Na,K-ATPase protein was barely detectable in the brain of homozygous mutants and strongly reduced in the brain of heterozygous mutants, likely as a consequence of endoplasmic reticulum retention and subsequent proteasomal degradation, as we demonstrate in transfected cells. In vivo analysis of cortical spreading depression (CSD, the phenomenon underlying migraine aura, revealed a decreased induction threshold and an increased velocity of propagation in the heterozygous FHM2 mouse. Since several lines of evidence involve a specific role of the glial α2 Na,K pump in active reuptake of glutamate from the synaptic cleft, we hypothesize that CSD facilitation in the FHM2 mouse model is sustained by inefficient glutamate clearance by astrocytes and consequent increased cortical excitatory neurotransmission. The demonstration that FHM2 and FHM1 mutations share the ability to facilitate induction and propagation of CSD in mouse models further support the role of CSD as a key migraine trigger.

  18. The agronomic characters of a high protein rice mutant

    International Nuclear Information System (INIS)

    Harn, C.; Won, J.L.; Choi, K.T.

    1975-01-01

    Mutant lines (M 5 -M 9 ) of macro-phenotypic traits from several varieties were screened for the protein content. Mutant 398 (M 9 ) is one of the high protein mutants selected from Hokwang. Three years' tests revealed that it has a high protein line under any condition of cultivation. Except for early maturity and short culmness, other agronomic and yield characters were similar to the original variety. There was no difference between the mutant 398 and its mother variety in grain shape and weight, and also the size and protein content of the embryo. The high protein content of the mutant is attributable to the increase of protein in the endosperm. About 150 normal-looking or a few days-earlier-maturing selections were made from Jinheung variety in the M 3 and screened for protein. Promising lines in terms of the plant type, yield and protein were obtained. (author)

  19. [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.

  20. Ascertainment of the effect of differential growth rates of mutants on observed mutant frequencies in X-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Knaap, A.G.A.C.; Simons, J.W.I.M.

    1983-01-01

    As it is not known to what extent differential growth rates of induced mutants lead to over- and under-representation of mutants in treated populations and thereby affect the determination of mutant frequencies, the mutation induction in X-irradiated L5178Y mouse lymphoma cells was determined via two methods. The first method involves the standard protocol which may suffer from the effect of differential growth rates, while the second method is based upon the fluctuation test in which the differential growth rates can be actually measured. It appeared that the standard protocol led to a mutant frequency that was similar to the mutant frequency determined in the fluctuation test. Therefore, the standard protocol appears to lead to only a minor under-estimation if any. Substantial heterogeneity in growth rates of induced mutants was observed, but the mutants with a selective advantage appear largely to compensate for the mutants that are lost because of selective disadvantage. It was calculated that the chance for isolating the same mutant twice from a treated population had been increased 2.2-fold because of the observed differential growth rates. (orig./AJ)

  1. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    Science.gov (United States)

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  2. Studies on an X-ray induced crinkled leaf mutant of Trichosanthes anguina L

    International Nuclear Information System (INIS)

    Datta, Subodh Kumar

    1986-01-01

    Crinkled leaf mutant isolated in the second generation after treatment with X-ray bred true in subsequent generation. The mutant was a late flowering type with increased percentage of PMC's with chromosomal abnormality and pollen grain sterility. TLC study on phenolic compounds in leaves showed that both the mother line and the mutant had equal number of spots but they differed from each other with respect to four spots. Spot Nos. 2 and 4 of the mother line were absent in the mutant but the latter had two new spots (spot Nos. 11 and 12). The mutant and the mother line also differed from each other in pollen grain morohology. (author)

  3. Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

    Directory of Open Access Journals (Sweden)

    Bijay S Jaiswal

    Full Text Available BACKGROUND: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L or NRAS(Q61K mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy. CONCLUSION/SIGNIFICANCE: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

  4. Isoenzymes performance of some rice varieties and their mutants

    International Nuclear Information System (INIS)

    Winarno, Ermin; Suliwarno, Ambyah; Ismachin, M.

    1992-01-01

    Isoenzymes performance of some rice varieties and their mutants. Genetics studies on alcohol dehydrogenase, malic enzyme, peroxidase, acid phosphase, and aminopeptidase isoenzymes were carried out on several groups of rice varieties and their mutant lines. The first groups consisted of Atomita I, Pelita I/1, A227/5, Mudgo, TN-1, and IR-26. The second group was Cisadane variety and its five mutants, namely OBS 18, OBS 208, OBS 297, OBS 306, and OBS 330. The third group was mutants line 627-10-3 and its mutants, namely 1063, 1066, 1067, 1076, and 1090. Isoenzymes extracts of the rice leaves were fractionated using polyacrylamide gel disc electrophoresis. The pattern of acid phosphate isoenzyme shows the specific character of rice mutants susceptible to brown plant hopper biotype 1. The gene(s) controlling malic enzyme in Cisadane's mutants is (are) estimated more resistant toward gamma irradiation than gene(s) responsible for controlling the other enzymes. Generally, the isoenzymes zymograms show that gene(s) controlling the mutants enzyme have undergone mutation. This case is shown by the changes of Rm value, as well as the amount and intensity of mutants bands. (authors). 7 refs., 7 figs

  5. ADAM2 interactions with mouse eggs and cell lines expressing α4/α9 (ITGA4/ITGA9 integrins: implications for integrin-based adhesion and fertilization.

    Directory of Open Access Journals (Sweden)

    Ulyana V Desiderio

    2010-10-01

    Full Text Available Integrins are heterodimeric cell adhesion molecules, with 18 α (ITGA and eight β (ITGB subunits forming 24 heterodimers classified into five families. Certain integrins, especially the α(4/α(9 (ITGA4/ITGA9 family, interact with members of the ADAM (a disintegrin and metalloprotease family. ADAM2 is among the better characterized and also of interest because of its role in sperm function. Having shown that ITGA9 on mouse eggs participates in mouse sperm-egg interactions, we sought to characterize ITGA4/ITGA9-ADAM2 interactions.An anti-β(1/ITGB1 function-blocking antibody that reduces sperm-egg binding significantly inhibited ADAM2 binding to mouse eggs. Analysis of integrin subunit expression indicates that mouse eggs could express at least ten different integrins, five in the RGD-binding family, two in the laminin-binding family, two in the collagen-binding family, and ITGA9-ITGB1. Adhesion assays to characterize ADAM2 interactions with ITGA4/ITGA9 family members produced the surprising result that RPMI 8866 cell adhesion to ADAM2 was inhibited by an anti-ITGA9 antibody, noteworthy because ITGA9 has only been reported to dimerize with ITGB1, and RPMI 8866 cells lack detectable ITGB1. Antibody and siRNA studies demonstrate that ITGB7 is the β subunit contributing to RPMI 8866 adhesion to ADAM2.These data indicate that a novel integrin α-β combination, ITGA9-ITGB7 (α(9β(7, in RPMI 8866 cells functions as a binding partner for ADAM2. ITGA9 had previously only been reported to dimerize with ITGB1. Although ITGA9-ITGB7 is unlikely to be a widely expressed integrin and appears to be the result of "compensatory dimerization" occurring in the context of little/no ITGB1 expression, the data indicate that ITGA9-ITGB7 functions as an ADAM binding partner in certain cellular contexts, with implications for mammalian fertilization and integrin function.

  6. Serrated leaf mutant in mungbean (Vigna radiata (L) Wilczek)

    International Nuclear Information System (INIS)

    Malik, I.A.; Ghulam, Sarwar; Yousaf, Ali; Saleem, M.

    1988-01-01

    Dry dormant seeds of mungbean (Vigna radiata (L) Wilczek) were treated with gamma rays (15, 30 and 60 kR). The serrated leaf mutation was noticed in M 2 of cultivar Pak 32 treated with 60 kR. Cf 14 plants, 3 showed the altered leaf structure and the others were normal. The feature of this mutant was the deep serration of leaflet margins. The mutant had large thick leaflets with prominent venation. The mutant bred true in the M 3 and successive generation. Details of the morphological characteristics of the mutant are presented. The mutant exhibited slower growth particularly during the early stages of development, flowered later and attained shorter height. There was an increase in the number of pods, in seed weight and in seed protein content, but number of seed per pod was considerably reduced. The seed coat colour showed a change from green to yellowish green. In the mutant's flowers the stamina were placed much below the stigma level and the stigma sometimes protruded the corolla. Outcrossing of 4% recorded in some of the mutant lines revealed a reduced cleistogamy. The low number of seeds per pod in the mutant could be due to reduced pollen fertility. The mutant behaved as monogenic recessive. The symbols SL/sl are proposed for this allelic pair. The mutant may have use as a green manure crop because of its large foliage and for the breeders as a genetic marker

  7. In vitro and in silico derived relative effect potencies of ah-receptor-mediated effects by PCDD/Fs and PCBs in rat, mouse, and guinea pig CALUX cell lines.

    Science.gov (United States)

    Ghorbanzadeh, Mehdi; van Ede, Karin I; Larsson, Malin; van Duursen, Majorie B M; Poellinger, Lorenz; Lücke-Johansson, Sandra; Machala, Miroslav; Pěnčíková, Kateřina; Vondráček, Jan; van den Berg, Martin; Denison, Michael S; Ringsted, Tine; Andersson, Patrik L

    2014-07-21

    For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO

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

    Directory of Open Access Journals (Sweden)

    Chun Fu

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

  9. Results of the use of induced mutants in maize breeding

    International Nuclear Information System (INIS)

    Balint, A.; Kovacs, Gezane; Hajos, Laszlone; Geczki, I.

    1979-01-01

    The investigated mutagens have the same effect on the increasing of protein content. In the case of WF9 mutants no essential improvement can be found compared with the untreated co trol selected for protein. ''Lines'' flowering 16-19 days earlier than controls were produced; the most effective agent of this production is the fast neutron. Mutation caused a significant change in their combining ability, but there were more negative variants than positive ones. Three hybrids with stronger stalk than that of MvSc 620 were obtained. Stalk standing ability of mutants did not improve. The flowering date of lines (male) is in r=+0.5672 +++ correlation to the yield of their test hybrid. Mutant lines in SC test cross seemed to be stable. The correlation of the yield of two years is r=+0.8659. The correlation of both the yield of test hybrids to the protein content of mutant lines (r=0.2307) and the flowering date of lines to their protein content (r=-0.3032) is loose. The earliest mutant line of WF9, which produced low crop (5000 kg/ha) when crossed with N6, gave a high-yielding hybrid when crossed with other lines. The average yield of eight combinations was 10050 kg/ha and the highest yield was 11680 kg/ha. (author)

  10. Changes in sensitivity to radiation and to bleomycin occurring during the life history of monolayer cultures of a mouse tumour cell line

    International Nuclear Information System (INIS)

    Twentyman, P.R.; Bleehen, N.M.

    1975-01-01

    The response to X-radiation and to bleomycin has been measured at a number of times during the life of monolayer cultures of EMT6 mouse tumour cells. Little change in radiation sensitivity was seen at any time and no loss of the shoulder to the survival curve occurred. Cultures in early plateau phase (where a considerable amount of cell proliferation is balanced by cell loss) showed a reduced sensitivity to bleomycin when compared with cells in exponential growth. However, after a longer period in plateau phase, when proliferation had virtually ceased, the sensitivity became greater than that of exponential phase cells. These findings are discussed with reference to the conflicting results of other workers. (author)

  11. Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples

    Energy Technology Data Exchange (ETDEWEB)

    Dullin, Christian, E-mail: christian.dullin@med.uni-goettingen.de [University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Monego, Simeone dal [Cluster in Biomedicine, AREA Science Park Basovizza, Trieste (Italy); Larsson, Emanuel [Elettra Sincrotrone Trieste, Strada Statale 14, km 163.5 in AREA Science Park, 34149 Basovizza (Trieste) (Italy); University of Trieste, Trieste (Italy); Linköping University, SE-581 83 Linkoeping (Sweden); Mohammadi, Sara [Elettra Sincrotrone Trieste, Strada Statale 14, km 163.5 in AREA Science Park, 34149 Basovizza (Trieste) (Italy); Krenkel, Martin [University of Göttingen, Göttingen (Germany); Garrovo, Chiara; Biffi, Stefania [IRCCS Burlo Garofolo, Trieste (Italy); Lorenzon, Andrea [Cluster in Biomedicine, AREA Science Park Basovizza, Trieste (Italy); Markus, Andrea [University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Napp, Joanna [University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen (Germany); University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Salditt, Tim [University of Göttingen, Göttingen (Germany); Accardo, Agostino [University of Trieste, Trieste (Italy); Alves, Frauke [University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen (Germany); University Medical Center Göttingen, Robert Koch Strasse 40, 37075 Göttingen (Germany); Tromba, Giuliana [Elettra Sincrotrone Trieste, Strada Statale 14, km 163.5 in AREA Science Park, 34149 Basovizza (Trieste) (Italy)

    2015-01-01

    This study presents an approach to increase the sensitivity of lung computed tomography (CT) imaging by utilizing in-line phase contrast CT in combination with single-distance phase-retrieval algorithms and a dedicated image-processing regime. As demonstrated here, functional CT imaging can be achieved for the assessment of both structural alterations in asthmatic mouse lung tissue and the accumulation pattern of instilled barium-sulfate-labelled macrophages in comparison with healthy controls. Functionalized computed tomography (CT) in combination with labelled cells is virtually non-existent due to the limited sensitivity of X-ray-absorption-based imaging, but would be highly desirable to realise cell tracking studies in entire organisms. In this study we applied in-line free propagation X-ray phase-contrast CT (XPCT) in an allergic asthma mouse model to assess structural changes as well as the biodistribution of barium-labelled macrophages in lung tissue. Alveolar macrophages that were barium-sulfate-loaded and fluorescent-labelled were instilled intratracheally into asthmatic and control mice. Mice were sacrificed after 24 h, lungs were kept in situ, inflated with air and scanned utilizing XPCT at the SYRMEP beamline (Elettra Synchrotron Light Source, Italy). Single-distance phase retrieval was used to generate data sets with ten times greater contrast-to-noise ratio than absorption-based CT (in our setup), thus allowing to depict and quantify structural hallmarks of asthmatic lungs such as reduced air volume, obstruction of airways and increased soft-tissue content. Furthermore, we found a higher concentration as well as a specific accumulation of the barium-labelled macrophages in asthmatic lung tissue. It is believe that XPCT will be beneficial in preclinical asthma research for both the assessment of therapeutic response as well as the analysis of the role of the recruitment of macrophages to inflammatory sites.

  12. ''Fushi'' - excellent mutant germplasm for peanut improvement

    International Nuclear Information System (INIS)

    Jiang, X.; Zhou, Y.

    1989-01-01

    Full text: The mutant line ''Fushi'' was selected following seed treatment of the variety ''Shi Xuan 64'' in 1960 with 32 P. Many good peanut varieties were developed using ''Fushi'' in cross-breeding (ref. Mutation Breeding Newsletter No. 30 (July 1987) p. 2-3). In the past 10 years, planting areas of these varieties added up to 3,3 million ha in South China, peanut production was increased by more than 500 000 t valued 500 million Yuan. (author)

  13. Comparative proteome analysis of three mouse lung adenocarcinoma CMT cell lines with different metastatic potential by two-dimensional gel electrophoresis and mass spectrometry

    DEFF Research Database (Denmark)

    Zhang, Kelan; Wrzesinski, Krzysztof; Stephen, J Fey

    2008-01-01

    and characterized in vivo to have different metastatic potential. In this study, the comprehensive protein expression profiles of three of these CMT cell lines at passage 5, 15 and 35 were analyzed by 2-DE separation followed by MS identification. As a result, 82 and 40 unique proteins were found...

  14. An early maturing rice mutant released as a variety

    International Nuclear Information System (INIS)

    Azam, M.A.; Imtiaz Uddin, Md.

    2001-01-01

    In the content of food grain production deficiency (about 1.0-1.5 million tons of rice per year according to the Bangladesh Bureau of Statistics, 1998) an induced mutation programme was undertaken in 1985. One moderate early maturing and high yielding rice mutant line (BINA6-84-4-115) has been developed by irradiating F 2 seeds of the cross 'BR4' x 'Iratom 38'. Three treatments viz., 250, 300 and 350 Gy were given to the F 2 seeds. Finally, this line was selected in M 6 generation for advanced yield trial. The line was evaluated in comparative trials with another mutant line BINA6-84-4-163. These two mutant lines had been selected earlier from 300 Gy originated lines. The two check varieties, 'BR 11' and 'BR 22' were also included in the trial, which was conducted in two consecutive T. aman seasons (July to December) during 1994 and 1995 at five locations in Bangladesh. From the results, it was evident that the mutant BINA6-84-4-115 did not differ much with the other mutant lines or check varieties in respect to plant height, number of effective tillers and panicle length but it was 10-18 days earlier than the other 3 entries. It produced a similar yield as the check BR 11 in 1994 and a higher yield than the check BR 11 and BR 22 in 1995. This mutant line gave the highest yield per day among all the entries. In addition to this, the grains are long, fine and possess a high L/B ratio, which are of high commercial value. This line has been released by the National Seed Board of Bangladesh in 1998 as a commercial variety under the name 'BINADHAN-4' for cultivation throughout Bangladesh

  15. Amplification of the Ect2 proto-oncogene and over-expression of Ect2 mRNA and protein in nickel compound and methylcholanthrene-transformed 10T1/2 mouse fibroblast cell lines

    International Nuclear Information System (INIS)

    Clemens, Farrah; Verma, Rini; Ramnath, Jamuna; Landolph, Joseph R.

    2005-01-01

    Occupational exposure of humans to mixtures of insoluble and soluble nickel (Ni) compounds correlates with increased incidences of lung, sinus, and pharyngeal tumors. Specific insoluble Ni compounds are carcinogenic to animals by inhalation and induce morphological and neoplastic transformation of cultured rodent cells. Our objectives were to (1) understand mechanisms of nickel ion-induced cell transformation, hence carcinogenesis and (2) develop biomarkers of nickel ion exposure and nickel ion-induced cell transformation. We isolated mRNAs from green nickel oxide (NiO), crystalline nickel monosulfide (NiS), and 3-methylcholanthrene (MCA) transformed C3H/10T1/2 Cl 8 cell lines, and determined by mRNA differential display that nine mRNA fragments were differentially expressed between Ni transformed and non-transformed 10T1/2 cell lines. Fragment R2-5 was expressed at higher steady-state levels in the transformed cell lines. R2-5 had 100% sequence identity to part of the coding region of Ect2, a mouse proto-oncogene encoding a GDP-GTP exchange factor. The 3.9-kb Ect2 transcript was expressed at 1.6- to 3.6-fold higher steady-state levels in four Ni transformed, and in two MCA-transformed, cell lines. Ect2 protein was expressed at 3.0- to 4.5-fold higher steady-state levels in Ni-transformed and in MCA-transformed cell lines. The Ect2 gene was amplified by 3.5- to 10-fold in Ni transformed, and by 2.5- to 3-fold in MCA transformed cell lines. Binding of nickel ions to enzymes of DNA synthesis likely caused amplification of the Ect2 gene. Ect2 gene amplification and over-expression of Ect2 mRNA and protein can cause microtubule disassembly and cytokinesis, contributing to induction and maintenance of morphological, anchorage-independent, and neoplastic transformation of these cell lines. Over-expression of Ect2 protein is a useful biomarker to detect exposure to nickel compounds and nickel ion-induced morphological and neoplastic cell transformation

  16. Assessing mouse behaviour throughout the light/dark cycle using automated in-cage analysis tools.

    Science.gov (United States)

    Bains, Rasneer S; Wells, Sara; Sillito, Rowland R; Armstrong, J Douglas; Cater, Heather L; Banks, Gareth; Nolan, Patrick M

    2018-04-15

    An important factor in reducing variability in mouse test outcomes has been to develop assays that can be used for continuous automated home cage assessment. Our experience has shown that this has been most evidenced in long-term assessment of wheel-running activity in mice. Historically, wheel-running in mice and other rodents have been used as a robust assay to determine, with precision, the inherent period of circadian rhythms in mice. Furthermore, this assay has been instrumental in dissecting the molecular genetic basis of mammalian circadian rhythms. In teasing out the elements of this test that have determined its robustness - automated assessment of an unforced behaviour in the home cage over long time intervals - we and others have been investigating whether similar test apparatus could be used to accurately discriminate differences in distinct behavioural parameters in mice. Firstly, using these systems, we explored behaviours in a number of mouse inbred strains to determine whether we could extract biologically meaningful differences. Secondly, we tested a number of relevant mutant lines to determine how discriminative these parameters were. Our findings show that, when compared to conventional out-of-cage phenotyping, a far deeper understanding of mouse mutant phenotype can be established by monitoring behaviour in the home cage over one or more light:dark cycles. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  17. Durable resistance to net blotch and agronomic performance in some barley mutants [Hordeum vulgare L.; Syria

    International Nuclear Information System (INIS)

    Arabi, M.I.E.

    2004-01-01

    Seeds from the net blotch (Pyrenophora teres) susceptible cultivar Thibaut were treated by gamma ray radiation and subsequently evaluated for reaction to the pathogen in the M2-M5 generations. Grain yield and agronomic characteristics of putative mutants were compared with Thibaut in two different locations. Genetic variation among some mutant lines/cv Thibaut was estimated using Amplified Fragment Length Polymorphism (AFLP) markers. Sixteen mutant lines and their mother cultivar Thibaut were analyzed with 14 EcoR1-Mse1 primer combinations. A total number of 504 AFLP bands were analyzed for each pair mutant/Thibaut. Narrow genetic variation among all genotypes was detected with an average of genetic similarity of 0.96. Cluster analysis with the entire AFLP data divided all genotypes into two major groups. The resistant mutant lines were grouped in one subcluster with 0.98 similarity index. Some resistant mutant lines to net blotch with good agronomic performances were produced [it

  18. Genetic mouse models of brain ageing and Alzheimer's disease.

    Science.gov (United States)

    Bilkei-Gorzo, Andras

    2014-05-01

    Progression of brain ageing is influenced by a complex interaction of genetic and environmental factors. Analysis of genetically modified animals with uniform genetic backgrounds in a standardised, controlled environment enables the dissection of critical determinants of brain ageing on a molecular level. Human and animal studies suggest that increased load of damaged macromolecules, efficacy of DNA maintenance, mitochondrial activity, and cellular stress defences are critical determinants of brain ageing. Surprisingly, mouse lines with genetic impairment of anti-oxidative capacity generally did not show enhanced cognitive ageing but rather an increased sensitivity to oxidative challenge. Mouse lines with impaired mitochondrial activity had critically short life spans or severe and rapidly progressing neurodegeneration. Strains with impaired clearance in damaged macromolecules or defects in the regulation of cellular stress defences showed alterations in the onset and progression of cognitive decline. Importantly, reduced insulin/insulin-like growth factor signalling generally increased life span but impaired cognitive functions revealing a complex interaction between ageing of the brain and of the body. Brain ageing is accompanied by an increased risk of developing Alzheimer's disease. Transgenic mouse models expressing high levels of mutant human amyloid precursor protein showed a number of symptoms and pathophysiological processes typical for early phase of Alzheimer's disease. Generally, therapeutic strategies effective against Alzheimer's disease in humans were also active in the Tg2576, APP23, APP/PS1 and 5xFAD lines, but a large number of false positive findings were also reported. The 3xtg AD model likely has the highest face and construct validity but further studies are needed. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Induction and characterization of Arabidopsis mutants by Ion beam

    International Nuclear Information System (INIS)

    Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S.

    2008-03-01

    This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and γ-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

  20. Mildew resistant and less lodging wheat mutants induced in Iran

    International Nuclear Information System (INIS)

    Naghedi-Ahmadi, I.

    1989-01-01

    ''Tabassi'' is a lodging and mildew susceptible cultivar. To induce mutations, seeds were gamma irradiated (50 to 150 Gy) in 1982 and selection for lodging resistance was carried out in M 2 . During field experiments with the mutant lines in 1985/86 there has been a heavy mildew epidemic under which mutant 63-5-I (derived from 50 Gy treatment) exhibited considerable resistance and as a consequence, higher yield. The control was 100% infected, the mutant only 40%. The mutant yielded 31% more grain, 7.5% less straw and 4.5% more protein than the control. Lodging of 63-5-I was only 60% in an experiment under rainfed conditions in the same season, resulting in a relative yield increase of about 11%. In 1986/87 there was no mildew epidemic and the mutant yielded the same as ''Tabassi''

  1. Induction and characterization of Arabidopsis mutants by Ion beam

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S. [Gyeongbuk Institute for Bio Industry, Andong (Korea, Republic of)

    2008-03-15

    This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and {gamma}-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

  2. 10. international mouse genome conference

    Energy Technology Data Exchange (ETDEWEB)

    Meisler, M.H.

    1996-12-31

    Ten years after hosting the First International Mammalian Genome Conference in Paris in 1986, Dr. Jean-Louis Guenet presided over the Tenth Conference at the Pasteur Institute, October 7--10, 1996. The 1986 conference was a satellite to the Human Gene Mapping Workshop and had approximately 50 attendees. The 1996 meeting was attended by 300 scientists from around the world. In the interim, the number of mapped loci in the mouse increased from 1,000 to over 20,000. This report contains a listing of the program and its participants, and two articles that review the meeting and the role of the laboratory mouse in the Human Genome project. More than 200 papers were presented at the conference covering the following topics: International mouse chromosome committee meetings; Mutant generation and identification; Physical and genetic maps; New technology and resources; Chromatin structure and gene regulation; Rate and hamster genetic maps; Informatics and databases; and Quantitative trait analysis.

  3. 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)

  4. Selection of gamma-ray induced salt tolerant rice mutants by in vitro mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Sub; Chun, Jae Beom; Lee, Kyung Jun; Kim, Jin Baek; Kim, Sang Hoon; Yun, Song Jong; Kang, Si Yong [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2010-06-15

    The present study had been performed to select the salt tolerant rice mutant lines through an in vivo and in vitro mutagenesis with a gamma-ray. The physiological responses such as MDA and chlorophyll of the selected salt mutant lines were investigated under salt stress. For the selection of the salt tolerant rice mutants by in vitro mutagenesis with gamma-ray, we conducted a second selection procedure with 1,500 mutant lines induced from the original cv. Dongan (wild-type, WT): Ist, selection under a nutrient solution with 171 mM NaCI: 2nd, selection under in vitro conditions. Based on a growth comparison of the entries, out of mutant lines, the putative 2 salt tolerant rice mutant lines, ST-495 and ST-532, were selected. The 2 ST-lines had a lower malonaldehyde (MDA) contents than wild-type (WT) during salt stress. The survival rate of the WT, ST-495 and ST-532 were 36.6%, 70% and 50% in 171 mM NaCI, respectively. The chlorophyll and carotenoid contents were decreased more in a WT plant than the two selected mutant lines. These rice mutant lines will be released for cultivation at the reclaimed land and used as a control plot for genetic research about salt tolerance.

  5. Selection of gamma-ray induced salt tolerant rice mutants by in vitro mutagenesis

    International Nuclear Information System (INIS)

    Kim, Dong Sub; Chun, Jae Beom; Lee, Kyung Jun; Kim, Jin Baek; Kim, Sang Hoon; Yun, Song Jong; Kang, Si Yong

    2010-01-01

    The present study had been performed to select the salt tolerant rice mutant lines through an in vivo and in vitro mutagenesis with a gamma-ray. The physiological responses such as MDA and chlorophyll of the selected salt mutant lines were investigated under salt stress. For the selection of the salt tolerant rice mutants by in vitro mutagenesis with gamma-ray, we conducted a second selection procedure with 1,500 mutant lines induced from the original cv. Dongan (wild-type, WT): Ist, selection under a nutrient solution with 171 mM NaCI: 2nd, selection under in vitro conditions. Based on a growth comparison of the entries, out of mutant lines, the putative 2 salt tolerant rice mutant lines, ST-495 and ST-532, were selected. The 2 ST-lines had a lower malonaldehyde (MDA) contents than wild-type (WT) during salt stress. The survival rate of the WT, ST-495 and ST-532 were 36.6%, 70% and 50% in 171 mM NaCI, respectively. The chlorophyll and carotenoid contents were decreased more in a WT plant than the two selected mutant lines. These rice mutant lines will be released for cultivation at the reclaimed land and used as a control plot for genetic research about salt tolerance

  6. Characterisation of Signalling by the Endogenous GPER1 (GPR30 Receptor in an Embryonic Mouse Hippocampal Cell Line (mHippoE-18.

    Directory of Open Access Journals (Sweden)

    Nicholas J Evans

    Full Text Available Estrogen can modulate neuronal development and signalling by both genomic and non-genomic pathways. Many of its rapid, non-genomic effects on nervous tissue have been suggested to be mediated via the activation of the estrogen sensitive G-protein coupled receptor (GPER1 or GPR30. There has been much controversy over the cellular location, signalling properties and endogenous activators of GPER1. Here we describe the pharmacology and signalling properties of GPER1 in an immortalized embryonic hippocampal cell line, mHippoE-18. This cell line does not suffer from the inherent problems associated with the study of this receptor in native tissue or the problems associated with heterologously expression in clonal cell lines. In mHippoE-18 cells, 17β-Estradiol can mediate a dose-dependent rapid potentiation of forskolin-stimulated cyclic AMP levels but does not appear to activate the ERK1/2 pathway. The effect of 17β-Estradiol can be mimicked by the GPER1 agonist, G1, and also by tamoxifen and ICI 182,780 which activate GPER1 in a variety of other preparations. The response is not mimicked by the application of the classical estrogen receptor agonists, PPT, (an ERα agonist or DPN, (an ERβ agonist, further suggesting that this effect of 17β-Estradiol is mediated through the activation of GPER1. However, after exposure of the cells to the GPER1 specific antagonists, G15 and G36, the stimulatory effects of the above agonists are replaced by dose-dependent inhibitions of forskolin-stimulated cyclic AMP levels. This inhibitory effect is mimicked by aldosterone in a dose-dependent way even in the absence of the GPER1 antagonists. The results are discussed in terms of possible "Biased Antagonism" whereby the antagonists change the conformation of the receptor resulting in changes in the agonist induced coupling of the receptor to different second messenger pathways.

  7. Systematic screening for skin, hair, and nail abnormalities in a large-scale knockout mouse program.

    Directory of Open Access Journals (Sweden)

    John P Sundberg

    Full Text Available The International Knockout Mouse Consortium was formed in 2007 to inactivate ("knockout" all protein-coding genes in the mouse genome in embryonic stem cells. Production and characterization of these mice, now underway, has generated and phenotyped 3,100 strains with knockout alleles. Skin and adnexa diseases are best defined at the gross clinical level and by histopathology. Representative retired breeders had skin collected from the back, abdomen, eyelids, muzzle, ears, tail, and lower limbs including the nails. To date, 169 novel mutant lines were reviewed and of these, only one was found to have a relatively minor sebaceous gland abnormality associated with follicular dystrophy. The B6N(Cg-Far2tm2b(KOMPWtsi/2J strain, had lesions affecting sebaceous glands with what appeared to be a secondary follicular dystrophy. A second line, B6N(Cg-Ppp1r9btm1.1(KOMPVlcg/J, had follicular dystrophy limited to many but not all mystacial vibrissae in heterozygous but not homozygous mutant mice, suggesting that this was a nonspecific background lesion. We discuss potential reasons for the low frequency of skin and adnexal phenotypes in mice from this project in comparison to those seen in human Mendelian diseases, and suggest alternative approaches to identification of human disease-relevant models.

  8. The niche-derived glial cell line-derived neurotrophic factor (GDNF induces migration of mouse spermatogonial stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Lisa Dovere

    Full Text Available In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF, a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  9. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    . To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...

  10. Myelination competent conditionally immortalized mouse Schwann cells

    NARCIS (Netherlands)

    Saavedra, José T.; Wolterman, Ruud A.; Baas, Frank; ten Asbroek, Anneloor L. M. A.

    2008-01-01

    Numerous mouse myelin mutants are available to analyze the biology of the peripheral nervous system related to health and disease in vivo. However, robust in vitro biochemical characterizations of players in peripheral nerve processes are still not possible due to the limited growth capacities of

  11. Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model.

    Science.gov (United States)

    Horemans, Tessa; Kerstens, Monique; Clais, Sofie; Struijs, Karin; van den Abbeele, Pieter; Van Assche, Tim; Maes, Louis; Cos, Paul

    2012-08-01

     The interest in non-antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic-resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection.  Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti-adhesive effect on H. pylori was determined first in a cell model using the mucus-producing gastric epithelial cell line NCI-N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day -2 to day 4 post-infection. Bacterial loads were determined by using quantitative real-time PCR and the standard plate count method.  The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 μg/mL. In the anti-adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti-adhesive effects and even caused an increase in bacterial load in the stomach.  The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection. © 2012 Blackwell Publishing Ltd.

  12. Photorepair mutants of Arabidopsis

    International Nuclear Information System (INIS)

    Jiang, C.Z.; Yee, J.; Mitchell, D.L.; Britt, A.B.

    1997-01-01

    UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system

  13. Construindo Marcas Mutantes

    Directory of Open Access Journals (Sweden)

    Elizete De Azevedo Kreutz

    2012-09-01

    Full Text Available O presente artigo é o resultado de estudos realizados desde 2000 e busca instrumentalizar os proñssionals para a construção de Marcas Mutantes, que é   uma tendência contemporânea nas estratégias comunicacionais e de branding. Embora esta estratégia ainda não esteja consolidada, observamos que a mesma tem obtido um crescimento constante e tem sido adotadas pelas mais diferentes categorias de marcas e não apenas por aquelas direcionadas aos jovens, ao esporte, ao entretenimento, como era no principia. Com base na Hermenêutica de Profundidade de Thompson (1995, alicerçada nas pesquisas bibliográficas, de intemet, entrevistas e análise semiótica, desenhamos um método de construção de Marcas Mutantes dividido em sete fases. Como resultado, esperamos que este estudo possa auxiliar na compreensão dos processos envolvidos, ao mesmo tempo que provoque a discussão sobreo mesmo e, por consequência, o seu aprimoramento.

  14. SU-C-204-05: Magnetic Resonance-Induced Adaptive Response to Orthovoltage Radiation Therapy in FSa and SA-NH Mouse Tumor Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Mendel, K; Miller, R; Murley, J; Sadinski, M; Grdina, D [University of Chicago, Chicago, IL (United States)

    2016-06-15

    Purpose: To assess the effect of pre-treatment Magnetic Resonance Imaging (MRI) on cell survival following orthovoltage radiation therapy. Methods: This in vitro study examined the survival of FSa cells (extracted from methylcholanthrene-induced fibrosarcoma in the flank of a C3H female mouse) and SA-NH cells (derived from a spontaneously arising murine sarcoma tumor) having undergone an MRI scan prior to radiation exposure. Cell cultures were kept at 37 C, in a humidified environment with 5% CO2, and were grown to confluence prior to the start of the experiment. Each cell culture underwent two, 25 minute MRIs spaced 24 hours apart using a standard brain imaging protocol. The cultures were exposed to a 2 Gy dose of radiation beginning 15 minutes after the end of each MRI scan. Irradiations were performed by a Philips RT250 X-ray generator at 250 kVp and 15 mA. All MR imaging was performed on a 1.5 T Philips Achieva scanner using a head and neck vasculature coil. Results: Cells given an MRI scan prior to radiation exhibited an increase in mean surviving fraction of 10.8% and 9.6% in FSa and SA-NH cells, respectively. The difference was found to be statistically significant in both cell types by a student two-tailed t test with P = 0.011 and P < 0.001 for FSa and SA-NH, respectively. Conclusion: MRI may cause an increase in radio-resistance in FSa and SA-NH cells. If this biological effect is found to be consistent across other cell types and voltage ranges, these results could help inform treatment planning by improving our understanding of the joint effects of MRI and ionizing radiation. This work was supported in part under NIH grant numbers T32 EB002103, NCI R01-CA 132998, DOE Low Dose Program/Project Grant DE-413 SC0001271. DJ Grdina is a paid consultant to Pinnacle Biologics. DJ Grdina and JS Murley are minority equity partners in Pinnacle Oncology LLC.

  15. SU-C-204-05: Magnetic Resonance-Induced Adaptive Response to Orthovoltage Radiation Therapy in FSa and SA-NH Mouse Tumor Cell Lines

    International Nuclear Information System (INIS)

    Mendel, K; Miller, R; Murley, J; Sadinski, M; Grdina, D

    2016-01-01

    Purpose: To assess the effect of pre-treatment Magnetic Resonance Imaging (MRI) on cell survival following orthovoltage radiation therapy. Methods: This in vitro study examined the survival of FSa cells (extracted from methylcholanthrene-induced fibrosarcoma in the flank of a C3H female mouse) and SA-NH cells (derived from a spontaneously arising murine sarcoma tumor) having undergone an MRI scan prior to radiation exposure. Cell cultures were kept at 37 C, in a humidified environment with 5% CO2, and were grown to confluence prior to the start of the experiment. Each cell culture underwent two, 25 minute MRIs spaced 24 hours apart using a standard brain imaging protocol. The cultures were exposed to a 2 Gy dose of radiation beginning 15 minutes after the end of each MRI scan. Irradiations were performed by a Philips RT250 X-ray generator at 250 kVp and 15 mA. All MR imaging was performed on a 1.5 T Philips Achieva scanner using a head and neck vasculature coil. Results: Cells given an MRI scan prior to radiation exhibited an increase in mean surviving fraction of 10.8% and 9.6% in FSa and SA-NH cells, respectively. The difference was found to be statistically significant in both cell types by a student two-tailed t test with P = 0.011 and P < 0.001 for FSa and SA-NH, respectively. Conclusion: MRI may cause an increase in radio-resistance in FSa and SA-NH cells. If this biological effect is found to be consistent across other cell types and voltage ranges, these results could help inform treatment planning by improving our understanding of the joint effects of MRI and ionizing radiation. This work was supported in part under NIH grant numbers T32 EB002103, NCI R01-CA 132998, DOE Low Dose Program/Project Grant DE-413 SC0001271. DJ Grdina is a paid consultant to Pinnacle Biologics. DJ Grdina and JS Murley are minority equity partners in Pinnacle Oncology LLC.

  16. Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

    Science.gov (United States)

    Gaub, S; Fisher, S E; Ehret, G

    2016-02-01

    Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  17. Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

    International Nuclear Information System (INIS)

    Bame, K.J.; Kiser, C.S.; Esko, J.D.

    1987-01-01

    The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate 35 SO 4 into acid-precipitable material. Some mutants did not incorporate 35 SO 4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects

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

    Science.gov (United States)

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

    2011-01-01

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

  19. Characteristics and use of wheat mutants tolerant or resistant to Septoria nodorum Berk

    International Nuclear Information System (INIS)

    Fossati, A.; Kleijer, G.; Fried, P.M.

    1983-01-01

    Mutation induction was used to obtain mutants tolerant or resistant to Septoria nodorum. This technique is valuable but many genotypes had to be treated because mutants could not be selected from all the genotypes. Short tolerant mutants could be obtained from 3 of the 15 treated tall tolerant lines. Induction of tolerance in susceptible lines of good agronomic value succeeded for 2 of 5 treated varieties. All these mutants showed a reduction in yield potential. One mutant showed partial resistance to S. nodorum. The disease development on the leaves and the spikes of this mutant was much slower than on the original variety. The characteristics of this mutant are discussed in detail. The genetics of tolerance proved to be polygenic and additive, which has consequences on the breeding method. A good way of obtaining a stable system would be the combination of high tolerance and partial resistance in the same cultivar. (author)

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

    Science.gov (United States)

    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

  1. Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Lalonde, R; Strazielle, C

    2011-04-15

    Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage. Copyright © 2010. Published by Elsevier B.V.

  2. Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

    Energy Technology Data Exchange (ETDEWEB)

    Strauss, W.M.; Dausman, J.; Beard, C.; Jaenisch, R. (Massachusetts Inst. of Technology, Cambridge (United States)); Johnson, C.; Lawrence, J.B. (Univ. of Massachusetts Medical School, Worcester (United States))

    1993-03-26

    Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.

  3. Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrP(Sc) in prion infected cell line via protein interaction.

    Science.gov (United States)

    Wang, Hui; Tian, Chan; Fan, Xue-Yu; Chen, Li-Na; Lv, Yan; Sun, Jing; Zhao, Yang-Jing; Zhang, Lu-bin; Wang, Jing; Shi, Qi; Gao, Chen; Chen, Cao; Shao, Qi-Xiang; Dong, Xiao-Ping

    2015-05-01

    Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP(Sc) level in a dose-dependent manner, but had minimal effect on the expression of PrP(C) in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP(Sc). We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP(Sc) in the brain tissues. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Proteostasis and ageing: insights from long-lived mutant mice.

    Science.gov (United States)

    Sands, William A; Page, Melissa M; Selman, Colin

    2017-10-15

    The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

  5. Isozyme differences in barley mutants

    Energy Technology Data Exchange (ETDEWEB)

    AI-Jibouri, A A.M.; Dham, K M [Department of Botany, Nuclear Research Centre, Baghdad (Iraq)

    1990-01-01

    Full text: Thirty mutants (M{sub 11}) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

  6. Isozyme differences in barley mutants

    International Nuclear Information System (INIS)

    AI-Jibouri, A.A.M.; Dham, K.M.

    1990-01-01

    Full text: Thirty mutants (M 11 ) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

  7. In vitro selection of high-infectious, leukemogenic virus from low-infectious, non-leukemogenic type C virus from a malignant ST/a mouse cell line

    DEFF Research Database (Denmark)

    Willumsen, B M

    1979-01-01

    ; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB...... nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non......-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC...

  8. Field Performance of Five Soybean Mutants Under Drought Stress Conditions and Molecular Analysis Using SSR Markers

    Directory of Open Access Journals (Sweden)

    Y Yuliasti

    2017-08-01

    Full Text Available The objectives of this research wereto evaluate (1 the performance of soybean mutant lines under drought stress conditions, and(2 the genetic diversity and relationship among the mutant lines using SSR markers.The field evaluation was conducted during the dry season of 2011 and 2012 at the experimental Farm of Mataram University, West Nusa Tenggara, Indonesia. The field experiment was set up in a randomized block design. Ten mutant lines and two control varieties were evaluated in four replications. Genetic distance among evaluated lines were determined based on allelic diversity analysis using 40 simple sequence repeat (SSR loci. Under drought stress conditions, two mutant lines, Kdl3 and Kdl8,showed a better performance compared to the other ones. The high yielding mutant lines were Kdl3and Kdl8, which yielded 1.75 t ha-1and 1.69 t ha-1, respectively, compared to the parent and national control, Panderman 1.43 t ha-1 and Muria 1.32 t ha-1. These mutant linesrequired 30.75 to 32days to flower and 79.75 to 83.75 day to harvest with relatively short plant height 28.25 and 23.35 cmrespectively. Those mutant characters were better than those of the other three mutants, the original parents, and the control soybean species. Since the evaluated soybean mutant lines yielded more under drought stress conditions than the standard varieties, they can be used and registered as drought-tolerant soybean mutants. Moreover, the evaluated soybean accessions showed a wide genetic distance. The accessions were clustered into two groups according to their genetic background, namelygroup I (the Panderman with three mutant lines and group II (the Muria with two mutant lines. Twenty-three out of 40 evaluated SSR loci, including AW31, BE806, CMAC7L, S080, S126, S57, S171, S224, S285, S294, S393, S294, S383, S511, S511, S520, S540, S547, S551, S571, S577, and S578, provided polymorphic alleles between the parents and their mutants and could be used to differentiate

  9. Methamphetamine-induced conditioned place preference in LG/J and SM/J mouse strains and an F45/F46 advanced intercross line.

    Science.gov (United States)

    Bryant, Camron D; Kole, Loren A; Guido, Michael A; Cheng, Riyan; Palmer, Abraham A

    2012-01-01

    The conditioned place preference (CPP) test is frequently used to evaluate the rewarding properties of drugs of abuse in mice. Despite its widespread use in transgenic and knockout experiments, there are few forward genetic studies using CPP to identify novel genes contributing to drug reward. In this study, we tested LG/J and SM/J inbred strains and the parents/offspring of 10 families of an F(45)/F(46) advanced intercross line (AIL) for methamphetamine-induced CPP (MA-CPP) once per week over 2 weeks. Both LG/J and SM/J mice exhibited significant MA-CPP that was not significantly different between the two strains. Furthermore, LG/J mice showed significantly less acute MA-induced locomotor activity as well as locomotor sensitization following subsequent MA injections. AIL mice (N = 105) segregating LG/J and SM/J alleles also demonstrated significant MA-CPP that was equal in magnitude between the first and second week of training. Importantly, MA-CPP in AIL mice did not correlate with drug-free or MA-induced locomotor activity, indicating that MA-CPP was not confounded by test session activity and implying that MA-CPP is genetically distinct from acute psychomotor sensitivity. We estimated the heritability of MA-CPP and locomotor phenotypes using midparent-offspring regression and maximum likelihood estimates derived from the kinship coefficients of the AIL pedigree. Heritability estimates of MA-CPP were low (0-0.21) and variable (SE = 0-0.33) which reflected our poor power to estimate heritability using only 10 midparent-offspring observations. In sum, we established a short-term protocol for MA-CPP in AIL mice that could reveal LG/J and SM/J alleles important for MA reward. The use of highly recombinant genetic populations like AIL should facilitate the identification of these genes and may have implications for understanding psychostimulant abuse in humans.

  10. A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line.

    Science.gov (United States)

    Takasugi, Mikako; Muta, Emi; Yamada, Koji; Arai, Hirofumi

    2018-02-01

    Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB 4 activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB 4 production by the cells was determined by HPLC with UV detection. LTB 4 was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB 4 production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB 4 detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB 4 production, but AACOCF 3 (phospholipase A 2 inhibitor) slightly increased LTB 4 production, suggesting that LTB 4 was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB 4 activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB 4 production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB 4 inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB 4 release suppression by food components such as flavonoids and the structure-activity relationship.

  11. Cellular determinants of the inverse cross sensitivity of mouse lymphoma L5178Y cell lines to ionizing radiation and hydrogen peroxide

    International Nuclear Information System (INIS)

    Kruszewski, M.

    1999-01-01

    The pair of L5178Y sublines (LY-R and LY-S) is exceptional among mammalian cell lines because of their unique inverse cross-sensitivity to ionizing radiation and hydrogen peroxide. The high sensitivity of LY-S cells to ionizing radiation is reasonably explained by the impairment of DNA double strand breaks rejoining. Although the enzymatic defect of LY-S cells is not yet identified, the more pronounced effect of DNA-dependent protein kinase (DNA-PK) inhibitor (OK-1035) on DNA damage repair after 8 Gy x-irradiation in LY-R cells than in LY-S cells, suggests that LY-S cells may be defective in DNA-PK activity or in the other enzymatic activities downstream from DNA-PK. An additional feature is a higher protection of DNA against ionizing radiation by nuclear proteins in LY-R cells. These data support the concept that nuclear matrix organization may contribute to the cellular susceptibility to DNA damaging agents. Ionizing radiation-sensitive LY-S cells suffer also more DNA base damage than ionizing radiation-resistant LY-R cells. However, the repair rates of the γ-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the lethal or mutagenic effects of ionizing radiation. In contrast, hydrogen peroxide (H 2 O 2 ) sensitive LY-R cells suffer more DNA base damage after H 2 O 2 treatment. This may be due to the lower activity of catalase and/or the lower level of glutathione and other monobromobimane-reactive thiols in LY-R cells than in LY-S cells. However, the main cause of LY-R cells' sensitivity to H 2 O 2 seems to be a higher iron ion content in these cells as compared to LY-S cells. A higher content of iron ions and a higher iron:copper ratio is found in isolated nuclei of LY-R cells than in those of LY-S cells. This is further confirmed by a higher ''labile iron'' pool in LY-R cells than in LY-S cells. Further evidence of different ions content in LY cells and its influence on nuclear matrix organization

  12. Primary tumor site is a useful predictor of cetuximab efficacy in the third-line or salvage treatment of KRAS wild-type (exon 2 non-mutant) metastatic colorectal cancer: a nationwide cohort study

    International Nuclear Information System (INIS)

    Chen, Kuo-Hsing; Shao, Yu-Yun; Chen, Ho-Min; Lin, Yu-Lin; Lin, Zhong-Zhe; Lai, Mei-Shu; Cheng, Ann-Lii; Yeh, Kun-Huei

    2016-01-01

    Previous studies have shown left-sided colorectal cancer (LCRC) and right-sided colorectal cancer (RCRC) exhibit different molecular and clinicopathological features. We explored the association between the primary tumor site and cetuximab efficacy in KRAS wild-type colorectal cancer (CRC). This study enrolled a cohort of patients, who had received cetuximab treatment after two or more lines of chemotherapy for KRAS wild-type (exon 2 nonmutant) metastatic CRC, from the databases of Taiwan Cancer Registry (2004–2010) and National Health Insurance (2004–2011). Survival data were obtained from the National Death Registry. Time to treatment discontinuation (TTD) and overall survival (OS) after the start of cetuximab treatment were compared between patients with LCRC (splenic flexure to rectum) and RCRC (cecum to hepatic flexure). A total of 969 CRC patients were enrolled. Among them, 765 (78.9 %) and 136 (14.0 %) patients had LCRC and RCRC, respectively. Patients with LCRC, compared to patients with RCRC, had longer TTD (median, 4.59 vs. 2.75 months, P = .0005) and OS (median, 12.62 vs. 8.07 months, P < .0001) after the start of cetuximab treatment. Multivariate analysis revealed a right-sided primary tumor