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Sample records for mutant proteins showed

  1. A fasciclin-like arabinogalactan-protein (FLA mutant of Arabidopsis thaliana, fla1, shows defects in shoot regeneration.

    Directory of Open Access Journals (Sweden)

    Kim L Johnson

    Full Text Available BACKGROUND: The fasciclin-like arabinogalactan-proteins (FLAs are an enigmatic class of 21 members within the larger family of arabinogalactan-proteins (AGPs in Arabidopsis thaliana. Located at the cell surface, in the cell wall/plasma membrane, they are implicated in many developmental roles yet their function remains largely undefined. Fasciclin (FAS domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms, but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants. Recent studies have started to elucidate the role of FLAs in Arabidopsis development. FLAs containing a single FAS domain are important for the integrity and elasticity of the plant cell wall matrix (FLA11 and FLA12 and FLA3 is involved in microspore development. FLA4/SOS5 with two FAS domains and two AGP domains has a role in maintaining proper cell expansion under salt stressed conditions. The role of other FLAs remains to be uncovered. METHOD/PRINCIPAL FINDINGS: Here we describe the characterisation of a T-DNA insertion mutant in the FLA1 gene (At5g55730. Under standard growth conditions fla1-1 mutants have no obvious phenotype. Based on gene expression studies, a putative role for FLA1 in callus induction was investigated and revealed that fla1-1 has a reduced ability to regenerate shoots in an in vitro shoot-induction assay. Analysis of FLA1p:GUS reporter lines show that FLA1 is expressed in several tissues including stomata, trichomes, the vasculature of leaves, the primary root tip and in lateral roots near the junction of the primary root. CONCLUSION: The results of the developmental expression of FLA1 and characterisation of the fla1 mutant support a role for FLA1 in the early events of lateral root development and shoot development in tissue culture, prior to cell-type specification.

  2. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    Full Text Available Yixuan Zeng,1,2,* Wenyuan Guo,1,* Guangqing Xu,3 Qinmei Wang,4 Luyang Feng,1,2 Simei Long,1 Fengyin Liang,1 Yi Huang,1 Xilin Lu,1 Shichang Li,5 Jiebin Zhou,5 Jean-Marc Burgunder,6 Jiyan Pang,5 Zhong Pei1,2 1Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Disease, The First Affiliated Hospital, Sun Yat-sen University, 2Guangzhou Center, Chinese Huntington’s Disease Network, 3Department of Rehabilitation, The First Affiliated Hospital, 4Key laboratory on Assisted Circulation, Ministry of Health, Department of Cardiovascular Medicine of the First Affiliated Hospital, 5School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 6Swiss Huntington’s Disease Center, Department of Neurology, University of Bern, Bern, Switzerland *These authors contributed equally to this work Abstract: Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt. Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a

  3. Induced mutants for cereal grain protein improvement

    International Nuclear Information System (INIS)

    1982-01-01

    Out of 17 papers and one summary presented, six dealing with the genetic improvement of seed protein using ionizing radiations fall within the INIS subject scope. Other topics discussed were non-radiation induced mutants used for cereal grain protein improvement

  4. Estimates of selection parameters in protein mutants of spring barley

    International Nuclear Information System (INIS)

    Gaul, H.; Walther, H.; Seibold, K.H.; Brunner, H.; Mikaelsen, K.

    1976-01-01

    Detailed studies have been made with induced protein mutants regarding a possible genetic advance in selection including the estimation of the genetic variation and heritability coefficients. Estimates were obtained for protein content and protein yield. The variation of mutant lines in different environments was found to be many times as large as the variation of the line means. The detection of improved protein mutants seems therefore possible only in trials with more than one environment. The heritability of protein content and protein yield was estimated in different sets of environments and was found to be low. However, higher values were found with an increasing number of environments. At least four environments seem to be necessary to obtain reliable heritability estimates. The geneticall component of the variation between lines was significant for protein content in all environmental combinations. For protein yield some environmental combinations only showed significant differences. The expected genetic advance with one selection step was small for both protein traits. Genetically significant differences between protein micromutants give, however, a first indication that selection among protein mutants with small differences seems also possible. (author)

  5. High-Protein Soybean Mutants by Using Irradiation Technique

    International Nuclear Information System (INIS)

    Yathaputanon, C.; Kumsueb, B.; Srisombun, S.

    2009-07-01

    Full text: Soybean variety improvement for high seed protein using induced mutation was initiated. Approximately 5,000 seeds of soybean variety Chiang Mai 60 were irradiated with gamma rays at the dose of 200 Grays at Kasetsart University. High-protein seed mutants in M2 to M4 generations were selected at Nakhon Ratchasima Field Crops Research Center during 2004-2008. The Pedigree method of selection was used. Kjeldahl method was used to analyze seed protein percentages. The M2 seeds protein content of the M2 generation was 45.2% while that of the original parent was 43.0%. M3s were seeded plant to row. In each row, the best four plants were selected for protein analysis. The average protein content of selected mutant lines was 3.9% while the check variety had average protein content of 42.4%. In the M4 generation, the result showed that the average protein contents of the selected mutant lines and the check variety were 42.8% and 42.0%, respectively. In the 2007-2008 trials, four promising mutants had and average protein content of 428%, while the check variety had and average protein content of 41.1%. The four mutants produced the mean grain yield of 2.20-2.42 t/Ha, which was 10.21% higher than that of Chiang Mai 60. The mutant lines produced both a high grain protein content and a high grain yield. They will be further tested their adaptability in the research centers and farmer fields

  6. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    Science.gov (United States)

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  7. Relations between protein production, protein quality and environmental factors in Pisum mutants

    International Nuclear Information System (INIS)

    Gottschalk, W.; Mueller, H.P.; Wolff, G.

    1975-01-01

    The seed protein content of 138 radiation-induced Pisum mutants was determined. The variability of this genetically well-defined material agrees approximately with that of the world collection of Pisum sativum. Some environmental factors to a great extent influence the protein production of the mutants and the initial line. Therefore, it is necessary to consider the relations between the genetically controlled protein production and its dependence upon the environmental factors. This is especially evident if the protein situation of the same genotypes cultivated under the moderate climatic conditions of middle Europe is compared with the subtropical conditions of India. A generally firm correlation between seed size and protein content could not be found in material regarding 148 different mutants of our assortment. Therefore, the selection of small-grained mutants does not result in a selection of protein-rich genotypes in Pisum sativum. Considering all the criteria positively and negatively influencing the protein production, a positive situation could be found in some mutants, especially in the fasciated ones. Furthermore, an improvement of the protein quality could be reached by a genetically conditioned alteration of the globulin-albumin ratio leading to an increase of some essential amino acids such as methionine and lysine. The combined action of mutant genes results in unexpected changes of the protein quantity as well as the quality of the recombinants in relation to their parental mutants. The comparison of some essential amino acids of our useful mutants with those of the varieties of other genera of the Leguminosae shows certain trends of biochemical alterations realized during evolutionary development of the family. (author)

  8. 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)

  9. Seed protein and nitrogen fixation in chickpea mutant variety Hyprosola

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, H E; Gibson, A H; Oram, R N [CSIRO, Division of Plant Industry, Canberra ACT (Australia); Shaikh, M A.Q. [Bangladesh Institute of Nuclear Agriculture, Mymensingh (Bangladesh)

    1989-01-01

    Full text: 'Hyprosola' is a high yielding, high protein mutant cultivar obtained after gamma irradiation from the variety 'Faridpur-1'. The mutant yields 45 % more protein per unit area. The essential amino acid index is unchanged. It is likely that the high nutritional value in 'Hyprosola' seed protein arises from an increase in the albumin:globulin ratio. Nitrogen fixation rates of the mutant during the first 7 weeks of growth were found to be similar to 'Faridpur-1'. Under field conditions, the mutant may be able to nodulate more rapidly and more extensively than the parent variety. (author)

  10. Use of wheat and maize protein mutants in breeding for improved protein quantity and quality

    International Nuclear Information System (INIS)

    Denic, M.; Dumanovic, J.; Misevic, D.; Konstantinov, K.; Fidler, D.; Stojanovic, Z.

    1984-01-01

    Selected offspring progenies (50 mutant lines) originating from mutation experiments with hexaploid wheat (cv. Bezostaya 1) were analysed for induced heritable variation in protein content, lysine content, grain yield and protein and lysine yields. Ten of these mutant lines were crossed with 11 local varieties. The protein and lysine contents were measured in the progenies of these crossings. The data showed better correlations of grain yield with protein and lysine yields than the protein and lysine contents with their corresponding yields. F 1 seeds showed higher lysine and protein contents than local varieties. Data with maize showed that: (1) the total endosperm protein content of modified opaque-2 types increases with an increase in the degree of normalization; (2) the lysine content in dry matter and protein in normalized o 2 kernels usually decreases with the increasing degree of normalization; (3) the lysine content in protein of modified o 2 kernels, is, in general, satisfactory up to the normalization of about 50% of endosperm. A desirable modification of o 2 endosperm within line A632o 2 was selected and crossed with o 2 lines. Most of the tested hybrids had a good protein quality, but endosperm modification was not evident in all hybrids. The o 2 gene was incorporated into high protein backgrounds. Besides a high protein content and quality, some of the hybrids tested had a comparable or higher yield than the o 2 check. (author)

  11. X-ray survival characteristics and genetic analysis for nine saccharomyces deletion mutants that show altered radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Game, John C.; Williamson, Marsha S.; Baccari, Clelia

    2004-01-07

    The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X-rays, we are screening these mutants to identify additional genes that show increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype co-segregates with the deletion allele and are obtaining multipoint survival-versus-dose assays in at least two haploid and one homozygous diploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1 and VID21/EAF1, and discuss their potential roles in repair. Eight of these genes have a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, has at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultra-violet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino-acids are also X-ray sensitive, seeming to confirm that methylation of the lysine-79 residue is required for effective repair of radiation damage.

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

    DEFF Research Database (Denmark)

    Hong, Kuk-Ki; Nielsen, Jens

    2013-01-01

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

  13. Nutrient value protein of mutant J3 of agaricus blazei by irradiation

    International Nuclear Information System (INIS)

    Jiang Zhihe; Lin Yong; Xiao Shuxia

    2003-01-01

    The studies of nutrient value protein of mutant J 3 of Agaricus blazei were carried out. The results showed that mutant J 3 had 9 kinds of essential amino acids, the compositions of essential amino acids is 46.0% of total amino acids. The amino acid score is 94.5, the essential amino acid index is 92.4, biological value is 89.0, the ratio coefficient of amino acids is 72.3, the nutrient value is 32.5. The chemical score is 74.18. These results proved that the protein of mutant J 3 had high nutrient value

  14. ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

    Directory of Open Access Journals (Sweden)

    Kavita Rajesh Pandey

    2016-09-01

    Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

  15. Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

    International Nuclear Information System (INIS)

    Bedian, V.; Summers, M.C.; Kauffman, S.A.

    1988-01-01

    We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

  16. Oncogenic Signaling by Leukemia-Associated Mutant Cbl Proteins

    Science.gov (United States)

    Nadeau, Scott; An, Wei; Palermo, Nick; Feng, Dan; Ahmad, Gulzar; Dong, Lin; Borgstahl, Gloria E. O.; Natarajan, Amarnath; Naramura, Mayumi; Band, Vimla; Band, Hamid

    2013-01-01

    Members of the Cbl protein family (Cbl, Cbl-b, and Cbl-c) are E3 ubiquitin ligases that have emerged as critical negative regulators of protein tyrosine kinase (PTK) signaling. This function reflects their ability to directly interact with activated PTKs and to target them as well as their associated signaling components for ubiquitination. Given the critical roles of PTK signaling in driving oncogenesis, recent studies in animal models and genetic analyses in human cancer have firmly established that Cbl proteins function as tumor suppressors. Missense mutations or small in-frame deletions within the regions of Cbl protein that are essential for its E3 activity have been identified in nearly 5% of leukemia patients with myelodysplastic/myeloproliferative disorders. Based on evidence from cell culture studies, in vivo models and clinical data, we discuss the potential signaling mechanisms of mutant Cbl-driven oncogenesis. Mechanistic insights into oncogenic Cbl mutants and associated animal models are likely to enhance our understanding of normal hematopoietic stem cell homeostasis and provide avenues for targeted therapy of mutant Cbl-driven cancers. PMID:23997989

  17. An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

    Science.gov (United States)

    Perez-Arnaiz, Patricia; Kaplan, Daniel L

    2016-11-20

    Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The characteristics and stability of a range of Cox's Orange Pippin apple mutants showing different growth habits

    International Nuclear Information System (INIS)

    Lacey, C.N.D.; Campbell, A.I.

    1979-01-01

    Seven hundred and fifty gamma-irradiated scions of Cox's Orange Pippin apple were grown to produce a V 1 generation and were then multiplied to produce a V 2 of 13158 individual trees. 272 obvious vegetative mutants, mainly dwarf or compact types, were found in this population and classified according to growth habit. These were propagated to produce clones of the mutant types (V 3 ) and a study of these clonal mutants as compared with their original (V 2 ) characteristics showed that while the vast majority of the selected mutants produced dwarf or compact clones, no clear indication of final cropping performance could be drawn from the original phenotype of the selected V 2 mutants. The majority of mutants produced were not of commercial value, and the main reasons for the rejection of V 3 clones depended, in many cases, on the phenotype of the V 2 selection. Thus while all types of V 2 mutant produced approximately the same proportion of acceptable trees, the reason for the rejection of the V 3 clones varies with the V 2 phenotype. Therefore selection can be carried out at an early stage in a mutation breeding programme to reduce the proportion of certain unwanted types such as mericlinal chimaeras as that otherwise are carried forward to yield trials. From the orchard trials of 82 mutant clones. 24 were short-listed for possible commercial introduction. All were derived from the less extreme mutant types. (Auth.)

  19. A herbicide-resistant ACCase 1781 Setaria mutant shows higher fitness than wild type.

    Science.gov (United States)

    Wang, T; Picard, J C; Tian, X; Darmency, H

    2010-10-01

    It is often alleged that mutations conferring herbicide resistance have a negative impact on plant fitness. A mutant ACCase1781 allele endowing resistance to the sethoxydim herbicide was introgressed from a resistant green foxtail (Setaria viridis (L.) Beauv) population into foxtail millet (S. italica (L.) Beauv.). (1) Better and earlier growth of resistant plants was observed in a greenhouse cabinet. (2) Resistant plants of the advanced BC7 backcross generation showed more vigorous juvenile growth in the field, earlier flowering, more tillers and higher numbers of grains than susceptible plants did, especially when both genotypes were grown in mixture, but their seeds were lighter than susceptible seeds. (3) Field populations originating from segregating hybrids had the expected allele frequencies under normal growth conditions, but showed a genotype shift toward an excess of homozygous resistant plants within 3 years in stressful conditions. Lower seed size, lower germination rate and perhaps unexplored differences in seed longevity and predation could explain how the resistant plants have the same field fitness over the whole life cycle as the susceptible ones although they produce more seeds. More rapid growth kinetics probably accounted for higher fitness of the resistant plants in adverse conditions. The likelihood of a linkage with a beneficial gene is discussed versus the hypothesis of a pleiotropic effect of the ACCase resistance allele. It is suggested that autogamous species like Setaria could not develop a resistant population without the help of a linkage with a gene producing a higher fitness.

  20. Spirogyra varians mutant generated by high dose gamma-irradiation shows increased antioxidant properties

    International Nuclear Information System (INIS)

    Lee, Hak-Jyung; Yoon, Minchul; Sung, Nak-Yun; Choi, Jong-il

    2012-01-01

    The aim of this study was to evaluate the antioxidant properties of a Spirogyra varians mutant (Mut) produced by gamma irradiation. Methanol extracts were prepared from Spirogyra varians wild-type and Mut plants, and their antioxidant activities and total phenolic content (TPC) were determined. Antioxidant parameters, including the 2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferric-reducing/antioxidant power, were higher in the Mut extract. Moreover, the TPC level was higher (P<0.05) in the Mut methanol extract. Therefore, these results suggest that gamma irradiation-induced S. varians Mut has superior antioxidant properties. - Highlights: ► The antioxidative properties of a Spirogyra varians mutant produced by gamma-irradiation was investiated. ► The antioxidant activities and total phenolic content levels were higher in mutant strain. ► These results suggest that gamma-irradiation induced algae mutant with superior antioxidant properties.

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

    OpenAIRE

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

    1991-01-01

    Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The...

  2. Adaptive response in Drosophila melanogaster heat shock proteins mutant strains

    International Nuclear Information System (INIS)

    Shaposhnikov, M.V.; Moskalev, A.A.; Turysheva, E.V.

    2007-01-01

    Complete text of publication follows. The members of the heat shock proteins (Hsp) family function as molecular chaperones and assist intracellular folding of newly synthesized proteins. Also it is possible that molecular chaperones are induced during adaptive response to oxidative stress and radiation. The aim of our research was to exam the role of heat shock proteins in adaptive response to oxidative stress after low dose rate gamma-irradiation in Drosophila melanogaster. Drosophilamelanogaster strains were kindly provided by Bloomington Drosophila Stock Center (University of state of Indiana, Bloomington, USA). We used wild type strain (CS), heat shock protein mutant strains (Hsp22, Hsp70, Hsp83), and heat shock factor mutant strain (Hsf). Strains were chronically exposured to adaptive dose of gamma-irradiation in dose rate of 0.17 cGy/h during all stages of life history (from the embrional stage to the stage of matured imago). The rate of absorbed dose was 60 cGy. For oxidative-stress challenge twodays old flies were starved in empty vials for 6 h and then transferred to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival data were collected after 26 h of treatment. Dead flies were counted daily. The obtained data were subjected to survival analysis by Kaplan and Meier method and presented as survival curves. Statistical analysis was held by non-parametric methods. To test the significance of the difference between the two age distributions Kolmogorov-Smirnov test was applied. Gehan-Braslow- Wilcoxon and Cox-Mantel tests were used for estimation of median life span differences. In addition the minimal and maximal life span, time of 90% death, and mortality rate doubling time (MRDT) were estimated. The obtained results will be discussed in presentation.

  3. Protein nutrient value of Agaricus bazei murrill mutant J3 induced by 60Co γ-irradiation in different generations

    International Nuclear Information System (INIS)

    Jiang Zhihe; Lin Yong; Xiao Shuxia

    2004-01-01

    Protein nutritional values of Agaricus bazei Murrill mutant J 3 and original strain J 1 were compared by non-biological evaluation methods. The results showed that five protein indexes in the fruitbodies of M 1 and M 6 generations of Agaricus bazei Murrill mutant J 3 were higher than that of original strain J 1 ; four protein indexes in M 4 and M 5 generations were higher than that of original strain J 1 ; and six protein indexes in M 2 and M 3 generations were higher than that of original strain J 1 . It was concluded that the protein nutritional values of Agaricus bazei Murrill mutant J 3 was better than that of original strain J 1 . Except the ratio scores of amino acids had little change in the different generations, the other indexes in strain J 3 Agaricus bazei Murrill showed that the heredity efficiencies of the proteins was rather stable. (authors)

  4. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

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

    Science.gov (United States)

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

    1991-01-01

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

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

    Science.gov (United States)

    Loging, W T; Reisman, D

    1999-11-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Induction of mutants with higher protein content in soy bean

    International Nuclear Information System (INIS)

    Hiraiwa, S.; Tanaka, S.; Nakamura, S.

    1976-01-01

    Two experiments were carried out to examine the effects of ionizing radiation on protein content of soy beans and to estimate the potential value of ionizing radiation in soy bean breeding. In the first experiment seeds of three cultivars were acutely irradiated by gamma ray doses of 8 krad and 16 krad. Seeds of M 3 plants were analysed for protein content by the Biuret method. Selection within each variety was made to isolate plants having about 2% higher protein content than the control populations. In the second experiment growing plants of a soy bean cultivar were irradiated with 2.9 krad and 6.1 krad, from germination to maturation, in a 60 Co gamma field. Seeds on individual lines in M 5 , M 6 and M 7 plants were determined for their protein contents by the Kjeldahl method and the upper 10% of the treated lines were selected for progeny testing. From the results of the analysis of variance for each trait, significant differences were observed between the control variety and selected lines for all the traits. Six out of 19 lines showed significant increases in both 100 seed weight and protein content, and four lines promising. The mean protein content of selected lines increased 0.68% compared with the control variety. Genetic gain expected from selection for high protein content was therefore considered at about 1%; however, the likelihood of a large increase in protein content would greatly increase if large populations derived from irradiated seeds were observed

  9. SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

    Directory of Open Access Journals (Sweden)

    Jacqueline Gire O’Rourke

    2013-07-01

    Full Text Available A key feature in Huntington disease (HD is the accumulation of mutant Huntingtin (HTT protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  10. Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

    Science.gov (United States)

    Molla, Muriel; Descroix, Vianney; Aïoub, Muhanad; Simon, Stéphane; Castañeda, Beatriz; Hotton, Dominique; Bolaños, Alba; Simon, Yohann; Lezot, Frédéric; Goubin, Gérard; Berdal, Ariane

    2010-11-01

    Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

  11. Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

    International Nuclear Information System (INIS)

    Hameed, A.; Iqbal, N.; Shah, T.M.

    2012-01-01

    A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

  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. A Sordaria macrospora mutant lacking the leu1 gene shows a developmental arrest during fruiting body formation.

    Science.gov (United States)

    Kück, Ulrich

    2005-10-01

    Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis ("cross-pathway control") have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body.

  14. Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

    Science.gov (United States)

    Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

    2015-07-01

    Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1 Mutant Zebrafish.

    Directory of Open Access Journals (Sweden)

    Brian P Grone

    Full Text Available Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1, are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

  16. High protein mutants of winter fodder barley induced by radiation and chemical mutagens

    Energy Technology Data Exchange (ETDEWEB)

    Yankulov, M.; Genchev, K.; Nikolov, Kh.

    1982-01-01

    Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley.

  17. High protein mutants of winter fodder barley induced by radiation and chemical mutagens

    International Nuclear Information System (INIS)

    Yankulov, M.; Genchev, K.; Nikolov, Kh.

    1982-01-01

    Several induced mutants of winter fodder barley with higher rpotein content are described. These mutants were produced by treating seeds of cvs. Vogelsaenger Gold, Ager and 468 with gamma-rays, sodium azide and ethyl methanesulfonate (alone and in combinations) and with ethylene and formamide. The gamma-ray induced mutants of winter fodder barley have 1-4% higher protein content. The mutant line 109 has, besides high protein content (17,37%), 5.96 lysine per 100 g protein, but its endosperm is wrinkeled. Mutants produced by chemical mutagens have 6-7% higher protein content than the initial cultivars. All induced mutants have 85-95 cm high stems, i.e. they are by 10-20 cm shorter than the initial cultivars. Some of these mutants are now resistant to the diseases Helminthosporium gramineum and Ustilago nuda. The recommended mutants could be successfully used in breeding programs for producing of higher protein content and quality in winter fodder barley

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

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

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

  19. Arabidopsis AtDjA3 null mutant shows increased sensitivity to abscisic acid, salt, and osmotic stress in germination and postgermination stages

    Directory of Open Access Journals (Sweden)

    Silvia eSalas-Muñoz

    2016-02-01

    Full Text Available DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid. The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher abscisic acid sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signalling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 μM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance.

  20. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  1. Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Barbieri, Christopher E.; He, Jintang; Gao, Yuqian; Shi, Tujin; Wu, Chaochao; Schepmoes, Athena A.; Fillmore, Thomas L.; Chae, Sung-Suk; Huang, Dennis; Mosquera, Juan Miguel; Qian, Wei-Jun; Smith, Richard D.; Srivastava, Sudhir; Kagan, Jacob; Camp, David G.; Rodland, Karin D.; Rubin, Mark A.; Liu, Tao

    2017-08-15

    Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification range from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present it is unknown if this also affects the activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.

  2. Evaluation of the protein quality of cereal mutants

    International Nuclear Information System (INIS)

    Eggum, B.O.

    1984-01-01

    Protein content, true protein digestibility, biological value, net protein utilization, and utilizable protein in several varieties of barley, wheat and rice were determined in nitrogen-balance trials with rats. It appeared that protein quality varied significantly between these three cereal grains, with the lowest values for wheat. However, the protein content was markedly higher in wheat; consequently, utilizable protein was highest in this cereal grain. The different varieties within barley, wheat and rice varied considerably in protein quality. This demonstrates a large variation in the potential for protein synthesis. The main problem with rice and barley is the low protein concentration, whereas with wheat the biggest problem seems to be the quality of the protein. As the lysine level in all cereal grains, expressed in percentage of the protein, cannot meet the requirements for either man or domestic animals efforts should be made to increase the lysine concentration in these food sources. (author)

  3. Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

    Science.gov (United States)

    Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Calculation of the redox potential of the protein azurin and some mutants

    NARCIS (Netherlands)

    van den Bosch, M; Swart, M; Snijders, JG; Berendsen, HJC; Mark, AE; Oostenbrink, C; van Gunsteren, WF; Canters, GW

    Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  6. Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

    OpenAIRE

    Davoodi, J.; Wakarchuk, W. W.; Surewicz, W. K.; Carey, P. R.

    1998-01-01

    The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second...

  7. Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

    Science.gov (United States)

    Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

    2016-08-01

    Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

  8. Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

    Directory of Open Access Journals (Sweden)

    Julie Jodoin

    2009-08-01

    Full Text Available Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD-associated prion protein (PrP mutants that are unable to generate cytosolic PrP (CyPrP. To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS-associated PrP mutants. These PrP mutants contained their respective methionine ((M or valine ((V at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V, D202N(V, P102L(V and Q217R(V retained, whereas the P102L(M, P105L(V, Y145stop(M and Q212P(M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

  9. Pyridones as NNRTIs against HIV-1 mutants: 3D-QSAR and protein informatics

    Science.gov (United States)

    Debnath, Utsab; Verma, Saroj; Jain, Surabhi; Katti, Setu B.; Prabhakar, Yenamandra S.

    2013-07-01

    CoMFA and CoMSIA based 3D-QSAR of HIV-1 RT wild and mutant (K103, Y181C, and Y188L) inhibitory activities of 4-benzyl/benzoyl pyridin-2-ones followed by protein informatics of corresponding non-nucleoside inhibitors' binding pockets from pdbs 2BAN, 3MED, 1JKH, and 2YNF were analysed to discover consensus features of the compounds for broad-spectrum activity. The CoMFA/CoMSIA models indicated that compounds with groups which lend steric-cum-electropositive fields in the vicinity of C5, hydrophobic field in the vicinity of C3 of pyridone region and steric field in aryl region produce broad-spectrum anti-HIV-1 RT activity. Also, a linker rendering electronegative field between pyridone and aryl moieties is common requirement for the activities. The protein informatics showed considerable alteration in residues 181 and 188 characteristics on mutation. Also, mutants' isoelectric points shifted in acidic direction. The study offered fresh avenues for broad-spectrum anti-HIV-1 agents through designing new molecules seeded with groups satisfying common molecular fields and concerns of mutating residues.

  10. Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

    Science.gov (United States)

    Ameziane-Le Hir, Sarah; Paboeuf, Gilles; Tascon, Christophe; Hubert, Jean-François; Le Rumeur, Elisabeth; Vié, Véronique; Raguénès-Nicol, Céline

    2016-07-26

    Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe.

  11. Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses.

    Science.gov (United States)

    Tsolmonbaatar, Ariunzaya; Hashida, Keisuke; Sugimoto, Yukiko; Watanabe, Daisuke; Furukawa, Shuhei; Takagi, Hiroshi

    2016-12-05

    During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1 P247S and PRO1 E415K gene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Directory of Open Access Journals (Sweden)

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  14. Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

    Science.gov (United States)

    de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

    2015-04-01

    Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.

    Science.gov (United States)

    Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun

    2014-07-23

    The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

    Science.gov (United States)

    Foulquier, Elodie; Pompeo, Frédérique; Freton, Céline; Cordier, Baptiste; Grangeasse, Christophe; Galinier, Anne

    2014-08-22

    The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

    Energy Technology Data Exchange (ETDEWEB)

    Mehlo, Luke, E-mail: LMehlo@csir.co.za [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa); Mbambo, Zodwa [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa); Microbiology Discipline, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000 (South Africa); Bado, Souleymane [Plant Breeding and Genetics Laboratory – Joint FAO/IAEA Agriculture and Biotechnology Laboratory, International Atomic Energy Agency Laboratories, A-2444 Seibersdorf (Austria); Lin, Johnson [Microbiology Discipline, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000 (South Africa); Moagi, Sydwell M.; Buthelezi, Sindisiwe; Stoychev, Stoyan; Chikwamba, Rachel [CSIR Biosciences, Meiring Naude Road, P.O. Box 395, Pretoria 0001 (South Africa)

    2013-09-15

    Highlights: • We analyse kafirin protein polymorphisms induced by gamma irradiation in sorghum. • One mutant with suppressed kafirins in the endosperm accumulated them in the germ. • Kafirin polymorphisms were associated with high levels of free amino acids. • Nutritional value of sorghum can be improved significantly by induced mutations. - Abstract: Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27 kDa γ-, 24 kDa α-A1 and the 22 kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals.

  18. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

    International Nuclear Information System (INIS)

    Mehlo, Luke; Mbambo, Zodwa; Bado, Souleymane; Lin, Johnson; Moagi, Sydwell M.; Buthelezi, Sindisiwe; Stoychev, Stoyan; Chikwamba, Rachel

    2013-01-01

    Highlights: • We analyse kafirin protein polymorphisms induced by gamma irradiation in sorghum. • One mutant with suppressed kafirins in the endosperm accumulated them in the germ. • Kafirin polymorphisms were associated with high levels of free amino acids. • Nutritional value of sorghum can be improved significantly by induced mutations. - Abstract: Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27 kDa γ-, 24 kDa α-A1 and the 22 kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals

  19. Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

    Directory of Open Access Journals (Sweden)

    Saara Laulumaa

    Full Text Available Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS. The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

  20. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    Energy Technology Data Exchange (ETDEWEB)

    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

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

    Directory of Open Access Journals (Sweden)

    Tali Gidalevitz

    2009-03-01

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

  2. Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

    Science.gov (United States)

    Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

    2014-01-01

    The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

  3. Induction of high yielding and high protein containing chickpea mutant variety through gamma radiation

    International Nuclear Information System (INIS)

    Hassan, S.; Javed, M.A.; Khan, A.J.; Tariq, M.

    1997-01-01

    Pure seeds of a blight susceptible but high yielding chickpea variety 6153 were irradiated at 20 Kr(0.2 kGy) dose of gamma radiation and the mutant line CMN-446-4 was selected in M3 generation on the basis of high yield and disease resistance. After confirmation of its resistance to blight in M4 and M5, the mutant line CMN-446-4 along with other promising chickpea mutants were evaluated in various yield trials at different locations. The mutant line CMN-446-4 was got evaluated in chickpea national uniform yield trial conducted over two locations in the country during 1993-94. The mutant line, on average, ranked 3rd by producing significantly higher yield of 1528 kg/ha as compared to the two checked varieties Punjab-91 and Paidar-91 which yielded 1316 and 1391 kg/ha respectively. The mutant CMN-446-4 has significantly greater percentage of protein content (25.22%) compared to its parental variety having (20.12%). (author)

  4. Comparative analysis of the protein compositions between wild type and body color mutant of helicoverpa armigera adult

    International Nuclear Information System (INIS)

    He Lihua; Chen Jin'e; Liu Yan; Wang Yongqiang; Liu Peigang; Meng Zhiqi

    2012-01-01

    To gain an in-depth understanding of the fineness and regulation mechanism of body color mutant of Helicoverpa armigera Hbner, the protein composition differences between adult of dominant mutant, recessive mutant and wild type were studied using the SDS-PAGE combined with MALDI-TOF-TOF/MS and bioinformatics analysis. The results indicated that the protein composition of the dominant mutant and wild type had little difference. However, there were obvious differences between the recessive mutant and wild-type. Three specific stripe were chosen for mass spectrometry and bioinformatics analysis, and two types of proteins related to energy metabolism and cytoskeleton were identified. These findings suggested that the two types of proteins may be associated with occurrence and regulation of body color mutant traits of H. armigera. (authors)

  5. Patients with polymyositis show changes in muscle protein charges

    DEFF Research Database (Denmark)

    Bartels, E M; Jacobsen, Søren; Rasmussen, L

    1989-01-01

    Polymyositis (PM) appears with indolent proximal muscle weakness and is an inflammatory disease with breakdown of muscle cells. In our study the protein charge concentrations of the contractile proteins in the A and I bands were determined, applying a microelectrode technique. Patients with PM sh...

  6. [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.

  7. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    International Nuclear Information System (INIS)

    Mathew, Shomita S.; Bridge, Eileen

    2007-01-01

    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci

  8. Conditioned taste aversion memory and c-Fos induction are disrupted in RIIbeta-protein kinase A mutant mice.

    Science.gov (United States)

    Koh, Ming Teng; Clarke, Sharon N D A; Spray, Kristina J; Thiele, Todd E; Bernstein, Ilene L

    2003-07-14

    The cAMP-dependent protein kinase (PKA) signaling pathway has been implicated in many forms of learning. The present studies examined conditioned taste aversion (CTA) learning, an amygdala-dependent task, in mice with a targeted disruption of a gene for a specific regulatory subunit of PKA (RIIbeta), which is selectively expressed in amygdala. Null mutant (RIIbeta(-/-)) mice and littermate controls (RIIbeta(+/+)) were tested for protein synthesis-independent short-term memory (STM) and protein synthesis-dependent long-term memory (LTM) for CTAs. The ability of the unconditioned stimulus (US) drug, LiCl, to induce c-Fos in regions thought to be important in this learning was also determined. RIIbeta(-/-) mice showed significant impairment in CTA memory when tested 24h after training (LTM). In contrast, STM was normal. With regard to the c-Fos response to LiCl, the US drug, significant elevations were evident in brainstem (nucleus of the solitary tract) and pontine (parabrachial nucleus) regions, in mutants as well as wild-type controls. However, in amygdala, elevations were seen in controls but were absent in the mutants. These findings suggest that disruption of PKA signaling interferes with LTM consolidation of CTA and that a possible mediator of this effect is interference with c-Fos expression in amygdala which may be necessary for CTA memory.

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

    Directory of Open Access Journals (Sweden)

    Karen L Posey

    2010-04-01

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

  10. Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

    Science.gov (United States)

    Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

    2014-07-01

    Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

  11. Restriction of phage T4 internal protein I mutants by a strain of Escherichia coli

    International Nuclear Information System (INIS)

    Black, L.W.; Abremski, K.

    1974-01-01

    Phage T4 internal protein I(IPI), a small (ca, 10,000 MW), basic protein injected into the host with the phage DNA, is not required for infection of most hosts, but mutants defective in IPI are restricted by at least one naturally occurring strain of Escherichia coli, CT 596 (CT). Phages lacking IPI (IPI - ) appear to inject their DNA and bind it to the membrane of CT cells as well as wild-type phage T4 does, but shutoff of host protein synthesis, initiation of T4 protein synthesis, and cell killing are abnormal in the IPI - mutant infected CT host. The injection of IPI appears to be important in allowing T4 DNA to carry out early steps involved in takeover of this host. Restriction of IPI - phage growth by CT cells appears to be due, at least in part, to a defective prophage it harbors which renders the host resistant to successful infection by phage T4 which lack IPI or rII functions. Bacteria cured of this prophage can be infected by mutants defective in these functions. The resistance of CT cells to other coliphages, and the question of T-even phage internal protein diversity are discussed. (U.S.)

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

    Science.gov (United States)

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

    2005-03-01

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

  13. Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

    Science.gov (United States)

    Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

    2016-04-01

    Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. © 2016 American Society of Plant Biologists. All rights reserved.

  14. Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

    Science.gov (United States)

    Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

    2016-01-01

    Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

  15. Droplet networks with incorporated protein diodes show collective properties

    Science.gov (United States)

    Maglia, Giovanni; Heron, Andrew J.; Hwang, William L.; Holden, Matthew A.; Mikhailova, Ellina; Li, Qiuhong; Cheley, Stephen; Bayley, Hagan

    2009-07-01

    Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.

  16. Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm "Ming" lethal egg mutant.

    Science.gov (United States)

    Chen, Anli; Gao, Peng; Zhao, Qiaoling; Tang, Shunming; Shen, Xingjia; Zhang, Guozheng; Qiu, Zhiyong; Xia, Dingguo; Huang, Yongping; Xu, Yunmin; He, Ningjia

    2013-02-25

    The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    DEFF Research Database (Denmark)

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived...... from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains......, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3...

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

    Science.gov (United States)

    Einav, Tal; Duque, Julia; Phillips, Rob

    2018-02-01

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

  19. Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.

    Directory of Open Access Journals (Sweden)

    Adrian W R Serohijos

    2008-02-01

    Full Text Available The absence of a functional ATP Binding Cassette (ABC protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR from apical membranes of epithelial cells is responsible for cystic fibrosis (CF. Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1. Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of

  20. Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

    Directory of Open Access Journals (Sweden)

    Pilar Eraso

    Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

  1. Characterization of PUD-1 and PUD-2, two proteins up-regulated in a long-lived daf-2 mutant.

    Science.gov (United States)

    Ding, Yue-He; Du, Yun-Guang; Luo, Shukun; Li, Yu-Xin; Li, Tie-Mei; Yoshina, Sawako; Wang, Xing; Klage, Karsten; Mitani, Shohei; Ye, Keqiong; Dong, Meng-Qiu

    2013-01-01

    C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar β-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid β-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against Aβ.

  2. Human T-Cell Leukemia Virus I Tax Protein Sensitizes p53-Mutant Cells to DNA Damage

    Science.gov (United States)

    Mihaylova, Valia T.; Green, Allison M.; Khurgel, Moshe; Semmes, Oliver J.; Kupfer, Gary M.

    2018-01-01

    Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53–containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective. PMID:18559532

  3. The 7B-1 mutant in tomato shows blue-light-specific resistance to osmotic stress and abscisic acid.

    Science.gov (United States)

    Fellner, Martin; Sawhney, Vipen K

    2002-03-01

    Germination of wild-type (WT) tomato ( Lycopersicon esculentum Mill.) seed is inhibited by mannitol (100-140 mM) in light, but not in darkness, suggesting that light amplifies the responsiveness of the seed to osmotic stress (M. Fellner, V.K. Sawhney (2001) Theor Appl Genet 102:215-221). Here we report that white light (W) and especially blue light (B) strongly enhance the mannitol-induced inhibition of seed germination, and that the effect of red light (R) is weak or nil. The inhibitory effect of mannitol could be completely overcome by fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, indicating that mannitol inhibits seed germination via ABA accumulation in seeds. The inhibition of WT seed germination by exogenous ABA was also amplified by W or B, but not by R. In a recessive, ABA-overproducing, 7B-1 mutant of tomato, seed germination and hypocotyl growth were resistant to inhibition by mannitol or exogenous ABA, both in W or B. Experiments with fluridone suggested that inhibition of hypocotyl growth by W or B is also partially via ABA accumulation. De-etiolation in the mutant was especially less in B compared to the WT, and there was no difference in hypocotyl growth between the two genotypes in R. Our data suggest that B amplifies the responsiveness of tomato seeds and hypocotyls to mannitol and ABA, and that W- or B-specific resistance of the 7B-1 mutant to osmotic stress or ABA is a consequence of a defect in B perception or signal transduction.

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

    Directory of Open Access Journals (Sweden)

    Daniele Vergara

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  6. A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

    Science.gov (United States)

    Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

    2016-07-01

    Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ritika Kar

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

  10. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-01-01

    Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

  11. Induced variation in protein mutants after multiple EMS and X-ray treatments

    International Nuclear Information System (INIS)

    Walther, H.; Seibold, K.H.

    1978-01-01

    Results from two experiments with spring barley in the M 3 and M 8 generations gave information on the efficiency of selected mutants, improved in protein yield by different mutagenic treatments. A selection rate of 1-2% was found to be realistic according to a 5% significance level. However, differences between mutagenic treatments with EMS and X-rays and between varieties were found to be not only incidental. To evaluate the results within a protein mutation breeding programme a bivariate selection model was applied and was found to allow clear decisions on mutagenic improvements in protein production, measured in g protein/m 2 . When substituting protein yield in g/seed for protein yield in g/m 2 in the early generations, all relations to protein and grain yield in g/m 2 were found to be low and negative. We conclude that this substituted selection character can be of only limited aid. But very high positive correlations exist between protein yield/m 2 , lysine yield/m 2 and grain yield/m 2 , which means that these selection characters would render a more reliable basis for selection in early generations. (author)

  12. Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies.

    Science.gov (United States)

    Noguchi, Hiroki; Mylemans, Bram; De Zitter, Elke; Van Meervelt, Luc; Tame, Jeremy R H; Voet, Arnout

    2018-03-18

    β-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel β-sheets arranged around a central axis. Recently we designed the first completely symmetrical β-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of β-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural β-propeller proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Wheat TILLING mutants show that the vernalization gene VRN1 down-regulates the flowering repressor VRN2 in leaves but is not essential for flowering.

    Directory of Open Access Journals (Sweden)

    Andrew Chen

    Full Text Available Most of the natural variation in wheat vernalization response is determined by allelic differences in the MADS-box transcription factor VERNALIZATION1 (VRN1. Extended exposures to low temperatures during the winter (vernalization induce VRN1 expression and promote the transition of the apical meristem to the reproductive phase. In contrast to its Arabidopsis homolog (APETALA1, which is mainly expressed in the apical meristem, VRN1 is also expressed at high levels in the leaves, but its function in this tissue is not well understood. Using tetraploid wheat lines with truncation mutations in the two homoeologous copies of VRN1 (henceforth vrn1-null mutants, we demonstrate that a central role of VRN1 in the leaves is to maintain low transcript levels of the VRN2 flowering repressor after vernalization. Transcript levels of VRN2 were gradually down-regulated during vernalization in both mutant and wild-type genotypes, but were up-regulated after vernalization only in the vrn1-null mutants. The up-regulation of VRN2 delayed flowering by repressing the transcription of FT, a flowering-integrator gene that encodes a mobile protein that is transported from the leaves to the apical meristem to induce flowering. The role of VRN2 in the delayed flowering of the vrn1-null mutant was confirmed using double vrn1-vrn2-null mutants, which flowered two months earlier than the vrn1-null mutants. Both mutants produced normal flowers and seeds demonstrating that VRN1 is not essential for wheat flowering, which contradicts current flowering models. This result does not diminish the importance of VRN1 in the seasonal regulation of wheat flowering. The up-regulation of VRN1 during winter is required to maintain low transcript levels of VRN2, accelerate the induction of FT in the leaves, and regulate a timely flowering in the spring. Our results also demonstrate the existence of redundant wheat flowering genes that may provide new targets for engineering wheat

  14. Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant.

    Science.gov (United States)

    Ueguchi-Tanaka, Miyako; Hirano, Ko; Hasegawa, Yasuko; Kitano, Hidemi; Matsuoka, Makoto

    2008-09-01

    The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.

  15. Enhanced photoprotection by protein-bound vs free xanthophyll pools: a comparative analysis of chlorophyll b and xanthophyll biosynthesis mutants.

    Science.gov (United States)

    Dall'Osto, Luca; Cazzaniga, Stefano; Havaux, Michel; Bassi, Roberto

    2010-05-01

    When light absorbed by plants exceeds the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly de-epoxidized to zeaxanthin in the so-called xanthophyll cycle. Zeaxanthin plays a key role in the protection of photosynthetic organisms against excess light, by promoting rapidly reversible (qE) and long-term (qI) quenching of excited chlorophylls, and preventing lipid oxidation. The photoprotective role of zeaxanthin, either free or bound to light-harvesting complexes (Lhcs), has been investigated by using mutants lacking Chl b (ch1) and/or specific xanthophyll species (npq, lut2). The ch1 mutation causes (1) the absence of Lhcb proteins; (2) strong reduction of the feedback de-excitation (qE); and (3) accumulation of xanthophylls as free pigments into thylakoids. Ch1 mutants showed extreme sensitivity to photo-oxidative stress in high light, due to higher singlet oxygen (¹O₂) release. The double mutant ch1npq1 was more sensitive to photo-oxidation than ch1, showing that zeaxanthin does protect lipids even when free in the membrane. Nevertheless, lack of zeaxanthin had a much stronger impact on the level of lipid peroxidation in Lhcs-containing plants (WT vs npq1) with respect to Lhc-less plants (ch1 vs ch1npq1), implying that its protective effect is enhanced by interaction with antenna proteins. It is proposed that the antioxidant capacity of zeaxanthin is empowered in the presence of PSII-LHCs-Zea complexes, while its effect on enhancement of qE only provides a minor contribution. Comparison of the sensitivity of WT vs npq1 plants to exogenous ¹O₂ suggests that besides the scavenging of ¹O₂, at least one additional mechanism is involved in chloroplast photoprotection.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-25

    DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  18. Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

    Science.gov (United States)

    Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

    2017-03-01

    In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Protein nutrient value evaluation of mutant strain J5 fruitbody Agaricus bazei murrill by 60Co γ-irradiation

    International Nuclear Information System (INIS)

    Weng Boqi; Huang Tingjun

    2004-01-01

    Protein nutrient value evaluation of mutant strain J 5 fruitbody Agaricus bazei Murrill by 60 Co γ-irradiation was studied. The results showed its total content of amino acids Agaricus bazei murrill was 48.20%; chemical score 58.10; amino acids score 88.60; necessary amino acids index 89.94; biologic value 86.29; nutrient index 29.15. All the index above were higher than that in original strain J 1 , but the ratio score of amino acids of J 5 fruitbody (39.48) was lower than strain J 1 . The results indicated that nutrient value of protein in J 5 was higher than in J 1 . (authors)

  20. 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.

  1. Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

    Science.gov (United States)

    Masso, Majid; Vaisman, Iosif I

    2008-09-15

    Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

  2. Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

    NARCIS (Netherlands)

    Masereeuw, R.; Notenboom, S.; Smeets, P.H.E.; Wouterse, A.C.; Russel, F.G.M.

    2003-01-01

    Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is

  3. Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

    DEFF Research Database (Denmark)

    Ermakova, Inessa; Boldyreff, Brigitte; Issinger, Olaf-Georg

    2003-01-01

    structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two...

  4. Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis

    International Nuclear Information System (INIS)

    Peterson, K.R.; Ganesan, A.K.; Mount, D.W.; Stanford Univ., CA)

    1986-01-01

    The SOS response is displayed following treatments which damage DNA or inhibit DNA replication. Two associated activities include enhanced capacity for DNA repair resulting from derepression of the recA, uvrA, uvrB and uvrD genes and increased mutagenesis due to derepression of recA, umuC and umuD. These changes are the consequence of the derepression of at least seventeen unlinked operons negatively regulated by LexA repressor. Following treatments that induce the SOS response, a signal molecule interacts with RecA protein, converting it to an activated form. Activated RecA protein facilitates the proteolytic cleavage of LexA repressor, which results in derepression of the regulon. The cell then enters a new physiological state during which time DNA repair processes are augmented. The lexA41 mutant of E. coli is a uv-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. The resultant protein is unstable. Lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistent with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the uv-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA - mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. 17 refs., 4 figs., 1 tab

  5. Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

    Science.gov (United States)

    Kumar, Jitendra; Gunapati, Samatha; Alok, Anshu; Lalit, Adarsh; Gadre, Rekha; Sharma, Naresh C; Roy, Joy K; Singh, Sudhir P

    2015-05-01

    Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

  6. Metabolic and molecular analyses of white mutant Vaccinium berries show down-regulation of MYBPA1-type R2R3 MYB regulatory factor.

    Science.gov (United States)

    Primetta, Anja K; Karppinen, Katja; Riihinen, Kaisu R; Jaakola, Laura

    2015-09-01

    MYBPA1-type R2R3 MYB transcription factor shows down-regulation in white mutant berries of Vaccinium uliginosum deficient in anthocyanins but not proanthocyanidins suggesting a role in the regulation of anthocyanin biosynthesis. Berries of the genus Vaccinium are among the best natural sources of flavonoids. In this study, the expression of structural and regulatory flavonoid biosynthetic genes and the accumulation of flavonoids in white mutant and blue-colored wild-type bog bilberry (V. uliginosum) fruits were measured at different stages of berry development. In contrast to high contents of anthocyanins in ripe blue-colored berries, only traces were detected by HPLC-ESI-MS in ripe white mutant berries. However, similar profile and high levels of flavonol glycosides and proanthocyanidins were quantified in both ripe white and ripe wild-type berries. Analysis with qRT-PCR showed strong down-regulation of structural genes chalcone synthase (VuCHS), dihydroflavonol 4-reductase (VuDFR) and anthocyanidin synthase (VuANS) as well as MYBPA1-type transcription factor VuMYBPA1 in white berries during ripening compared to wild-type berries. The profiles of transcript accumulation of chalcone isomerase (VuCHI), anthocyanidin reductase (VuANR), leucoanthocyanidin reductase (VuLAR) and flavonoid 3'5' hydroxylase (VuF3'5'H) were more similar between the white and the wild-type berries during fruit development, while expression of UDP-glucose: flavonoid 3-O-glucosyltransferase (VuUFGT) showed similar trend but fourfold lower level in white mutant. VuMYBPA1, the R2R3 MYB family member, is a homologue of VmMYB2 of V. myrtillus and VcMYBPA1 of V. corymbosum and belongs to MYBPA1-type MYB family which members are shown in some species to be related with proanthocyanidin biosynthesis in fruits. Our results combined with earlier data of the role of VmMYB2 in white mutant berries of V. myrtillus suggest that the regulation of anthocyanin biosynthesis in Vaccinium species could differ

  7. Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

    Science.gov (United States)

    Jernejc, Katarina; Bencina, Mojca

    2003-08-29

    Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

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

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Meggio, F

    1996-01-01

    Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition...... of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha......191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A, K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type...

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

    International Nuclear Information System (INIS)

    Wang Qishan; Bag, Jnanankur

    2006-01-01

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

  10. Photosystem II repair and plant immunity: Lessons learned from Arabidopsis mutant lacking the THYLAKOID LUMEN PROTEIN 18.3

    Directory of Open Access Journals (Sweden)

    Sari eJärvi

    2016-03-01

    Full Text Available Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415-425. Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

  11. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

    Directory of Open Access Journals (Sweden)

    Humaira Adnan

    Full Text Available Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD, to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon, to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin containing genetically inactivated (± an N-terminal polyleucine tail A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF mutant (5-10x, F508delCFTR Golgi maturation (<10x, cell surface expression (20x and chloride transport (2x in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC in N370SGCC Gaucher Disease fibroblasts (3x, another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

  12. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

    Science.gov (United States)

    Adnan, Humaira; Zhang, Zhenbo; Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth; Lingwood, Clifford

    2016-01-01

    Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

  13. Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

    Science.gov (United States)

    Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2013-07-01

    The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

  14. PROXiMATE: a database of mutant protein-protein complex thermodynamics and kinetics.

    Science.gov (United States)

    Jemimah, Sherlyn; Yugandhar, K; Michael Gromiha, M

    2017-09-01

    We have developed PROXiMATE, a database of thermodynamic data for more than 6000 missense mutations in 174 heterodimeric protein-protein complexes, supplemented with interaction network data from STRING database, solvent accessibility, sequence, structural and functional information, experimental conditions and literature information. Additional features include complex structure visualization, search and display options, download options and a provision for users to upload their data. The database is freely available at http://www.iitm.ac.in/bioinfo/PROXiMATE/ . The website is implemented in Python, and supports recent versions of major browsers such as IE10, Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  15. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

    Directory of Open Access Journals (Sweden)

    Victoria A Lawson

    Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

  16. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein.

    Science.gov (United States)

    Michalko, Jaroslav; Glanc, Matouš; Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

    The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.

  17. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Directory of Open Access Journals (Sweden)

    Serena Leone

    Full Text Available MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  18. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Science.gov (United States)

    Leone, Serena; Picone, Delia

    2016-01-01

    MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  19. Effects of mutants in bHLH region on structure stability and protein-DNA binding energy in DECs.

    Science.gov (United States)

    Kong, Yi; Wang, Zhen; Jia, Yanfei; Li, Ping; Hao, Shuhua; Wang, Yunshan

    2017-07-01

    The human DEC subfamily contains two highly conserved members belonging to basic helix-loop-helix (bHLH) transcription factors. This conserved family is spread widely among various species with the function of regulating various crucial molecular signaling pathways. Due to the significance of DECs for important biological processes, their relationship with diseases and the lack of experimentally proven structures, we have implemented a comparative modeling for the bHLH region of DECs as homodimers with themselves and heterodimers with HES-1. Three mutants with predicted roles in reducing intramolecular binding (H57A, R65A, and LL7879AA in DEC1 and LL7071AA in DEC2) were investigated on DEC monomers. Molecular dynamics (MD) simulations were also employed to evaluate the behavior of the mutant molecules in aqueous solution. The monomer was divided into subregions for accurate investigation. The fluctuation in the basic region of mutants was higher than that of wild-type molecules. The binding energy value between protein and DNA obviously increased in the homodimer harboring R65A mutants, which led to more unstable status between protein and DNA. Thus, the mutant R65A interfered DNA-binding affinity. A study on the spatial structures of wild-type and mutant DECs may facilitate functional prediction for mutation effects and dynamic behavior under various conditions and may ultimately help in targeted drug design.

  20. A yigP mutant strain is a small colony variant of E. coli and shows pleiotropic antibiotic resistance.

    Science.gov (United States)

    Xia, Hui; Tang, Qiongwei; Song, Jie; Ye, Jiang; Wu, Haizhen; Zhang, Huizhan

    2017-12-01

    Small colony variants (SCVs) are a commonly observed subpopulation of bacteria that have a small colony size and distinctive biochemical characteristics. SCVs are more resistant than the wild type to some antibiotics and usually cause persistent infections in the clinic. SCV studies have been very active during the past 2 decades, especially Staphylococcus aureus SCVs. However, fewer studies on Escherichia coli SCVs exist, so we studied an E. coli SCV during an experiment involving the deletion of the yigP locus. PCR and DNA sequencing revealed that the SCV was attributable to a defect in the yigP function. Furthermore, we investigated the antibiotic resistance profile of the E. coli SCV and it showed increased erythromycin, kanamycin, and d-cycloserine resistance, but collateral sensitivity to ampicillin, polymyxin, chloramphenicol, tetracycline, rifampin, and nalidixic acid. We tried to determine the association between yigP and the pleiotropic antibiotic resistance of the SCV by analyzing biofilm formation, cellular morphology, and coenzyme Q (Q 8 ) production. Our results indicated that impaired Q 8 biosynthesis was the primary factor that contributed to the increased resistance and collateral sensitivity of the SCV. This study offers a novel genetic basis for E. coli SCVs and an insight into the development of alternative antimicrobial strategies for clinical therapy.

  1. Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

    Science.gov (United States)

    Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

    2017-07-01

    The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

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

    Directory of Open Access Journals (Sweden)

    Haiyan Mao

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

  3. Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein.

    Science.gov (United States)

    Cunningham, C; Davison, A J; MacLean, A R; Taus, N S; Baines, J D

    2000-01-01

    Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.

  4. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

    International Nuclear Information System (INIS)

    Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia; Pegg, Anthony E.; Ealick, Steven E.

    2008-01-01

    The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction

  5. Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

    Science.gov (United States)

    Al-Ramahi, Ismael; Panapakkam Giridharan, Sai Srinivas; Chen, Yu-Chi; Patnaik, Samarjit; Safren, Nathaniel; Hasegawa, Junya; de Haro, Maria; Wagner Gee, Amanda K; Titus, Steven A; Jeong, Hyunkyung; Clarke, Jonathan; Krainc, Dimitri; Zheng, Wei; Irvine, Robin F; Barmada, Sami; Ferrer, Marc; Southall, Noel; Weisman, Lois S; Botas, Juan; Marugan, Juan Jose

    2017-12-26

    The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

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

    Science.gov (United States)

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

    1999-04-01

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

  7. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Kumar, S.

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi 80 cya + and phi 80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants

  8. Structural characterization of V57D and V57P mutants of human cystatin C, an amyloidogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Orlikowska, Marta; Szymańska, Aneta [University of Gdansk, Sobieskiego 18/19, 80-952 Gdansk (Poland); Borek, Dominika; Otwinowski, Zbyszek [University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8816 (United States); Skowron, Piotr; Jankowska, Elżbieta, E-mail: elaj@chem.univ.gda.pl [University of Gdansk, Sobieskiego 18/19, 80-952 Gdansk (Poland)

    2013-04-01

    Val57 point mutants of human cystatin C, which were designed to assess the influence of changes in the properties of the L1 loop on the dimerization propensity, were structurally characterized. Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in β-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations.

  9. The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

    Science.gov (United States)

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

    2014-01-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

  10. Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

    NARCIS (Netherlands)

    Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

    1995-01-01

    Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

  11. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

    Directory of Open Access Journals (Sweden)

    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

  12. An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

    Science.gov (United States)

    Mitobe, Jiro; Sinha, Ritam; Mitra, Soma; Nag, Dhrubajyoti; Saito, Noriko; Shimuta, Ken; Koizumi, Nobuo; Koley, Hemanta

    2017-07-01

    Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

  13. Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein

    Directory of Open Access Journals (Sweden)

    Marc Ehrlich

    2015-07-01

    Full Text Available Frontotemporal dementia (FTD is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.

  14. Alkaline transition of pseudoazurin Met16X mutant proteins: protein stability influenced by the substitution of Met16 in the second sphere coordination.

    Science.gov (United States)

    Abdelhamid, Rehab F; Obara, Yuji; Kohzuma, Takamitsu

    2008-01-01

    Several blue copper proteins are known to change the active site structure at alkaline pH (alkaline transition). Spectroscopic studies of Met16Phe, Met16Tyr, Met16Trp, and Met16Val pseudoazurin variants were performed to investigate the second sphere role through alkaline transition. The visible electronic absorption and resonance Raman spectra of Met16Phe, Met16Tyr, and Met16Trp variants showed the increasing of axial component at pH approximately 11 like wild-type PAz. The visible electronic absorption and far-UV CD spectra of Met16Val demonstrated that the destabilization of the protein structure was triggered at pH>11. Resonance Raman (RR) spectra of PAz showed that the intensity-weighted averaged Cu-S(Cys) stretching frequency was shifted to higher frequency region at pH approximately 11. The higher frequency shift of Cu-S(Cys) bond is implied the stronger Cu-S(Cys) bond at alkaline transition pH approximately 11. The visible electronic absorption and far-UV CD spectra of Met16X PAz revealed that the Met16Val variant is denatured at pH>11, but Met16Phe, Met16Tyr, and Met16Trp mutant proteins are not denatured even at pH>11. These observations suggest that Met16 is important to maintain the protein structure through the possible weak interaction between methionine -SCH3 part and coordinated histidine imidazole moiety. The introduction of pi-pi interaction in the second coordination sphere may be contributed to the enhancement of protein structure stability.

  15. 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

  16. Nature of mutants induced by ionizing radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by. gamma. -rays or. cap alpha. -particles showing that the majority have deletions of all or part of the hprt gene

    Energy Technology Data Exchange (ETDEWEB)

    Thacker, J

    1986-05-01

    DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionizing radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the ..gamma..-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. 16 references, 4 figures, 1 table.

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

    Directory of Open Access Journals (Sweden)

    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

  18. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  19. Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.

    Science.gov (United States)

    Noll, Meenakshi; Battaile, Kevin P; Bateman, Raynard; Lax, Timothy P; Rathbun, Keany; Reifsteck, Carol; Bagby, Grover; Finegold, Milton; Olson, Susan; Grompe, Markus

    2002-07-01

    Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.

  20. Comparison of radiation-induced proteins in two revertants with those in a radiation sensitive mutant of Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Hu Qian; Tanaka, Atsushi; Kobayashi, Yasuhiko; Nozawa, Ranko; Watanabe, Hiroshi.

    1992-01-01

    In order to search proteins responsible for DNA repair, the intracellular proteins in gamma-irradiated cells of D.radiodurans R1 and its three mutants were analyzed by means of two-dimensional gel electrophoresis. Two new proteins, P35 (M.W. 35 kDa) and P92 (M.W. 92 kDa), were found in the cells of the radioresistant revertant (KH8401-1-5) and D.radiodurans R1. However, only P92 was detected in the moderately radioresistant revertant (KH8401-10-7). Both proteins were not detected in the cells of the radiosensitive mutant (KH8401). On the other hand, one protein, P25 (M.W. 25 kDa), which was detected either before or after gamma-irradiation, was only found in KH8401-10-7. These results suggest that these special induced proteins, P35 and P92, might be directly responsible for radiation resistance. The analyses of similar proteins in the cells of E.coli B/r and Deinobactor grandis KS0460 were also carried out. (author)

  1. Characterization of a crp* mutant of the E. coli cAMP receptor protein

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1987-01-01

    One of the crp* mutants previously isolated to activate lac promoter in vivo has been characterized with regard to its biochemical properties. CRP*592 shows a more open conformation than CRP as indicated by its sensitivity to proteolytic attack. Dithionitrobenzoic acid mediated intersubunit crosslinking of CRP requires cAMP; this reaction occurs with unliganded CRP*592. Binding of CRP to its site on the lac promoter and activation of abortive initiation is effected by cAMP but not by cGMP. CRP*592 can activate abortive initiation in the presence of cAMP or cGMP and also at a high CRP*592 concentration in the absence of cyclic nucleotide. DNase I footprinting shows that cAMP-CRP* binds to its site on lac P + while unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lac P + only in the presence of RNA polymerase. While cGMP binds to CRP it cannot replace cAMP in effecting the conformation necessary for site specific promoter binding; the weakly active unliganded CRP*592 can be shifted to a functional conformation by cAMP, cGMP and RNA polymerase

  2. Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls.

    Science.gov (United States)

    Saitoh, Aya; Takase, Tomoyuki; Kitaki, Hiroyuki; Miyazaki, Yuji; Kiyosue, Tomohiro

    2015-01-01

    Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.

  3. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Science.gov (United States)

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

    International Nuclear Information System (INIS)

    Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

    1986-01-01

    The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-[ 14 C]-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  6. 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

  7. Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders

    OpenAIRE

    Gery, Sigal; Gueller, Saskia; Chumakova, Katya; Kawamata, Norihiko; Liu, Liqin; Koeffler, H. Phillip

    2007-01-01

    Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk i...

  8. Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue shifted intermediate

    NARCIS (Netherlands)

    Derix, N.M.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; van der Horst, M.A.; Hellingwerf, K.J.; Boelens, R.|info:eu-repo/dai/nl/070151407; Kaptein, R.|info:eu-repo/dai/nl/074334603; van Nuland, N.A.J.

    2003-01-01

    The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB

  9. Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

    Science.gov (United States)

    Yoshihara, Ryouhei; Li, ZhengHao; Ishimori, Keisuke; Kuwabara, Kazuki; Hatakeyama, Shin; Tanaka, Shuuitsu

    2017-08-01

    To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1 KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1 KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1 KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1 KO strain. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

    Science.gov (United States)

    Varela, Mariana; Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong; Palmarini, Massimo

    2016-06-01

    Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc

  11. Molybdenum x-ray absorption studies of the mutant Kp nifV of nitrogenase MO-FE protein

    International Nuclear Information System (INIS)

    Eidsness, M.K.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer, S.P.

    1985-01-01

    The nifV mutant nitrogenase enzyme of Klebsiella pheumoniae exhibits altered substrate reducing activity. This nitrogenase mutant cannot fix N 2 in vivo but can reduce C 2 H 2 to C 2 H 4 . The nifV mutant enzyme differs further from the wild-type enzyme by CO inhibition of its H 2 evolution activity, up to 80%. The NifV - phenotype (NifV - Kp1) has been shown to be associated with the iron-molybdenum cofactor (FeMoco) in the Mo Fe protein which is generally accepted as the site for substrate reduction. An X-Ray absorption study of the Mo site in this mutant may reveal a difference in its FeMoco structure. The authors report here a comparison of Mo X-Ray absorption data from the nitrogenase enzymes of the wild-type and NifV - strains in three different forms: (1) as isolated, (2) dye-oxidized, and (3) fixing enzyme systems. Mo edge structure of NifV - Kp1 and wild-type enzymes are nearly identical. Small shifts to higher energies are observed in the oxidized and fixing states. Mo EXAFS of NifV - Kp1 and wild-type in the ''as isolated'' state appear indistinguishable. Curve fitting results best describe the molybdenum in FeMoco as bound by 4-5 S atoms at 2.36 A ,3 Fe atoms at 2.69 A, and 0-2 O(N) atoms at 2.19 A. The spectral similarity of these results concerning the nifV mutant FeMoco structure is discussed

  12. Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice.

    Science.gov (United States)

    Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P

    1996-09-01

    The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

  13. Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

    Science.gov (United States)

    Tanaka, Tetsuya S; Ikenishi, Kohji

    2002-02-01

    An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

  14. Mutant p53 protein in serum could be used as a molecular marker in human breast cancer.

    Science.gov (United States)

    Balogh, G A; Mailo, D A; Corte, M M; Roncoroni, P; Nardi, H; Vincent, E; Martinez, D; Cafasso, M E; Frizza, A; Ponce, G; Vincent, E; Barutta, E; Lizarraga, P; Lizarraga, G; Monti, C; Paolillo, E; Vincent, R; Quatroquio, R; Grimi, C; Maturi, H; Aimale, M; Spinsanti, C; Montero, H; Santiago, J; Shulman, L; Rivadulla, M; Machiavelli, M; Salum, G; Cuevas, M A; Picolini, J; Gentili, A; Gentili, R; Mordoh, J

    2006-04-01

    p53 wild-type is a tumor suppressor gene involved in DNA gene transcription or DNA repair mechanisms. When damage to DNA is unrepairable, p53 induces programmed cell death (apoptosis). The mutant p53 gene is the most frequent molecular alteration in human cancer, including breast cancer. Here, we analyzed the genetic alterations in p53 oncogene expression in 55 patients with breast cancer at different stages and in 8 normal women. We measured by ELISA assay the serum levels of p53 mutant protein and p53 antibodies. Immunohistochemistry and RT-PCR using specific p53 primers as well as mutation detection by DNA sequencing were also evaluated in breast tumor tissue. Serological p53 antibody analysis detected 0/8 (0%), 0/4 (0%) and 9/55 (16.36%) positive cases in normal women, in patients with benign breast disease and in breast carcinoma, respectively. We found positive p53 mutant in the sera of 0/8 (0.0%) normal women, 0/4 (0%) with benign breast disease and 29/55 (52.72%) with breast carcinoma. Immunohistochemistry evaluation was positive in 29/55 (52.73%) with mammary carcinoma and 0/4 (0%) with benign breast disease. A very good correlation between p53 mutant protein detected in serum and p53 accumulation by immunohistochemistry (83.3% positive in both assays) was found in this study. These data suggest that detection of mutated p53 could be a useful serological marker for diagnostic purposes.

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

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

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

  16. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    Science.gov (United States)

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  17. Deficiency of toxin-binding protein activity in mutants of sugarcane clone H54-775 as it relates to disease resistance

    International Nuclear Information System (INIS)

    Strobel, G.A.; Steiner, G.W.; Byther, R.

    1975-01-01

    Three mutants selected from a population of sugarcane clone H54-775 that had been irradiated with 3 kR γ-radiation all lacked toxin-binding protein activity. This activity previously had been shown to be essential for eye spot disease susceptibility and was demonstrated in the susceptible parent clone H54-775. In one mutant, the biochemical, immunochemical, and electrophoretic mobilities of the toxin-binding protein were all modified

  18. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Science.gov (United States)

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  19. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  20. An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

    Directory of Open Access Journals (Sweden)

    Jiro Mitobe

    2017-07-01

    Full Text Available Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

  1. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    International Nuclear Information System (INIS)

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s)

  2. Mutations in ribosomal proteins, RPL4 and RACK1, suppress the phenotype of a thermospermine-deficient mutant of Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kakehi

    Full Text Available Thermospermine acts in negative regulation of xylem differentiation and its deficient mutant of Arabidopsis thaliana, acaulis5 (acl5, shows excessive xylem formation and severe dwarfism. Studies of two dominant suppressors of acl5, sac51-d and sac52-d, have revealed that SAC51 and SAC52 encode a transcription factor and a ribosomal protein L10 (RPL10, respectively, and these mutations enhance translation of the SAC51 mRNA, which contains conserved upstream open reading frames in the 5' leader. Here we report identification of SAC53 and SAC56 responsible for additional suppressors of acl5. sac53-d is a semi-dominant allele of the gene encoding a receptor for activated C kinase 1 (RACK1 homolog, a component of the 40S ribosomal subunit. sac56-d represents a semi-dominant allele of the gene for RPL4. We show that the GUS reporter activity driven by the CaMV 35S promoter plus the SAC51 5' leader is reduced in acl5 and restored by sac52-d, sac53-d, and sac56-d as well as thermospermine. Furthermore, the SAC51 mRNA, which may be a target of nonsense-mediated mRNA decay, was found to be stabilized in these ribosomal mutants and by thermospermine. These ribosomal proteins are suggested to act in the control of uORF-mediated translation repression of SAC51, which is derepressed by thermospermine.

  3. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis

    DEFF Research Database (Denmark)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei

    2016-01-01

    In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylako...... was restored by supplementation with Mn2+, but not Ca2+. Furthermore, PAM71 suppressed the Mn2+-sensitive phenotype of the yeast mutant Δpmr1. Therefore, PAM71 presumably functions in Mn2+ uptake into thylakoids to ensure optimal PSII performance....

  4. The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

    Science.gov (United States)

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

    2014-05-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

  5. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fabao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); You, Xiaona [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Chi, Xiumei [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Wang, Tao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Niu, Junqi, E-mail: junqiniu@yahoo.com.cn [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-02-07

    Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

  6. Alterations in gene expression in mutant amyloid precursor protein transgenic mice lacking Niemann-Pick type C1 protein.

    Directory of Open Access Journals (Sweden)

    Mahua Maulik

    Full Text Available Niemann-Pick type C (NPC disease, a rare autosomal recessive disorder caused mostly by mutation in NPC1 gene, is pathologically characterized by the accumulation of free cholesterol in brain and other tissues. This is accompanied by gliosis and loss of neurons in selected brain regions, including the cerebellum. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer's disease, including the presence of neurofibrillary tangles and increased levels of amyloid precursor protein (APP-derived β-amyloid (Aβ peptides in vulnerable brain neurons. To evaluate the role of Aβ in NPC disease, we determined the gene expression profile in selected brain regions of our recently developed bigenic ANPC mice, generated by crossing APP transgenic (Tg mice with heterozygous Npc1-deficient mice. The ANPC mice exhibited exacerbated neuronal and glial pathology compared to other genotypes [i.e., APP-Tg, double heterozygous (Dhet, Npc1-null and wild-type mice]. Analysis of expression profiles of 86 selected genes using real-time RT-PCR arrays showed a wide-spectrum of alterations in the four genotypes compared to wild-type controls. The changes observed in APP-Tg and Dhet mice are limited to only few genes involved mostly in the regulation of cholesterol metabolism, whereas Npc1-null and ANPC mice showed alterations in the expression profiles of a number of genes regulating cholesterol homeostasis, APP metabolism, vesicular trafficking and cell death mechanism in both hippocampus and cerebellum compared to wild-type mice. Intriguingly, ANPC and Npc1-null mice, with some exceptions, exhibited similar changes, although more genes were differentially expressed in the affected cerebellum than the relatively spared hippocampus. The altered gene profiles were found to match with the corresponding protein levels. These results suggest that lack of Npc1 protein can alter the expression profile of selected transcripts as well as proteins, and

  7. Fibrillary glomerulonephritis associated with monoclonal gammopathy of undetermined significance showing lambda-type Bence Jones protein.

    Science.gov (United States)

    Nagao, Tomoaki; Okura, Takafumi; Miyoshi, Ken-Ichi; Watanabe, Sanae; Manabe, Seiko; Kurata, Mie; Irita, Jun; Fukuoka, Tomikazu; Higaki, Jitsuo

    2005-09-01

    A 79-year-old woman was admitted to our hospital because of leg edema due to a nephrotic syndrome. Urinary and serum immunoelectrophoresis showed positive for the lambda type of Bence Jones protein. A bone marrow aspiration test revealed mild plasmacytosis (6.4% of the total cells). These findings confirmed her diagnosis of monoclonal gammopathy of undetermined significance (MGUS). Her renal biopsy specimen revealed mild mesangial cell proliferation and an increase in the mesangial matrix. Immunofluorescence studies showed positive staining for IgG, IgA, C3, and kappa and lambda light chains in the capillary wall and mesangium area. Electron microscopy showed that the electron deposits in the thickened basement membrane were formed by randomly arranged 16- to 18-nm nonbranching fibrils. A Congo red stain for amyloid was negative. These findings corresponded with the diagnosis of fibrillary glomerulonephritis. Therefore, this case showed a rare combination of fibrillary glomerulonephritis and MGUS.

  8. Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

    Science.gov (United States)

    Sharma, Shruti; Gupta, Ravi; Deswal, Renu

    2017-05-01

    In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

    Science.gov (United States)

    Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Zhendre, Vanessa; Henry, Sarah; Thévenot, Julie; Dufourc, Erick J; Alves, Isabel D; Peruch, Frédéric

    2014-01-01

    The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles. © 2013.

  10. The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic ProteinsW⃞

    Science.gov (United States)

    Kubis, Sybille; Baldwin, Amy; Patel, Ramesh; Razzaq, Azam; Dupree, Paul; Lilley, Kathryn; Kurth, Joachim; Leister, Dario; Jarvis, Paul

    2003-01-01

    The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins. PMID:12897258

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

    International Nuclear Information System (INIS)

    Wang Qishan; Bag, Jnanankur

    2008-01-01

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

  12. Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders.

    Science.gov (United States)

    Gery, Sigal; Gueller, Saskia; Chumakova, Katya; Kawamata, Norihiko; Liu, Liqin; Koeffler, H Phillip

    2007-11-01

    Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)-expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches.

  13. Effect of varying temperature on growth, morphology and soluble protein content of div I and div II mutant strains of bacillus sub tills

    International Nuclear Information System (INIS)

    Ahmed, A.; Sabri, A.N.

    2004-01-01

    In B.subtilis, cell division is controlled by div-genes which have been mapped on its circular chromosome. In the present work, div-mutant strains 1A316(div II), 1A317 and 1A318 (div I) were studied. These strains exhibited temperature sensitive cell division mutations. Colony morphology, cell morphology, staining behavior, growth rate and protein content of PY79 (wild type) and div-mutant strains (1A316, 1A317, 1A318) was studied at different temperatures ( 25 deg. Centi grade and 42 deg. with varying incubation periods(4, 16, 24, 48, 72,96 hrs). div-mutants differ from wild type (PY79) in colony morphology. Colony margin in PY79 was entire while in the div strains it is undulate. Staining behavior of cells as well as cell morphology i.e., cell size, cell types, formation of filaments/minicells were affected by high temperature. At higher temperature (42 deg. Centi grade), div-mutants undergo more severe lysis and degeneration as compare to wild type (PY79). Defective spores were produced by div-mutants at 25 deg. Centi grade and 42 deg. Centi grade. Tetrazolium overlay test was performed at 37 deg. Centi grade and 42 deg. Centi grade to check the spore germination ability of wild type and div-mutants. In 1A318, defective spores were produced at 37 deg. Centi grade, div-mutant was checked after 24 and 96 hrs at different temperatures (25, 37 and 42 deg. Centi grade). At all temperatures protein content were more in PY79 as compare to div-mutants. Also at 25 and 42 deg. Centi grade, protein content was more as compare to 37 deg. Centi grade. Protein contents was reduced at sporulation stages. Thus cell division mutations affect cell morphology, sporulation and germination processes in B.subtilis and thus are multifaceted mutations. (author)

  14. Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases

    OpenAIRE

    Adnan, Humaira; Zhang, Zhenbo; Park, Hyun-Joo; Tailor, Chetankumar; Che, Clare; Kamani, Mustafa; Spitalny, George; Binnington, Beth; Lingwood, Clifford

    2016-01-01

    Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subu...

  15. Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

    Science.gov (United States)

    Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

    2012-05-01

    Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Study on seed storage protein of offspring 60Co γ-rays induced mutants of soybean Jindou-24

    International Nuclear Information System (INIS)

    Li Huifeng; Li Guiquan; Li Ling

    2008-01-01

    Seed of soybean variety Jindou-24 were irradiated with 60 Co γ-rays and the offspring of mutants were used to research the relative contents of glycinin (11S) and β-conglycinin (7S), the composition of its subunits, the contents of different subunits and the rules of its variance by using SDS-PAGE technique. the results indicated that the content of different subunits or the same subunit of different varieties varied greatly in the offspring of mutants. Significant difference existed in the contents of different subunits of the same variety or the same subunits of different varieties. The content of 11S negatively correlated with that of 7S, and positively correlated with 11S/7S. Cluster analysis showed the mutants were divided into three groups (average 11S/7S rate 1.70, 1.25 and 0.79) according to the rate of 11S/7S. The results could provide theoretical guidance to breed new soybean varieties with the character of high quality and other special purpose. (authors)

  17. Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus

    International Nuclear Information System (INIS)

    Shang, Guijun; Cang, Huaixing; Liu, Zhijie; Gao, Wei; Bi, Ruchang

    2010-01-01

    Recombinant calcineurin B-like protein 1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K + , osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298 K with PEG 2000 MME as a precipitant diffracted to 2.90 Å resolution. The crystal belonged to space group P2 1 2 1 2, with unit-cell parameters a = 99.87, b = 114.42, c = 63.80 Å, α = β = γ = 90.00° and three molecules per asymmetric unit

  18. G2019S LRRK2 mutant fibroblasts from Parkinson’s disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy

    International Nuclear Information System (INIS)

    Yakhine-Diop, Sokhna M.S.; Bravo-San Pedro, José M.; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo

    2014-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP + )-induced damage in fibroblasts from Parkinson’s patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP + induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP + were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome–lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD

  19. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  20. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

    CSIR Research Space (South Africa)

    Mehlo, L

    2013-09-01

    Full Text Available in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification...

  1. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

    Science.gov (United States)

    Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

  2. Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

    Science.gov (United States)

    Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

    2006-03-01

    Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

  3. Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex

    DEFF Research Database (Denmark)

    Sørensen, Charlotte B; Andresen, Brage S; Jensen, Uffe B

    2003-01-01

    vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection...... of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated...... with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P

  4. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

    Science.gov (United States)

    Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David

    2012-01-01

    A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702

  5. Biological safety assessment of mutant variant of Allium sativum leaf agglutinin (mASAL), a novel antifungal protein for future transgenic application.

    Science.gov (United States)

    Ghosh, Prithwi; Roy, Amit; Chakraborty, Joydeep; Das, Sampa

    2013-12-04

    Genetic engineering has established itself to be an important tool for crop improvement. Despite the success, there is always a risk of food allergy induced by alien gene products. The present study assessed the biosafety of mutant Allium sativum leaf agglutinin (mASAL), a potent antifungal protein generated by site directed mutagenesis of Allium sativum leaf agglutinin (ASAL). mASAL was cloned in pET28a+ and expressed in E. coli, and the safety assessment was carried out according to the FAO/WHO guideline (2001). Bioinformatics analysis, pepsin digestion, and thermal stability assay showed the protein to be nonallergenic. Targeted sera screening revealed no significant IgE affinity of mASAL. Furthermore, mASAL sensitized Balb/c mice showed normal histopathology of lung and gut tissue. All results indicated the least possibility of mASAL being an allergen. Thus, mASAL appears to be a promising antifungal candidate protein suitable for agronomical biotechnology.

  6. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

    Directory of Open Access Journals (Sweden)

    Ioannis Sitaras

    Full Text Available Evolution of Avian Influenza (AI viruses--especially of the Highly Pathogenic Avian Influenza (HPAI H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1, using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  7. Transcriptional switching by the MerR protein: Activation and repression mutants implicate distinct DNA and mercury(II) binding domains

    International Nuclear Information System (INIS)

    Shewchuk, L.M.; Helmann, J.D.; Ross, W.; Park, S.J.; Summers, A.O.; Walsh, C.T.

    1989-01-01

    Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a - r - ) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a - r + ) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variable deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation

  8. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) prov...

  9. CAN THE STABILITY OF PROTEIN MUTANTS BE PREDICTED BY FREE-ENERGY CALCULATIONS

    NARCIS (Netherlands)

    YUNYU, S; MARK, AE; WANG, CX; HUANG, FH; BERENDSEN, HJC; VANGUNSTEREN, WF

    The use of free energy simulation techniques in the study of protein stability is critically evaluated. Results from two simulations of the thermostability mutation Asn218 to Ser218 in Subtilisin are presented. It is shown that components of the free energy change can be highly sensitive to the

  10. Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

    NARCIS (Netherlands)

    Michels, AA; Kanon, B; Bensaude, O; Kampinga, HH

    1999-01-01

    Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272,

  11. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity

    Directory of Open Access Journals (Sweden)

    Peter W. Lindinger

    2015-09-01

    Full Text Available Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  12. Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

    Science.gov (United States)

    Lindinger, Peter W; Christe, Martine; Eberle, Alex N; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J I; Fearnley, Ian M; Walker, John E

    2015-09-01

    Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

  13. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination

    International Nuclear Information System (INIS)

    Madiraju, M.V.; Templin, A.; Clark, A.J.

    1988-01-01

    A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine). This mutation, originally named srf-803, was renamed recA803. Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected. This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803. The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules. Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules. The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA. We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background. Implications of this hypothesis for the function of RecF protein in recombination are discussed

  14. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

    Science.gov (United States)

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration.

  15. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    Science.gov (United States)

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  16. Major urinary protein (MUP) profiles show dynamic changes rather than individual 'barcode' signatures.

    Science.gov (United States)

    Thoß, M; Luzynski, K C; Ante, M; Miller, I; Penn, D J

    2015-06-30

    House mice ( Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This 'barcode hypothesis' requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent ('major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous ('minor') bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis ('dynamic changes' hypothesis).

  17. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  18. Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

    International Nuclear Information System (INIS)

    Vaezeslami, Soheila; Jia, Xiaofei; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H.

    2008-01-01

    A water network stabilizes the structure of cellular retionic acid binding protein II. The structural integrity of cellular retinoic acid-binding protein II (CRABPII) has been investigated using the crystal structures of CRABPII mutants. The overall fold was well maintained by these CRABPII mutants, each of which carried multiple different mutations. A water-mediated network is found to be present across the large binding cavity, extending from Arg111 deep inside the cavity to the α2 helix at its entrance. This chain of interactions acts as a ‘pillar’ that maintains the integrity of the protein. The disruption of the water network upon loss of Arg111 leads to decreased structural integrity of the protein. A water-mediated network can be re-established by introducing the hydrophilic Glu121 inside the cavity, which results in a rigid protein with the α2 helix adopting an altered conformation compared with wild-type CRABPII

  19. Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

    Science.gov (United States)

    Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

    2015-04-01

    Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

  20. 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...

  1. Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

    Science.gov (United States)

    Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

    2003-02-01

    Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

  2. An expanded evaluation of protein function prediction methods shows an improvement in accuracy

    NARCIS (Netherlands)

    Jiang, Yuxiang; Oron, Tal Ronnen; Clark, Wyatt T.; Bankapur, Asma R.; Andrea, D' Daniel; Lepore, Rosalba; Funk, Christopher S.; Kahanda, Indika; Verspoor, Karin M.; Ben-Hur, Asa; Koo, Da Chen Emily; Penfold-Brown, Duncan; Shasha, Dennis; Youngs, Noah; Bonneau, Richard; Lin, Alexandra; Sahraeian, Sayed M.E.; Martelli, Pier Luigi; Profiti, Giuseppe; Casadio, Rita; Cao, Renzhi; Zhong, Zhaolong; Cheng, Jianlin; Altenhoff, Adrian; Skunca, Nives; Dessimoz, Christophe; Dogan, Tunca; Hakala, Kai; Kaewphan, Suwisa; Mehryary, Farrokh; Salakoski, Tapio; Ginter, Filip; Fang, Hai; Smithers, Ben; Oates, Matt; Gough, Julian; Törönen, Petri; Koskinen, Patrik; Holm, Liisa; Chen, Ching Tai; Hsu, Wen Lian; Bryson, Kevin; Cozzetto, Domenico; Minneci, Federico; Jones, David T.; Chapman, Samuel; BKC, Dukka; Khan, Ishita K.; Kihara, Daisuke; Ofer, Dan; Rappoport, Nadav; Stern, Amos; Cibrian-Uhalte, Elena; Denny, Paul; Foulger, Rebecca E.; Hieta, Reija; Legge, Duncan; Lovering, Ruth C.; Magrane, Michele; Melidoni, Anna N.; Mutowo-Meullenet, Prudence; Pichler, Klemens; Shypitsyna, Aleksandra; Li, Biao; Zakeri, Pooya; ElShal, Sarah; Tranchevent, Léon Charles; Das, Sayoni; Dawson, Natalie L.; Lee, David; Lees, Jonathan G.; Sillitoe, Ian; Bhat, Prajwal; Nepusz, Tamás; Romero, Alfonso E.; Sasidharan, Rajkumar; Yang, Haixuan; Paccanaro, Alberto; Gillis, Jesse; Sedeño-Cortés, Adriana E.; Pavlidis, Paul; Feng, Shou; Cejuela, Juan M.; Goldberg, Tatyana; Hamp, Tobias; Richter, Lothar; Salamov, Asaf; Gabaldon, Toni; Marcet-Houben, Marina; Supek, Fran; Gong, Qingtian; Ning, Wei; Zhou, Yuanpeng; Tian, Weidong; Falda, Marco; Fontana, Paolo; Lavezzo, Enrico; Toppo, Stefano; Ferrari, Carlo; Giollo, Manuel; Piovesan, Damiano; Tosatto, Silvio C.E.; Pozo, del Angela; Fernández, José M.; Maietta, Paolo; Valencia, Alfonso; Tress, Michael L.; Benso, Alfredo; Carlo, Di Stefano; Politano, Gianfranco; Savino, Alessandro; Rehman, Hafeez Ur; Re, Matteo; Mesiti, Marco; Valentini, Giorgio; Bargsten, Joachim W.; Dijk, van Aalt-Jan; Gemovic, Branislava; Glisic, Sanja; Perovic, Vladmir; Veljkovic, Veljko; Veljkovic, Nevena; Almeida-e-Silva, Danillo C.; Vencio, Ricardo Z.N.; Sharan, Malvika; Vogel, Jörg; Kansakar, Lakesh; Zhang, Shanshan; Vucetic, Slobodan; Wang, Zheng; Sternberg, Michael J.E.; Wass, Mark N.; Huntley, Rachael P.; Martin, Maria J.; O'Donovan, Claire; Robinson, Peter N.; Moreau, Yves; Tramontano, Anna; Babbitt, Patricia C.; Brenner, Steven E.; Linial, Michal; Orengo, Christine A.; Rost, Burkhard; Greene, Casey S.; Mooney, Sean D.; Friedberg, Iddo; Radivojac, Predrag

    2016-01-01

    Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role

  3. CELLS OVEREXPRESSING HSP27 SHOW ACCELERATED RECOVERY FROM HEAT-INDUCED NUCLEAR-PROTEIN AGGREGATION

    NARCIS (Netherlands)

    KAMPINGA, HH; BRUNSTING, JF; STEGE, GJJ; KONINGS, AWT; LANDRY, J

    1994-01-01

    Protein denaturation/aggregation upon cell exposure to heat shock is a likely cause of cell death. in the nucleus, protein aggregation has often been correlated to inhibition of nuclear located processes and heat-induced cell killing. in Chinese hamster 023 cells made thermotolerant by a prior

  4. Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Gregor Ilc

    2013-08-01

    Full Text Available The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

  5. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing......) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains...... and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either...

  6. Comparisons of protein, lipid, phenolics, γ-oryzanol, vitamin E, and mineral contents in bran layer of sodium azide-induced red rice mutants.

    Science.gov (United States)

    Jeng, Toong Long; Ho, Pei Tzu; Shih, Yi Ju; Lai, Chia Chi; Wu, Min Tze; Sung, Jih Min

    2011-06-01

    The bran part of red rice grain is concentrated with many phytochemicals, including proanthocyanidins, oryzanol and vitamin E, that exert beneficial effects on human health, but it contains low levels of essential minerals such as Fe and Zn. In the present study, the protein, lipid, phytochemicals and mineral contents in bran samples were compared among red rice SA-586 and its NaN₃-induced mutants. The plant heights of NaN₃-induced mutants were decreased. The contents of protein, lipid, total phenolics, total flavonoids, total anthocyanins, total proanthocyanidins, total γ-oryzanol, total tocopherols and total tocotrienols also varied among the tested mutants. The brans of mutants M-18, M-56 and M-50 contained more proanthocyanidins, γ-oryzanol, vitamin E than that of SA-586, respectively. M-54 accumulated more Fe content (588.7 mg kg⁻¹ bran dry weight) than SA-586 (100.1 mg kg⁻¹ bran dry weight). The brans of M-18, M-50 and M-56 are good sources of proanthocyanidins, vitamin E and γ-oryzanol, respectively, while the bran of M-54 is rich in Fe. Thus these mutants could be used to produce high-value phytochemicals or Fe byproducts from bran during rice grain milling or as genetic resources for rice improvement programs. Copyright © 2011 Society of Chemical Industry.

  7. Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability*

    Science.gov (United States)

    Liu, Yiting; Lee, Sang Y.; Neely, Elizabeth; Nandar, Wint; Moyo, Mthabisi; Simmons, Zachary; Connor, James R.

    2011-01-01

    A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability. PMID:21349849

  8. Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato

    International Nuclear Information System (INIS)

    Oelmueller, R.; Kendrick, R.E.; Briggs, W.R.

    1989-01-01

    Polyclonal antibodies against pea phytochrome detect 2 protein bands (about 116 and 120 kDa) on blots of crude protein extracts and protein of microsomal preparations of dark-grown tomato seedlings. Both protein bands are undetectable in Western blots of the aurea mutant extracts. Neither protein band is detectable after isogenic wild-type seedlings are illuminated with 3 h of red light, either in the crude extract or in the membrane fraction of the irradiated seedlings; this result is consistent with the hypothesis that both bands are phytochrome. When dark-grown wild-type seedlings are illuminated with 3 h of red light or blue light against a red light background, the transcript levels for chlorophyll a/b-binding proteins of photosystem I and II, plastocyanin, and the subunit II of photosystem I increase. In all cases, the same fluence rate of blue light is much more effective than red light alone, a result that indicates the involvement of a blue/UV-A light photoreceptor in addition to the involvement of the far-red-absorbing form of phytochrome, Pfr. The aurea mutant responds neither to red light nor to blue light. Thus, no Pfr-independent induction of the four transcripts by a blue/UV-A light photoreceptor can be measured in the aurea mutant

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

    Science.gov (United States)

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

    2016-12-01

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

  10. Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water

    Energy Technology Data Exchange (ETDEWEB)

    Riback, Joshua A.; Bowman, Micayla A.; Zmyslowski, Adam M.; Knoverek, Catherine R.; Jumper, John M.; Hinshaw, James R.; Kaye, Emily B.; Freed, Karl F.; Clark, Patricia L.; Sosnick, Tobin R.

    2017-10-12

    A substantial fraction of the proteome is intrinsically disordered, and even well-folded proteins adopt non-native geometries during synthesis, folding, transport, and turnover. Characterization of intrinsically disordered proteins (IDPs) is challenging, in part because of a lack of accurate physical models and the difficulty of interpreting experimental results. We have developed a general method to extract the dimensions and solvent quality (self-interactions) of IDPs from a single small-angle x-ray scattering measurement. We applied this procedure to a variety of IDPs and found that even IDPs with low net charge and high hydrophobicity remain highly expanded in water, contrary to the general expectation that protein-like sequences collapse in water. Our results suggest that the unfolded state of most foldable sequences is expanded; we conjecture that this property was selected by evolution to minimize misfolding and aggregation.

  11. Na(v)1.8 channelopathy in mutant mice deficient for myelin protein zero is detrimental to motor axons

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Alvarez Herrero, Susana; Pinchenko, Volodymyr

    2011-01-01

    Myelin protein zero mutations were found to produce Charcot-Marie-Tooth disease phenotypes with various degrees of myelin impairment and axonal loss, ranging from the mild 'demyelinating' adult form to severe and early onset forms. Protein zero deficient homozygous mice ( ) show a severe and prog......Myelin protein zero mutations were found to produce Charcot-Marie-Tooth disease phenotypes with various degrees of myelin impairment and axonal loss, ranging from the mild 'demyelinating' adult form to severe and early onset forms. Protein zero deficient homozygous mice ( ) show a severe...... and progressive dysmyelinating neuropathy from birth with compromised myelin compaction, hypomyelination and distal axonal degeneration. A previous study using immunofluorescence showed that motor nerves deficient of myelin protein zero upregulate the Na(V)1.8 voltage gated sodium channel isoform, which...... is normally present only in restricted populations of sensory axons. The aim of this study was to investigate the function of motor axons in protein zero-deficient mice with particular emphasis on ectopic Na(V)1.8 voltage gated sodium channel. We combined 'threshold tracking' excitability studies...

  12. Immunogenicity in African Green Monkeys of M Protein Mutant Vesicular Stomatitis Virus Vectors and Contribution of Vector-Encoded Flagellin

    Directory of Open Access Journals (Sweden)

    Marlena M. Westcott

    2018-03-01

    Full Text Available Recombinant vesicular stomatitis virus (VSV is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R and M51R VSV that produces flagellin (M51R-F as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (108 pfu and low-dose (105 pfu vaccination. A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production (IgM, IgG and neutralizing antibody at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity.

  13. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    Science.gov (United States)

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  14. Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k.

    Science.gov (United States)

    Condezo, Gabriela N; Marabini, Roberto; Ayora, Silvia; Carazo, José M; Alba, Raúl; Chillón, Miguel; San Martín, Carmen

    2015-09-01

    Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the

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

    Science.gov (United States)

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

    2017-05-01

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

  16. Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

    Directory of Open Access Journals (Sweden)

    Kumagai Yoshinori

    2010-12-01

    Full Text Available Abstract Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium, several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

  17. 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

  18. 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.

  19. Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

    Science.gov (United States)

    Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

    2016-01-01

    Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

  20. Abscisic Acid–Responsive Guard Cell Metabolomes of Arabidopsis Wild-Type and gpa1 G-Protein Mutants[C][W

    Science.gov (United States)

    Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M.

    2013-01-01

    Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography–multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼350 million guard cell protoplasts from ∼30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca2+-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1. PMID:24368793

  1. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Yoon, Hyunjin; Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; Mcclelland, Michael; Heffron, Fred; Smith, Richard D.

    2009-03-11

    In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will

  3. The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

    Science.gov (United States)

    Piekarowicz, Katarzyna; Machowska, Magdalena; Dratkiewicz, Ewelina; Lorek, Daria; Madej-Pilarczyk, Agnieszka; Rzepecki, Ryszard

    2017-08-01

    LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

  4. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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

  6. Calcium-Induced Activation of a Mutant G-Protein-Coupled Receptor Causes In Vitro Transformation of NIH/3T3 Cells

    Directory of Open Access Journals (Sweden)

    Ana O. Hoff

    1999-12-01

    Full Text Available The calcium-sensing receptor (CaR is a G-proteincoupled receptor that is widely expressed, has tissuespecific functions, regulates cell growth. Activating mutations of this receptor cause autosomal dominant hypocalcemia, a syndrome characterized by hypocalcemia and hypercalciuria. The identification of a family with an activating mutation of the CaR (Thr151 Met in which hypocalcemia cosegregates with several unusual neoplasms led us to examine the transforming effects of this mutant receptor. Transfection of NIH/3T3 cells with the mutant but not the normal receptor supported colony formation in soft agar at subphysiologic calcium concentrations. The mutant CaR causes a calcium-dependent activation of the extracellular signal-regulated protein kinase (ERK 1/2 and Jun-N-terminal kinase/stress-activated (JNK/ SAPK pathways, but not P38 MAP kinase. These findings contribute to a growing body of information suggesting that this receptor plays a role in the regulation of cellular proliferation, that aberrant activation of the mutant receptor in this family may play a role in the unusual neoplastic manifestations.

  7. Salt-soluble proteins from wheat-derived foodstuffs show lower allergenic potency than those from raw flour.

    Science.gov (United States)

    de Gregorio, Marta; Armentia, Alicia; Díaz-Perales, Araceli; Palacín, Arantxa; Dueñas-Laita, Antonio; Martín, Blanca; Salcedo, Gabriel; Sánchez-Monge, Rosa

    2009-04-22

    Salt-soluble proteins from wheat flour have been described as main allergens associated with both baker's asthma and food allergy. However, most studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheat-derived foodstuffs. Salt extracts from different commercial wheat-derived products were obtained and their allergenic properties investigated by IgE-immunodetection, ELISA assays, and skin prick test. The IgE-binding capacity of salt-soluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that of raw flour, the reduction being less dramatic in noncooked pastas and biscuits. Several wheat-derived foodstuffs showed major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulin proteins, respectively. These proteins, as well as most flour and bread salt-soluble proteins, were hydrolyzed when subjected to simulated gastrointestinal digestion. However, the digested products still exhibited a residual IgE-binding capacity. Therefore, processing of wheat flour to obtain derived foodstuffs decreases the IgE binding-capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion further inactivates some heat-resistant IgE-binding proteins.

  8. 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

  9. 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.

  10. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

    Science.gov (United States)

    Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

    2011-01-01

    In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

  11. Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

    Science.gov (United States)

    Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

    2015-01-01

    Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

  12. Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

    Science.gov (United States)

    Lach, Marcel; Künzle, Matthias; Beck, Tobias

    2017-12-11

    The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Elucidating the Mechanism of Gain of Toxic Function From Mutant C1 Inhibitor Proteins in Hereditary Angioedema

    Science.gov (United States)

    2017-10-01

    antibodies to 5 specifically blot wild-type C1INH in the pathologic polymers.. A FLAG tag was placed into the wild-type C1INH cDNA located immediately...resulted in decreased secretion of the 3x-FLAG-WT-C1INH when cotransfected with the mutant cDNA . This was an important confirmation of our...C1INH plus mutant C1INH cDNA in the presence or absence of a lactacystin, a proteasome inhibitor. As shown in figure 2, blocking degradation of

  14. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3 Mutants in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Imilce A Rodriguez-Fernandez

    Full Text Available The Adaptor Protein (AP-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with

  15. The N54-αs Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

    Science.gov (United States)

    Cleator, John H; Wells, Christopher A; Dingus, Jane; Kurtz, David T; Hildebrandt, John D

    2018-05-01

    Ser54 of G s α binds guanine nucleotide and Mg 2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α i/o , this results from persistent binding of α to βγ , whereas for small GTP binding proteins and α s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of G α subunits. Unexpectedly, WT- α s or N54- α s coexpressed with α 1B -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- α s was more effective than the mutant. One explanation for this result would be that α s , like Ser47 α i/o , blocks receptor activation by sequestering βγ ; implying that N54- α S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- α s was more effective than the mutant in inhibiting βγ activation of phospholipase C β 2. Further, in vitro synthesized N54- α s bound biotinylated- βγ with lower apparent affinity than did WT- α s The Cys54 mutation also decreased βγ binding but less effectively than N54- α s Substitution of the conserved Ser in α o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.

    Science.gov (United States)

    Ceci, Pierpaolo; Chiancone, Emilia; Kasyutich, Oksana; Bellapadrona, Giuliano; Castelli, Lisa; Fittipaldi, Maria; Gatteschi, Dante; Innocenti, Claudia; Sangregorio, Claudio

    2010-01-11

    A comparative analysis of the magnetic properties of iron oxide nanoparticles grown in the cavity of the DNA-binding protein from starved cells of the bacterium Listeria innocua, LiDps, and of its triple-mutant lacking the catalytic ferroxidase centre, LiDps-tm, is presented. TEM images and static and dynamic magnetic and electron magnetic resonance (EMR) measurements reveal that, under the applied preparation conditions, namely alkaline pH, high temperature (65 degrees C), exclusion of oxygen, and the presence of hydrogen peroxide, maghemite and/or magnetite nanoparticles with an average diameter of about 3 nm are mineralised inside the cavities of both LiDps and LiDps-tm. The magnetic nanoparticles (MNPs) thus formed show similar magnetic properties, with superparamagnetic behaviour above 4.5 K and a large magnetic anisotropy. Interestingly, in the EMR spectra an absorption at half-field is observed, which can be considered as a manifestation of the quantum behaviour of the MNPs. These results indicate that Dps proteins can be advantageously used for the production of nanomagnets at the interface between molecular clusters and traditional MNPs and that the presence of the ferroxidase centre, though increasing the efficiency of nanoparticle formation, does not affect the nature and fine structure of the MNPs. Importantly, the self-organisation of MNP-containing Dps on HRTEM grids suggests that Dps-enclosed MNPs can be deposited on surfaces in an ordered fashion.

  17. Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC

    DEFF Research Database (Denmark)

    Chaib, Imane; Karachaliou, Niki; Pilotto, Sara

    2017-01-01

    Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) ...

  18. pH-dependent absorption spectra of rhodopsin mutant E113Q: On the role of counterions and protein

    Science.gov (United States)

    Xie, Peng; Zhou, Panwang; Alsaedi, Ahmed; Zhang, Yan

    2017-03-01

    The absorption spectra of bovine rhodopsin mutant E113Q in solutions were investigated at the molecular level by using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. The calculations suggest the mechanism of the absorption variations of E113Q at different pH values. The results indicate that the polarizations of the counterions in the vicinity of Schiff base under protonation and unprotonation states of the mutant E113Q would be a crucial factor to change the energy gap of the retinal to tune the absorption spectra. Glu-181 residue, which is close to the chromophore, cannot serve as the counterion of the protonated Schiff base of E113Q in dark state. Moreover, the results of the absorption maximum in mutant E113Q with the various anions (Cl-, Br-, I- and NO3-) manifested that the mutant E113Q could have the potential for use as a template of anion biosensors at visible wavelength.

  19. Uncoupling in Secondary Transport Proteins. A Mechanistic Explanation for Mutants of lac Permease with an Uncoupled Phenotype

    NARCIS (Netherlands)

    Lolkema, J.S.; Poolman, B.

    1995-01-01

    The kinetic behavior of a H+-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (E.S.H) of the carrier also one of the binary complexes (E.S or E.H) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the

  20. Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

    LENUS (Irish Health Repository)

    Tudor, E L

    2010-05-19

    Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

  1. Evolution of plant cell wall: Arabinogalactan-proteins from three moss genera show structural differences compared to seed plants.

    Science.gov (United States)

    Bartels, Desirée; Baumann, Alexander; Maeder, Malte; Geske, Thomas; Heise, Esther Marie; von Schwartzenberg, Klaus; Classen, Birgit

    2017-05-01

    Arabinogalactan-proteins (AGPs) are important proteoglycans of plant cell walls. They seem to be present in most, if not all seed plants, but their occurrence and structure in bryophytes is widely unknown and actually the focus of AGP research. With regard to evolution of plant cell wall, we isolated AGPs from the three mosses Sphagnum sp., Physcomitrella patens and Polytrichastrum formosum. The moss AGPs show structural characteristics common for AGPs of seed plants, but also unique features, especially 3-O-methyl-rhamnose (trivial name acofriose) as terminal monosaccharide not found in arabinogalactan-proteins of angiosperms and 1,2,3-linked galactose as branching point never found in arabinogalactan-proteins before. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

  3. The Small Heat Shock Protein α-Crystallin B Shows Neuroprotective Properties in a Glaucoma Animal Model

    Directory of Open Access Journals (Sweden)

    Fabian Anders

    2017-11-01

    Full Text Available Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP, followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, β, and γ of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.

  4. Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A; Boudko, Sergei P; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R; Rashmir-Raven, Ann M; Nagata, Kazuhiro; Winand, Nena J; Bächinger, Hans Peter

    2012-06-22

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

  5. Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

    2012-01-01

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

  6. Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

    Science.gov (United States)

    Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

    2015-08-01

    The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

  7. Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

    Science.gov (United States)

    Berman, Andrea J; Gooding, Anne R; Cech, Thomas R

    2010-10-01

    The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

  8. Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

    DEFF Research Database (Denmark)

    Hammer, Karin; Jensen, Kaj Frank; Poulsen, Peter

    1987-01-01

    Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of c......II or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation....... In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing...

  9. Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

    Science.gov (United States)

    Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

    2016-09-12

    Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    Science.gov (United States)

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

  11. Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

    DEFF Research Database (Denmark)

    Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

    2018-01-01

    PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

  12. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    International Nuclear Information System (INIS)

    Kokjohn, T.A.; Miller, R.V.

    1987-01-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants

  13. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    Energy Technology Data Exchange (ETDEWEB)

    Kokjohn, T.A.; Miller, R.V.

    1987-04-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants.

  14. The protein kinase MBK-1 contributes to lifespan extension in daf-2 mutant and germline-deficient Caenorhabditis elegans.

    Science.gov (United States)

    Mack, Hildegard I D; Zhang, Peichuan; Fonslow, Bryan R; Yates, John R

    2017-05-25

    In Caenorhabditis elegans , reduction of insulin/IGF-1 like signaling and loss of germline stem cells both increase lifespan by activating the conserved transcription factor DAF-16 (FOXO). While the mechanisms that regulate DAF-16 nuclear localization in response to insulin/IGF-1 like signaling are well characterized, the molecular pathways that act in parallel to regulate DAF-16 transcriptional activity, and the pathways that couple DAF-16 activity to germline status, are not fully understood at present. Here, we report that inactivation of MBK-1, the C. elegans ortholog of the human FOXO1-kinase DYRK1A substantially shortens the prolonged lifespan of daf-2 and glp-1 mutant animals while decreasing wild-type lifespan to a lesser extent. On the other hand, lifespan-reduction by mutation of the MBK-1-related kinase HPK-1 was not preferential for long-lived mutants. Interestingly, mbk-1 loss still allowed for DAF-16 nuclear accumulation but reduced expression of certain DAF-16 target genes in germline-less, but not in daf-2 mutant animals. These findings indicate that mbk-1 and daf-16 functionally interact in the germline- but not in the daf-2 pathway. Together, our data suggest mbk-1 as a novel regulator of C. elegans longevity upon both, germline ablation and DAF-2 inhibition, and provide evidence for mbk-1 regulating DAF-16 activity in germline-deficient animals.

  15. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  16. Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

    Science.gov (United States)

    Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

    2013-01-01

    To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

  17. Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform.

    Science.gov (United States)

    Sharma, Shiwani; Burdon, Kathryn P; Dave, Alpana; Jamieson, Robyn V; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E

    2008-01-01

    Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for

  18. Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hang [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110122 (China); Hsiao, Yung-Chin [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chiang, Sum-Fu [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Wu, Chia-Chun; Lin, Yu-Tsun [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Liu, Hsuan [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Zhao, Hong [Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122 (China); Chen, Jinn-Shiun [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chang, Yu-Sun [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan (China); and others

    2016-08-24

    The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ({sup 592}IGDFGLATVK{sup 601} and {sup 592}IGDFGLATEK{sup 601}, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies

  19. A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins[W][OA

    Science.gov (United States)

    Yamamoto, Yuko; Hirai, Takaaki; Yamamoto, Eiji; Kawamura, Mayuko; Sato, Tomomi; Kitano, Hidemi; Matsuoka, Makoto; Ueguchi-Tanaka, Miyako

    2010-01-01

    To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1P99S interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1P99A has smaller Ka (association) and Kd (dissociation) values for GA4 than does wild-type GID1. This suggests that the GID1P99A lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1P99A. Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants. PMID:21098733

  20. A rice gid1 suppressor mutant reveals that gibberellin is not always required for interaction between its receptor, GID1, and DELLA proteins.

    Science.gov (United States)

    Yamamoto, Yuko; Hirai, Takaaki; Yamamoto, Eiji; Kawamura, Mayuko; Sato, Tomomi; Kitano, Hidemi; Matsuoka, Makoto; Ueguchi-Tanaka, Miyako

    2010-11-01

    To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants.

  1. The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2010-01-01

    The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

  2. Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

    International Nuclear Information System (INIS)

    Morton, Craig J.; Cameron, Rachel; Lawrence, Lynne J.; Lin Bo; Lowe, Melinda; Luttick, Angela; Mason, Anthony; McKimm-Breschkin, Jenny; Parker, Michael W.; Ryan, Jane; Smout, Michael; Sullivan, Jayne; Tucker, Simon P.; Young, Paul R.

    2003-01-01

    Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors

  3. Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

    Science.gov (United States)

    Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

    2017-07-01

    The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

  4. Role of xanthophylls in light harvesting in green plants: a spectroscopic investigation of mutant LHCII and Lhcb pigment-protein complexes.

    Science.gov (United States)

    Fuciman, Marcel; Enriquez, Miriam M; Polívka, Tomáš; Dall'Osto, Luca; Bassi, Roberto; Frank, Harry A

    2012-03-29

    The spectroscopic properties and energy transfer dynamics of the protein-bound chlorophylls and xanthophylls in monomeric, major LHCII complexes, and minor Lhcb complexes from genetically altered Arabidopsis thaliana plants have been investigated using both steady-state and time-resolved absorption and fluorescence spectroscopic methods. The pigment-protein complexes that were studied contain Chl a, Chl b, and variable amounts of the xanthophylls, zeaxanthin (Z), violaxanthin (V), neoxanthin (N), and lutein (L). The complexes were derived from mutants of plants denoted npq1 (NVL), npq2lut2 (Z), aba4npq1lut2 (V), aba4npq1 (VL), npq1lut2 (NV), and npq2 (LZ). The data reveal specific singlet energy transfer routes and excited state spectra and dynamics that depend on the xanthophyll present in the complex.

  5. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  6. ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

    Science.gov (United States)

    Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

    2017-11-04

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. 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

  8. New sub-family of lysozyme-like proteins shows no catalytic activity: crystallographic and biochemical study of STM3605 protein from Salmonella Typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Michalska, Karolina; Brown, Roslyn N.; Li, Hui; Jedrzejczak, Robert; Niemann, George; Heffron, Fred; Cort, John R.; Adkins, Joshua N.; Babnigg, Gyorgy; Joachimiak, Andrzej

    2013-03-01

    Phage viruses that infect prokaryotes integrate their genome into the host chromosome; thus, microbial genomes typically contain genetic remnants of both recent and ancient phage infections. Often phage genes occur in clusters of atypical G+C content that reflect integration of the foreign DNA. However, some phage genes occur in isolation without other phage gene neighbors, probably resulting from horizontal gene transfer. In these cases, the phage gene product is unlikely to function as a component of a mature phage particle, and instead may have been co-opted by the host for its own benefit. The product of one such gene from Salmonella enterica serovar Typhimurium, STM3605, encodes a protein with modest sequence similarity to phage-like lysozyme (N-acetylmuramidase) but appears to lack essential catalytic residues that are strictly conserved in all lysozymes. Close homologs in other bacteria share this characteristic. The structure of the STM3605 protein was characterized by X-ray crystallography, and functional assays showed that it is a stable, folded protein whose structure closely resembles lysozyme. However, this protein is unlikely to hydrolyze peptidoglycan. Instead, STM3605 is presumed to have evolved an alternative function because it shows some lytic activity and partitions to micelles.

  9. 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)

  10. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.

    Science.gov (United States)

    Chiti, Fabrizio; Calamai, Martino; Taddei, Niccolo; Stefani, Massimo; Ramponi, Giampietro; Dobson, Christopher M

    2002-12-10

    Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.

  11. Phylogenetic Reconstruction Shows Independent Evolutionary Origins of Mitochondrial Transcription Factors from an Ancient Family of RNA Methyltransferase Proteins.

    Science.gov (United States)

    Aj Harris; Goldman, Aaron David

    2018-04-25

    Here, we generate a robust phylogenetic framework for the rRNA adenine N(6)-methyltransferase (RAMTase) protein family that shows a more ancient and complex evolutionary history within the family than previously reported. RAMTases occur universally by descent across the three domains of life, and typical orthologs within the family perform methylation of the small subunits of ribosomal RNA (rRNA). However, within the RAMTase family, two different groups of mitochondrial transcription factors, mtTFB1 and mtTFB2, have evolved in eukaryotes through neofunctionalization. Previous phylogenetic analyses have suggested that mtTFB1 and mtTFB2 comprise sister clades that arose via gene duplication, which occurred sometime following the endosymbiosis event that produced the mitochondrion. Through dense and taxonomically broad sampling of RAMTase family members especially within bacteria, we found that these eukaryotic mitochondrial transcription factors, mtTFB1 and mtTFB2, have independent origins in phylogenetically distant clades such that their divergence most likely predates the last universal common ancestor of life. The clade of mtTFB2s comprises orthologs in Opisthokonts and the clade of mtTFB1s includes orthologs in Amoebozoa and Metazoa. Thus, we clearly demonstrate that the neofunctionalization producing the transcription factor function evolved twice independently within the RAMTase family. These results are consistent with and help to elucidate outcomes from prior experimental studies, which found that some members of mtTFB1 still perform the ancestral rRNA methylation function, and the results have broader implications for understanding the evolution of new protein functions. Our phylogenetic reconstruction is also in agreement with prior studies showing two independent origins of plastid RAMTases in Viridiplantae and other photosynthetic autotrophs. We believe that this updated phylogeny of RAMTases should provide a robust evolutionary framework for ongoing

  12. Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

    Science.gov (United States)

    Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

    1999-01-01

    The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

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

    Science.gov (United States)

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

    2017-09-15

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

  14. Analysis of AtCry1 and Mutants

    Science.gov (United States)

    Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

    Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

  15. Modeling and Docking Studies on Novel Mutants (K71L and T204V of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2014-04-01

    Full Text Available The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO were mutated. 3D mutant models (K71L and T204V using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255, as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.

  16. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

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

  17. Betabaculovirus F proteins showed different efficiencies when rescuing the infectivity of gp64-null Autographa californica nucleopolyhedrovirus

    NARCIS (Netherlands)

    Yin, F.; Wang, M.; Ying, T.; Deng, F.; Vlak, J.M.; Hu, Z.; Wang, H.

    2013-01-01

    The Agrotis segetum granulovirus (AgseGV) F protein was previously identified as the first betabaculovirus F protein with functional homology to Autographa californica nucleopolyhedrovirus (AcMNPV) GP64. In the current study, F proteins from Xestia c-nigrum granulovirus (XecnGV), Cydia pomonella

  18. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    Directory of Open Access Journals (Sweden)

    Xiaolin eWu

    2015-01-01

    Full Text Available ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5, deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs, late embryogenesis abundant (LEA proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

  19. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  20. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    International Nuclear Information System (INIS)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He; Su, Xiao-Dong

    2007-01-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  1. Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: Impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of inoculation.

    Science.gov (United States)

    Lawrence, Paul; Pacheco, Juan; Stenfeldt, Carolina; Arzt, Jonathan; Rai, Devendra K; Rieder, Elizabeth

    2016-05-01

    A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL) inoculation. Interestingly, JMJD6-FMDV pathogenesis was equivalent to A24-WT administered by IEL route. In contrast, JMJD6-FMDV aerosol-infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6(+) cells while A24-WT was occasionally found in JMJD6(+) cells. In vitro, chemical uptake inhibitors demonstrated JMJD6-FMDV entered cells via clathrin-coated pit endocytosis. In vivo, JMJD6-FMDV exhibited preference for JMJD6(+) cells, but availability of this alternative receptor likely depends on route of inoculation. Copyright © 2016. Published by Elsevier Inc.

  2. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

    Science.gov (United States)

    Metts, J; West, J; Doares, S H; Matthysse, A G

    1991-02-01

    Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

  3. Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

    Science.gov (United States)

    Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

    2016-08-08

    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

  4. The Evidence for the Spread and Seeding Capacities of the Mutant Huntingtin Protein in in Vitro Systems and Their Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    Maria Masnata

    2017-11-01

    Full Text Available Neurodegenerative disorders are not only characterized by specific patterns of cell loss but the presence and accumulation of various pathological proteins—both of which correlate with disease evolution. There is now mounting evidence to suggest that these pathological proteins present with toxic, at times prion-like, properties and can therefore seed pathology in neighboring as well remotely connected healthy neurons as they spread across the brain. What is less clear, at this stage, is how much this actually contributes to, and drives, the core pathogenic events. In this review, we present a comprehensive, up-to-date summary of the reported in vitro studies that support the spreading and seeding capacities of pathological proteins, with an emphasis on mutant huntingtin protein in the context of Huntington's disease, although in vivo work remains to be performed to validate this theory in this particular disease. We have further reviewed these findings in light of their potential implications for the development of novel therapeutic approaches.

  5. Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

    KAUST Repository

    LaMonte, Gregory; Orjuela-Sanchez, Pamela; Wang, Lawrence; Li, Shangzhong; Swann, Justine; Cowell, Annie; Zou, Bing Yu; Abdel- Haleem Mohamed, Alyaa; Villa-Galarce, Zaira; Moreno, Marta; Tong-Rios, Carlos; Vinetz, Joseph; Lewis, Nathan; Winzeler, Elizabeth A

    2017-01-01

    The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

  6. Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

    KAUST Repository

    LaMonte, Gregory

    2017-10-03

    The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

  7. Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins.

    Science.gov (United States)

    Malamud, Mariano; Carasi, Paula; Bronsoms, Sílvia; Trejo, Sebastián A; Serradell, María de Los Angeles

    2017-04-01

    The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.

  8. Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

    Science.gov (United States)

    Kishida, Ken; Nagaretani, Hiroyuki; Kondo, Hidehiko; Kobayashi, Hideki; Tanaka, Sachiyo; Maeda, Norikazu; Nagasawa, Azumi; Hibuse, Toshiyuki; Ohashi, Koji; Kumada, Masahiro; Nishizawa, Hitoshi; Okamoto, Yoshihisa; Ouchi, Noriyuki; Maeda, Kazuhisa; Kihara, Shinji; Funahashi, Tohru; Matsuzawa, Yuji

    2003-06-20

    Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

  9. Coat protein deletion mutants elicit more severe symptoms than wild-type virus in multiple cereal hosts

    Science.gov (United States)

    The coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. This study demonstrates that deletion of CP amino acids 58 to 84, but not 36 to 57, from WSMV genome induced severe ...

  10. Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

    Directory of Open Access Journals (Sweden)

    Catarina Moreirinha

    2018-03-01

    Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

  11. 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)

  12. 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)

  13. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

    Directory of Open Access Journals (Sweden)

    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  14. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Holečková, Nela; Goldová, Jana; Doubravová, Linda; Benada, Oldřich; Kofroňová, Olga; Halada, Petr; Branny, Pavel

    2016-01-01

    Roč. 16, OCT 24 (2016), s. 247 ISSN 1471-2180 R&D Projects: GA ČR GAP302/12/0256; GA ČR GAP207/12/1568; GA MŠk LH12055 Institutional support: RVO:61388971 Keywords : Signal transduction * Protein phosphatase * Protein kinase Subject RIV: EE - Microbiology, Virology Impact factor: 2.644, year: 2016

  15. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  16. Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani.

    Science.gov (United States)

    Ghosh, Prithwi; Roy, Amit; Hess, Daniel; Ghosh, Anupama; Das, Sampa

    2015-10-26

    Mutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani. The effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL. Knowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae. Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein. Taken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.

  17. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

    Directory of Open Access Journals (Sweden)

    Kazuhiro eYamamoto

    2014-04-01

    Full Text Available Several missense mutations in the protein kinase Cγ (γPKC gene have been found to cause spinocerebellar ataxia type 14 (SCA14, an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate, a type of phorbol ester. Wild-type (WT γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in

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

    Directory of Open Access Journals (Sweden)

    Nil Guzel

    2017-01-01

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

  19. 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.

  20. Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins

    DEFF Research Database (Denmark)

    Maragakis, Paul; Lindorff-Larsen, Kresten; Eastwood, Michael P

    2008-01-01

    . Molecular dynamics (MD) simulation provides a complementary approach to the study of protein dynamics on similar time scales. Comparisons between NMR spectroscopy and MD simulations can be used to interpret experimental results and to improve the quality of simulation-related force fields and integration......A molecular-level understanding of the function of a protein requires knowledge of both its structural and dynamic properties. NMR spectroscopy allows the measurement of generalized order parameters that provide an atomistic description of picosecond and nanosecond fluctuations in protein structure...... methods. However, apparent systematic discrepancies between order parameters extracted from simulations and experiments are common, particularly for elements of noncanonical secondary structure. In this paper, results from a 1.2 micros explicit solvent MD simulation of the protein ubiquitin are compared...

  1. Study in mice shows that an aggressive type of breast cancer is linked to an inflammatory protein

    Science.gov (United States)

    Aberrant expression of an inflammatory protein, nitric oxide synthase 2 (NOS2), may enhance the progression and metastasis of an aggressive and less common form of breast cancer, known as the estrogen receptor-negative type of disease.

  2. Evaluation of tall rice mutant

    International Nuclear Information System (INIS)

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

    1989-01-01

    One tall mutant (Mut NS1) of rice variety Nizersail was put to multilocation on-farm trial. It showed improvement over the parent in respect of by earlier maturity and higher grain yield at all locations and thus it appears as an improved mutant of Nizersail. (author). 6 refs

  3. Promising semi-dwarf mutant in wheat variety K68

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, D [Banaras Hindu Univ. (India). Dept. of Genetics and Plant Breeding

    1977-04-01

    A semi-dwarf mutant (HUW-SDf 1) was induced from common wheat Var. K68 through the exposure of /sup 60/Co ..gamma..-rays at 15 kR. This mutant along with other induced mutants and control was assessed for yield components, yield and grain quality (M/sub 4/ generation); internode length reduction pattern and the yielding ability at three levels of nitrogen (M/sub 5/ generation). The mutant was significantly shorter in height and almost equal in tillers per plant and grains per spike to K68. However, it showed marked reduction in spike length and spikelets per spike. On the other hand, it possessed significantly higher (50.04 g) 1000-grain weight against control (41.15 g). The mutant gave 56.0% higher yield than the control. Grain quality studies indicated that the mutant possessed significantly higher (14.15%) total protein than K68. It was equally as good as K68 in lysine content. Pelshenke value (62.5 min) of the mutant indicated medium hard nature of gluten as compared to hard nature (198.0) of the control. The mutant showed 24.0% reduction in total culm length compared to K68. Reduction occurred due to maximum and almost equal reduction in 5th and 4th internodes (ca 34.0%) followed by 3rd, 2nd and 1st. The mutant showed similar yield and yield response to increasing nitrogen levels (80 to 160 kg per ha.) as for current commercial semi-dwarf varieties.

  4. Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

    Science.gov (United States)

    Fernández-García, Aurora; Alvarez-García, Gema; Marugán-Hernández, Virginia; García-Lunar, Paula; Aguado-Martínez, Adriana; Risco-Castillo, Verónica; Ortega-Mora, Luis M

    2013-07-01

    Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, Presult, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

  5. Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss.

    Science.gov (United States)

    Scekic-Zahirovic, Jelena; Sendscheid, Oliver; El Oussini, Hajer; Jambeau, Mélanie; Sun, Ying; Mersmann, Sina; Wagner, Marina; Dieterlé, Stéphane; Sinniger, Jérome; Dirrig-Grosch, Sylvie; Drenner, Kevin; Birling, Marie-Christine; Qiu, Jinsong; Zhou, Yu; Li, Hairi; Fu, Xiang-Dong; Rouaux, Caroline; Shelkovnikova, Tatyana; Witting, Anke; Ludolph, Albert C; Kiefer, Friedemann; Storkebaum, Erik; Lagier-Tourenne, Clotilde; Dupuis, Luc

    2016-05-17

    FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  6. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    Science.gov (United States)

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein AVR4

    NARCIS (Netherlands)

    Hoorn, Van der R.A.L.; Roth, R.; Wit, De P.J.G.M.

    2001-01-01

    The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9,

  8. Peroxisomal matrix protein import - Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor pex5p

    NARCIS (Netherlands)

    Kiel, JAKW; Veenhuis, M

    2000-01-01

    In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that

  9. Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

    Directory of Open Access Journals (Sweden)

    Amy eCurran

    2011-08-01

    Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

  10. Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

    DEFF Research Database (Denmark)

    Jensen, L T; Ladefoged, S; Birkelund, S

    1995-01-01

    characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells...

  11. Fitness and virulence of a coxsackievirus mutant that can circumnavigate the need for phosphatidylinositol 4-kinase class III beta

    NARCIS (Netherlands)

    Thibaut, Hendrik Jan; van der Schaar, Hilde M; Lanke, Kjerstin H W; Verbeken, Erik; Andrews, Martin; Leyssen, Pieter; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Coxsackieviruses require phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as

  12. Reducing dietary protein in pond production of hybrid striped bass - study shows a significant reduction is possible in digestible protein level in commercial diets

    Science.gov (United States)

    In previous work, we demonstrated that diets containing 40% digestible protein (DP) (45% crude protein) and 18 %lipid supplemented with Met and Lys resulted in superior performance and nutrient retentions in hybrid striped bass compared to less energy-dense diets when rearing hybrid striped bass at ...

  13. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

    Directory of Open Access Journals (Sweden)

    Thomas L Prince

    Full Text Available The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

  14. Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Noritake, Hidenao [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kimura, Wataru; Wu, Yi-Xin [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kobayashi, Yoshimasa [Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Uezato, Tadayoshi [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Miura, Naoyuki, E-mail: nmiura@hama-med.ac.jp [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

  15. Improvement of seed protein in rice through mutation breeding

    International Nuclear Information System (INIS)

    Monyo, J.H.; Sugiyama, T.

    1978-01-01

    Mutants selected from an M 4 generation on the basis of high yield potential and high grain protein content were grown in a preliminary yield trial. The two parent varieties, Faya Theresa and Kihogo Red, were grown as controls. Thirteen mutants originating from Faya Theresa and three mutants derived from Kihogo Red were found to be equal or superior in yield to the controls. The best improvement in protein content through mutagen treatment was 44% increase in a Faya Theresa mutant and 35% increase in a Kihogo Red mutant. The protein values obtained for the same mutants in the M 3 , M 4 and M 5 generations were found to be consistently higher than in the check varieties. Correlations between grain yield and yield attributes including seed protein per cent and protein per grain showed that these were mutagen-dependent. Grain yield showed negative correlations with protein per cent and protein per grain. However, a few mutants were found which combined high grain yield and high protein per cent. The correlation between protein per cent and protein per grain was positive and very highly significant. It was concluded that despite the high negative correlations between grain protein per cent and grain yield screening for high protein per cent in high-yielding mutants provides a great scope for the identification of the correlation breakers which combine high grain yield potential and high protein content. (author)

  16. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  17. Variation of several agronomic and biochemical traits in γ-ray induced mutant soybeans

    International Nuclear Information System (INIS)

    Shim, Kyo Moon; Kim, Sun Hyung; Kim, Nam Soo; Son, Hi Sup; Rhee, Hae Ik

    1996-01-01

    Two soybean cultivars(Paldalkong and Bangsakong) were irradiated with gamma-ray to reduce seed size, which is an important trait for soybean sprout and the derived mutant soybeans were analyzed in several agronomic and biochemical traits. There were high levels of variations in quantitative traits among the mutants. Several mutant lines showed higher yield and smaller seed than their parents. Qualitative traits such as seed coat color or pubescent color were also changed in a few lines. Biochemical variations were also observed among the mutants. In seed storage protein analysis, many mutant lines showed reduced intensities in β-subunits in 7S globulin than their parents and an additional band in the acidic subunit at 31KDa. Two mutant lines derived from Paldalkong showed an additional band and a shifted band of protease inhibitor by electrophoretic analysis. Variations in isozymes and RAPD were also observed among the mutants. Six isozymes(Adh, Est, Gdh, Idh, Mdh and Pgm) among eleven isozymes showed some variations and six out of ten primers showed polymorphic amplified DNA fragments among the mutants. (author)

  18. Degradation of p53 by human Alphapapillomavirus E6 proteins shows a stronger correlation with phylogeny than oncogenicity.

    Directory of Open Access Journals (Sweden)

    Leiping Fu

    2010-09-01

    Full Text Available Human Papillomavirus (HPV E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types to include members of all known HPV species groups within the Alphapapillomavirus genus.We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71 were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.These data suggest that the alpha-HPV E6 proteins' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.

  19. Dissection of β-barrel Outer Membrane Protein Assembly Pathways through Characterizing BamA POTRA 1 Mutants of Escherichia coli

    Science.gov (United States)

    Bennion, Drew; Charlson, Emily S.; Coon, Eric; Misra, Rajeev

    2010-01-01

    Summary BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains, respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalyzed by the trans-membrane β-barrel domain of Bam A. PMID:20598079

  20. Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice.

    Science.gov (United States)

    Yamanegi, Koji; Yamada, Naoko; Nakasho, Keiji; Nishiura, Hiroshi

    2018-01-01

    We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71 high /TER119 low ) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71 high /TER119 high ) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen. Copyright © 2017 Elsevier GmbH. All rights reserved.

  1. Plasma proteomics shows an elevation of the anti-inflammatory protein APOA-IV in chronic equine laminitis

    Directory of Open Access Journals (Sweden)

    Steelman Samantha M

    2012-09-01

    Full Text Available Abstract Background Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed “founder”. Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated this broad hypothesis by using DIGE to assess global differences in the plasma proteome between horses with chronic laminitis and controls. Results We identified 16 differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. Clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin (PT clotting time, although most other indices were within normal ranges. Upregulation of the intestinal apolipoprotein APOA-IV in horses with chronic laminitis was confirmed by western blot. Conclusions Our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. Gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis.

  2. Mutants of alfalfa mosaic virus

    International Nuclear Information System (INIS)

    Roosien, J.

    1983-01-01

    In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

  3. Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    A monomeric mutant of Azami-Green from G. fascicularis was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal belonged to space group P1 and diffracted X-rays to 2.20 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 Å, α = 90.96, β = 103.41, γ = 101.79°. The Matthews coefficient (V M = 2.10 Å 3 Da −1 ) indicated that the crystal contained two mAG molecules per asymmetric unit

  4. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O

    1998-01-01

    signals for transport into the endoplasmic reticulum. Surprisingly, Ic is encoded by TFS1, which has previously been isolated as a high-copy suppressor of cdc25-1. CDC25 encodes the putative GTP exchange factor for Ras1p/Ras2p in yeast. In an attempt to rationalize this finding, we looked...... degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  5. Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

    Science.gov (United States)

    Suzuki, Keisuke; Mizushima, Hiroto; Abe, Hiroyuki; Iwamoto, Ryo; Nakamura, Haruki; Mekada, Eisuke

    2015-05-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

    Directory of Open Access Journals (Sweden)

    Ram H Nagaraj

    Full Text Available Methylglyoxal (MGO is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12 is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM, R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

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

    Science.gov (United States)

    Johnson, E M; Sears, B B

    1990-06-01

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

  8. Dwarf mutant of rice variety Seratus Malam

    International Nuclear Information System (INIS)

    Mugiono, P. S.; Soemanggono, A.M.R.

    1989-01-01

    Full text: Seeds of 'Seratus Malam', a local tall upland variety with long panicles and high yield potential were irradiated with 10-50 krad gamma rays in 1983. From 50,000 M 2 plants, 130 semidwarf mutants and 1 dwarf mutant were selected. The dwarf mutant M-362 was obtained from the 10 krad treatment. The mutant shows about 50% reduction in plant height, but also in number of productive tillers. Thus the yield per plant is also significantly less. However, the mutant gene is not allelic to DGWG and therefore may be useful in cross breeding. (author)

  9. The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

    2017-12-01

    To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

  10. A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus and snowdrop lectin shows oral toxicity to target insects

    Directory of Open Access Journals (Sweden)

    Fitches Elaine

    2006-03-01

    Full Text Available Abstract Background Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake. Results A gene encoding a toxin, ButaIT, from the red scorpion (Mesobuthus tamulus was synthesised and assembled into expression constructs. One construct contained ButaIT alone, whereas the other contained ButaIT fused N-terminally to a GNA polypeptide (ButaIT/GNA. Both recombinant proteins were produced using the yeast Pichia pastoris as an expression host, and purified. Recombinant ButaIT and ButaIT/GNA were acutely toxic when injected into larvae of tomato moth (Lacanobia oleracea, causing slow paralysis, leading to mortality or decreased growth. ButaIT/GNA was chronically toxic when fed to L. oleracea larvae, causing decreased survival and weight gain under conditions where GNA alone was effectively non-toxic. Intact ButaIT/GNA was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. Proteolysis of the fusion protein was also observed. ButaIT/GNA was significantly more toxic that GNA alone when fed to the homopteran Nilaparvata lugens (rice brown planthopper in liquid artificial diet. Conclusion The ButaIT/GNA recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of GNA as a carrier to transport potentially toxic peptides and proteins across the insect gut. Although ButaIT has been claimed to be lepidopteran

  11. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

    Science.gov (United States)

    Ship, Noam J.; Zamble, Deborah B.

    2005-01-01

    The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

  12. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  15. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

    Directory of Open Access Journals (Sweden)

    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  16. The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defence and Scavenging Oxidative Stress Secreted Proteins

    Directory of Open Access Journals (Sweden)

    Guillermo Nogueira-Lopez

    2018-04-01

    Full Text Available In Nature, almost every plant is colonized by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an opportunistic plant endophyte. Even though many plants are colonized by this symbiont, the exact mechanisms by which Trichoderma masks its entrance into its plant host remain unknown, but likely involve the secretion of different families of proteins into the apoplast that may play crucial roles in the suppression of plant immune responses. In this study, we investigated T. virens colonization of maize roots under hydroponic conditions, evidencing inter- and intracellular colonization by the fungus and modifications in root morphology and coloration. Moreover, we show that upon host penetration, T. virens secretes into the apoplast an arsenal of proteins to facilitate inter- and intracellular colonization of maize root tissues. Using a gel-free shotgun proteomics approach, 95 and 43 secretory proteins were identified from maize and T. virens, respectively. A reduction in the maize secretome (36% was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. Furthermore, T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector-like proteins. Levels of peroxidase activity were reduced in the inoculated roots, suggesting a strategy used by T. virens to manipulate host immune responses. The results provide an insight into the crosstalk in the apoplast which is essential to maintain the T. virens-plant interaction.

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

    Science.gov (United States)

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

    2003-03-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

  19. A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

    Science.gov (United States)

    Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

    2013-01-01

    Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

  20. Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Taddei Kevin

    2010-01-01

    Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

  1. Photosynthetic Characteristics of Flag Leaves in Rice White Stripe Mutant 6001 During Senescence Process

    Directory of Open Access Journals (Sweden)

    Xiao-hui ZHEN

    2014-11-01

    Full Text Available Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate (Pn in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate of Pn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2+-ATPase, Mg2+-ATPase and photophosphorylation activities exhibited the similar trends as the Pn. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.

  2. Selection of high hectolitre weight mutants of winter wheat

    International Nuclear Information System (INIS)

    Crowley, C.; Jones, P.

    1989-01-01

    Grain quality in wheat includes hectolitre weight (HLW) besides protein content and thousand-grain weight (TGW). The British winter wheat variety ''Guardian'' has a very high yield potential. Although the long grain of ''Guardian'' results in a desirable high TGW the HLW is too low. To select mutants exhibiting increased HLW the character was first analyzed to identify traits that could more easily be screened for using M 2 seeds. In comparison of 6 wheat cultivars, correlation analyses with HLW resulted in coefficients of -0.86 (grain length, L:P 2 seeds for shorter, less prolate grains. Mutagenesis was carried out using EMS sulphonate (1.8 or 3.6%), sodium azide (2 or 20 mM) or X-rays (7.5 or 20 kR). 69 M 2 grains with altered shape were selected. Examination of the M 3 progeny confirmed 6 grain-shape mutants, most of them resulting from EMS treatment (Table). Two of the mutants showed TGW values significantly below the parental variety, but three mutants exhibited HLW and TGW values significantly greater than those of the parental variety. Microplot yield trails on selected M 3 lines are in progress. The influence of physical grain characteristics on HLW offers prospects for mechanical fractionation of large M 2 populations. The application of gravity separators (fractionation on the basis of grain density) and sieves (fractionation on the basis of grain length) in screening mutants possessing improved grain quality is being investigated

  3. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

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

    Science.gov (United States)

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

    2016-11-01

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

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

    Science.gov (United States)

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

    2012-04-01

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

  6. Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

    Science.gov (United States)

    Rezelj, Veronica V; Överby, Anna K; Elliott, Richard M

    2015-05-01

    Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a

  7. Biochemical characterization and cellular effects of CADASIL mutants of NOTCH3.

    Directory of Open Access Journals (Sweden)

    He Meng

    Full Text Available Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.

  8. Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Eriksen, Kathrine Krageskov; Hauser, Frank; Schiøtt, Morten

    2000-01-01

    After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino aci...... knock-out mutants, where the DLGR-2 gene is interrupted by a P element insertion, die around the time of hatching. This finding, together with the expression data, strongly suggests that DLGR-2 is exclusively involved in development....

  9. Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

    Science.gov (United States)

    Ghanavatian, Parisa; Khalifeh, Khosrow; Jafarian, Vahab

    2016-12-01

    Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala 19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    Science.gov (United States)

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-07-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

  11. Reduced anti-oxidative stress activities of DJ-1 mutants found in Parkinson's disease patients

    International Nuclear Information System (INIS)

    Takahashi-Niki, Kazuko; Niki, Takeshi; Taira, Takahiro; Iguchi-Ariga, Sanae M.M.; Ariga, Hiroyoshi

    2004-01-01

    DJ-1 is a multi-functional protein that plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in onset of Parkinson's disease. We have previously reported that L166P, a mutant DJ-1 found in Parkinson's disease patients, had no activity to prevent hydrogen peroxide (H 2 O 2 )-induced cell death. In this study, we analyzed other mutants of DJ-1 found in Parkinson's disease patients, including M26I, R98Q, and D149A, as well as L166P. We first found that all of the mutants made heterodimers with wild-type DJ-1, while all of the mutants except for L166P made homodimers. We then found that M26I and L166P, both of which are derived from homozygous mutations of the DJ-1 gene, were unstable and that their stabilities were recovered, in part, in the presence of proteasome inhibitor, MG132. NIH3T3 cell lines stably expressing these mutants of DJ-1 showed that cell lines of L166P and C106S, a mutant for protease activity (-) of DJ-1, had no activity to scavenge even endogenously producing reactive oxygen species. These cell lines also showed that all of the mutants had reduced activities to eliminate exogenously added H 2 O 2 and that these activities, except for that of D149A, were parallel to those preventing H 2 O 2 -induced cell death

  12. physical, chemical, technological and biological properties of some mutant oil seeds induced by gamma radiation

    International Nuclear Information System (INIS)

    Ali, H.G.M.

    2003-01-01

    The present study has been undertaken to evaluated sesame, sunflower and safflower seeds induced by gamma rays, as plant breeding unit, plant research department, radioisotope application division, nuclear research center, atomic energy authority Inshas. the obtained results indicate the following : chemical composition of mutant seeds: the radiation mutation caused a significant increase in both oil and ash content total carbohydrates showed a significant decreased in sesame seeds. radiation mutation induced significant increase in oil and protein content of sunflower and safflower seeds. while the total carbohydrate showed a significant decrease. physiochemical properties of oils extracted mutant seeds: the radiation mutation had no real effect on the refractive index and A.V of oils extracted from control and mutant sesame, sunflower and safflower seeds. while it caused a slight increase in red color and P.V. of sesame oil, the thiobarbituric acid (TBA) value of mutant sesame oil was not alter upon radiation mutation, but it induced a slight decrease in TBA of mutant sunflower and safflower oils. the unsaponifiable matter percentage of oils extracted from mutant sesame, sunflower and safflower seeds were slightly increased by radiation mutation .radiation mutation of seeds had no real effect on the total SFA and USFA of sesame oil. however, radiation mutation induced a remarkable changes in fatty acid profiles of sunflower and safflower oil as total SFA decreased, while USFA increased. Uric acid was only detected in oil extracted from mutant sunflower seeds

  13. High lysine and high yielding mutants in wheat (Triticum aestivum) L

    International Nuclear Information System (INIS)

    Mohammad, T.; Mahmood, F.; Ahmad, A.; Sattar, A.; Khan, I.

    1988-01-01

    The dry seeds of the variety Lu-26 were irradiated with 20 krad of gamma rays. In M 2 about 300 mutant plants were selected for short stature, rust resistance and other desirable traits. As a result of further selection, in M 6 , eight superior lines were finally identified. The agronomic characteristics of these mutants, the parent variety (Lu-26) and a standard check variety (Pak-81) are shown. The selected mutants and commercially grown cultivars (Lu-26 and Pak-81) were studied for total protein content and amino acid pattern. The mutants WM-89-1, WM-6-17 and WM-81-2 showing high yield also contained comparatively high amounts of methionine and lysine. The lysine contents were 565, 410, and 370 mg/100g in the mutants WM-89-1, WM-6-17 and WM-81-2, respectively against a range value of 210-370 mg/100g in other mutants and 250-320 in the commercial cultivars. The mutant WM-81-2 was comparable to WM-56-1-5 in lysine content. The results of these experiments show a possibility of developing varieties having high yield and high amounts of essential amino acids such as lysine and methionine

  14. Studies on reduced height mutants in rice

    International Nuclear Information System (INIS)

    Narahari, P.; Bhagwat, S.G.

    1984-01-01

    Two cross-bred derivatives of the mutant TR5xTR17 and TR21 continued to show promise and were advanced to wider scale testing. TR5 was found to carry a semi-dwarfing gene different from that in IR8. New semi-dwarf mutants were screened from M 2 through M 4 from two separate radiation experiments. The gibberellin response of seedlings of mutant and tester strains was evaluated and crosses of tester stocks and mutant semi-dwarfs were made for genetic analyses. (author)

  15. 'Let the phage do the work': Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

    International Nuclear Information System (INIS)

    Teschke, Carolyn M.; Parent, Kristin N.

    2010-01-01

    The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine. DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

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

    Science.gov (United States)

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

    2011-12-01

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

  17. Mutation induction of protein variability in wheat and rice

    International Nuclear Information System (INIS)

    Narahari, P.; Bhatia, C.R.; Gopalakrishna, T.; Mitra, R.K.

    1976-01-01

    No high protein mutants of wheat have been obtained without depression of grain yield after screening a few thousand lines. The best wheat mutant identified in our programme so far is an erectoid mutant that has consistently shown about 1.5-2% points increase in protein over Kalyan sona for the last four years. Grain yield of the mutant is about 89% of the parent. No significant variation in amino composition is noted in the mutant. Preliminary analysis of over 200 macro mutants in three varieties of rice has resulted in identification of mutants with high protein content (10-22%) compared with 8.0 to 8.5% in the high yielding controls. The amino-acid composition of some of the mutant kernels do not show great deviation from the controls. All the high protein percentage mutants are lower in grain yield. Despite very high F 1 sterility in a cross involving the high protein genotype GMPR-51 and high yielding IR-8, several fertile F 2 plants resembling IR-8 have been isolated which on preliminary analysis have shown still higher protein content than GMPR-51, suggesting a transgressive mode of inheritance of this trait. (author)

  18. Evolved osmotolerant Escherichia coli mutants frequently exhibit defective N-acetylglucosamine catabolism and point mutations in cell shape-regulating protein MreB.

    Science.gov (United States)

    Winkler, James D; Garcia, Carlos; Olson, Michelle; Callaway, Emily; Kao, Katy C

    2014-06-01

    Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    Science.gov (United States)

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

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

    International Nuclear Information System (INIS)

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

    2005-01-01

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

  1. Analysis of nutritional quality of low phytic acid mutants in maize

    International Nuclear Information System (INIS)

    Yuan Ming'an; Luo Hongbing; Wang Zhonghua; Chen Jinhong; Mei Shufang; Shu Xiaoli; Wu Dianxing

    2008-01-01

    Major nutritional quality and components of eight low phytic acid (lpa) mutants and their corresponding wild types were studied. Compared to their corresponding wild types, the content of inorganic P (Pi) was all increased several times, while the content of total P (TP) in mutants was almost the same as their wild types. The contents of crude lipid and amylose were similar, but total starch was significantly different. Crude protein in some of mutants was increased significantly. Most of amino acids were increased, and essential amino acid-Lysine was increased except mutants derived from Q319 and X178. Mineral macronutrients and micronutrients were similar. All results showed that the lpa mutation in maize could enhance the nutritional quality and bioactivities. (authors)

  2. 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

  3. 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

  4. Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

    Directory of Open Access Journals (Sweden)

    Angèle Nalbandian

    Full Text Available Mutations in the valosin containing protein (VCP gene cause hereditary Inclusion body myopathy (hIBM associated with Paget disease of bone (PDB, frontotemporal dementia (FTD, more recently termed multisystem proteinopathy (MSP. Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem

  5. 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

  6. Biological changes in Barley mutants resistant to powdery mildew disease

    International Nuclear Information System (INIS)

    Amer, I. M.; Fahim, M. M.; Moustafa, N. A.

    2012-12-01

    physiological studies showed that all kinds of chlorophyll (a), (b) and (a + b) content in infected plant were decreased while, the carotenes pigment were increased. Infection generally reduced total sugars content of all resistant mutants. Infected resistant mutant showed more phenols content and peroxidase, polyphenoloxidase activities than healthy ones of the mutants. (Author)

  7. Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant.

    Science.gov (United States)

    Bruijn, L I; Beal, M F; Becher, M W; Schulz, J B; Wong, P C; Price, D L; Cleveland, D W

    1997-07-08

    Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515-518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

  8. Assessment of Lupin Induced Mutants for Quality Traits and Susceptibility to Callosbruchus chinensis and Heliothis armigera insects

    International Nuclear Information System (INIS)

    Ragab, A.I.; Boshra, S.A.; Mehany, A.L.; Darwish, A.A.; Kharrab, M.M.

    2008-01-01

    This study was conducted to assess 23 induced mutants and two parental varieties Giza1 and Giza2 in the three generations (M3, M4, and M5) for seed quality traits, and susceptibility to two insects i.e C. chinnensis and H.armigera. The obtained results exhibited highly significant decrease for alkaloid content of mutants 20 and 23 as compared with the two local varieties. Most of mutants and Giza 2 showed marked increase for protein content as compared with Giza1, however, the increase did not reach the level of significance for the most mutants as compared with Giza2 in the three generations. Except of M4 generation. marked resistance for infestations with C.chinensis and H.armigera was obtained for mutants 1, 5 and 11 in the three generations. However, for total infestation with the two insects, resistance was obtained in mutants 4 and 10. Except of mutant lines 1, 5 and 11, all mutants showed higher loss percentage than that of the local varieties

  9. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  10. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

    Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

  11. Evaluation of Chemical Changes in Some Soybean Mutants Induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Abd-Elkalik, K.; Mekkawy, S.H.; El-Demerdash, H.M.

    2010-01-01

    The Egyptian soybean cultivar Giza-22 was used to induce genetic variability by using gamma rays at dose levels of 100, 150 and 200 Gy. Sixteen mutants (including parental cultivar) were evaluated in M3 generation for their agronomic traits and chemical analysis was done in seeds of M3 generation. Four mutants A21 (150 Gy), A22, A23 and A24 (200 Gy) showed superiority in their agronomic traits compared with parental cultivar. The results of chemical analysis of seeds of M3 generation showed that, oil and energy contents were unaffected by irradiation treatments while protein contents were significantly increased at doses 150 and 200 Gy. Phenolic and tannin contents in seeds of M3 generation showed no significant changes in their percentages due to irradiation treatments. Gamma irradiation significantly increased in linoleic acid content in most of the mutants compared with the control (Giza-22), whereas, there were decreases in linolenic acid content. Investigation of amino acid composition of mutants of M3 generation revealed significant increases in the essential amino acids in most mutants induced by gamma irradiation at 150 and 200 Gy. It could be suggested that the use of gamma irradiation can induce an improvement of the oil and protein composition in soybean

  12. Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation

    International Nuclear Information System (INIS)

    Verbenko, V.N.; Akhmedov, A.T.; Kalinin, V.L.

    1986-01-01

    By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gam 444 ad Gam 445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 deg C and are induced more intensively during heat shock (in comparison to the parental) wild-tupe strain AB parallel 57). When the missense htp R15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into genome of the Gam 444 mutant, its enhanced radiation-resistance disappeared but could not be restored upon introduction of pKV3 plasmid bearing the htpR, gene. These data show that heat shock Protens are participating in the enhanced radioresistance of Gam mutants

  13. Overexpression of a Protein Phosphatase 2C from Beech Seeds in Arabidopsis Shows Phenotypes Related to Abscisic Acid Responses and Gibberellin Biosynthesis1

    Science.gov (United States)

    Reyes, David; Rodríguez, Dolores; González-García, Mary Paz; Lorenzo, Oscar; Nicolás, Gregorio; García-Martínez, José Luis; Nicolás, Carlos

    2006-01-01

    A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis. PMID:16815952

  14. The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

    Science.gov (United States)

    1996-08-01

    that my statement of work (SOW) for the current project omitted many of the tasks that had to be carried out in order to get the lab up and running...that we knew that we could stabilize wild-type p53 in ML-1 cells along with the possibility of being able to get an excellent elutriation profile with...nuclear protein extract was immunoprecipitated with PAb421 cross-linked to ProteinA -Sepharose beads and analysed by SDS-PAGE Western blot analysis with

  15. Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus.

    Science.gov (United States)

    Vigeolas, Hélène; Duby, Francéline; Kaymak, Esra; Niessen, Guillaume; Motte, Patrick; Franck, Fabrice; Remacle, Claire

    2012-11-30

    This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  17. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants

    NARCIS (Netherlands)

    Felipe Farias, Davi; Peijnenburg, A.A.C.M.; Grossi-de-Sá, Maria F.; Carvalho, A.F.U.

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular

  18. Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

    International Nuclear Information System (INIS)

    Pirsel, M.; Slezarikova, V.

    1977-01-01

    A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

  19. Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein

    Czech Academy of Sciences Publication Activity Database

    Doležal, Michal; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2015-01-01

    Roč. 9, č. 2 (2015), s. 229-233 ISSN 1874-2718 Institutional support: RVO:61388963 Keywords : isotopic labeling * matrix protein * M-PMV * myristoylation * resonance assignment * reverse labeling Subject RIV: CE - Biochemistry Impact factor: 0.687, year: 2015

  20. Assignment of 1H, 13C, and 15N resonances of WT matrix protein and its R55F mutant from Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Vlach, J.; Lipov, J.; Veverka, V.; Rumlová, Michaela; Ruml, T.; Hrabal, R.

    2005-01-01

    Roč. 31, - (2005), s. 381-382 ISSN 0925-2738 R&D Projects: GA ČR GA203/03/0490 Institutional research plan: CEZ:AV0Z4055905 Keywords : Mason-Pfizer monkey virus * NMR resonance assignment * matrix protein Subject RIV: CE - Biochemistry Impact factor: 2.180, year: 2005

  1. 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

  2. Novel G Protein-Coupled Oestrogen Receptor GPR30 Shows Changes in mRNA Expression in the Rat Brain over the Oestrous Cycle

    Directory of Open Access Journals (Sweden)

    Emma J. Spary

    2012-02-01

    Full Text Available Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and β in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS, ventrolateral medulla (VLM and periaqueductal gray (PAG GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

  3. The application of shortened upper leaf mutant in barley breeding

    International Nuclear Information System (INIS)

    Jin Hua

    2004-01-01

    The shortened upper leaf mutant was induced from Fuji Nigo by γ-ray irradiation. Fuji Nigo, the mutant, cross-cut F 1 , F 2 and back-cross F 1 , F 2 were used to analyze mutant heredity by comparative study. The yield, chlorophyll content, light intensity, dry matter of mutant were investigated. The results showed that (1) the mutant character was controlled by a couple of nuclear genes which were partial dominance; (2) the transmittance of the mutant colony was better than that of Fuji Nigo and bottom dry matter was much more than that of Fuji Nigo; (3) under the condition of high fertilizer and high plant population , the yield of mutant was higher than that of Fuji Nigo; (4) the content of chlorophyll a in the mutant was higher than that in Fuji Nigo

  4. Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

    Science.gov (United States)

    Guan, Su Hua; Belsham, Graham J

    2017-04-01

    Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

  5. Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform

    OpenAIRE

    Sharma, Shiwani; Burdon, Kathryn P.; Dave, Alpana; Jamieson, Robyn V.; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E.

    2008-01-01

    Purpose Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junction...

  6. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS

    OpenAIRE

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-01-01

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/M...

  7. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    Directory of Open Access Journals (Sweden)

    Liu Xiangtao

    2011-02-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE, and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA and indirect sandwich-ELISA (IS-ELISA showed both recombinant baculoviruses (with or without T-cell epitopes were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ. Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P P Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection.

  8. Emery-Dreifuss Muscular Dystrophy-Associated Mutant Forms of Lamin A Recruit the Stress Responsive Protein Ankrd2 into the Nucleus, Affecting the Cellular Response to Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Silvia Angori

    2017-05-01

    Full Text Available Background: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2 is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. Methods: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS release and cell viability. Results: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. Conclusions: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.

  9. Semi-dwarf mutants for rice improvement

    International Nuclear Information System (INIS)

    Othman, Ramli; Osman, Mohammad; Ibrahim, Rusli

    1990-01-01

    Full text: MARDI and the National University of Malaysia embarked on a programme to induce resistance against blast in rice in 1978. MARDI also obtained semi dwarf mutants of cvs 'Mahsuri', 'Muda', 'Pongsu seribu' and 'Jarum Mas', which are under evaluation. The popular local rice variety 'Manik' was subjected to gamma irradiation (15-40 krad) and 101 promising semidwarf mutants have been obtained following selection in M 2 -M 6 . 29 of them show grain yields of 6.0-7.3 t/ha, compared with 5.7t for 'Manik'. Other valuable mutants were found showing long grain, less shattering, earlier maturity, and glutinous endosperm. One mutant, resistant to brown plant hopper yields 6.3t/ha. (author)

  10. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    Science.gov (United States)

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  11. Induction of Mutants in Durum Wheat

    International Nuclear Information System (INIS)

    AL-Ubaidi, M.; Ibrahim, I.; AL-Hadithi, A.

    2002-01-01

    This investigation presents a breeding program for induction and development of a new genotype of durum wheat, resistant to lodging with high yield, by irradiation durum wheat hybrids (F2) with gamma rays 100 Gy, during 1990-1997 cultivation seasons. This program involves: induction of variability, selection evaluation of the mutants at three locations: Twaitha (Baghdad) Latifya ( Babylon) and Swari (Kutt). All mutants showed resistance to lodging and there was a significant reduction in plant height. Mutant SIXIZ-22 surpassed other mutants and its origin in lodging resistance and plant height (83.5,82.8 and 89.4 cm) in the three locations at generation M5 and M6, respectively. Also, there were significant differences between mutant and their origin in the number of spikes/M 2 and grain yild during the two successive generation. On the other hand, mutant IZxCO-105 surpassed other mutants in the number of spikes/M 2 (231.8,242.3 and 292) and grain yield (4336,3376 and 5232 kg/ha) in all testing location, respectively . (authors) 14 refs., 4 tabs

  12. 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

  13. The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

    DEFF Research Database (Denmark)

    Escobar-Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela

    2017-01-01

    radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein...... consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric...

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  15. 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

  16. The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions.

    Science.gov (United States)

    Bartho, Joseph D; Bellini, Dom; Wuerges, Jochen; Demitri, Nicola; Toccafondi, Mirco; Schmitt, Armin O; Zhao, Youfu; Walsh, Martin A; Benini, Stefano

    2017-01-01

    AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    NARCIS (Netherlands)

    Yang, X.; Kraan, P.M. van der; Dolder, J. van den; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2007-01-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and

  19. An analysis of partial efficiencies of energy utilisation of different macronutrients by barramundi (Lates calcarifer) shows that starch restricts protein utilisation in carnivorous fish.

    Science.gov (United States)

    Glencross, Brett D; Blyth, David; Bourne, Nicholas; Cheers, Susan; Irvin, Simon; Wade, Nicholas M

    2017-02-01

    This study examined the effect of including different dietary proportions of starch, protein and lipid, in diets balanced for digestible energy, on the utilisation efficiencies of dietary energy by barramundi (Lates calcarifer). Each diet was fed at one of three ration levels (satiety, 80 % of initial satiety and 60 % of initial satiety) for a 42-d period. Fish performance measures (weight gain, feed intake and feed conversion ratio) were all affected by dietary energy source. The efficiency of energy utilisation was significantly reduced in fish fed the starch diet relative to the other diets, but there were no significant effects between the other macronutrients. This reduction in efficiency of utilisation was derived from a multifactorial change in both protein and lipid utilisation. The rate of protein utilisation deteriorated as the amount of starch included in the diet increased. Lipid utilisation was most dramatically affected by inclusion levels of lipid in the diet, with diets low in lipid producing component lipid utilisation rates well above 1·3, which indicates substantial lipid synthesis from other energy sources. However, the energetic cost of lipid gain was as low as 0·65 kJ per kJ of lipid deposited, indicating that barramundi very efficiently store energy in the form of lipid, particularly from dietary starch energy. This study defines how the utilisation efficiency of dietary digestible energy by barramundi is influenced by the macronutrient source providing that energy, and that the inclusion of starch causes problems with protein utilisation in this species.

  20. nitrosoguanidine-induced cadmium resistant mutants of Aspergillus

    Indian Academy of Sciences (India)

    Unknown

    nitrosoguanidine-induced cadmium resistant mutants of. Aspergillus niger. SAMAR ... gens and UV irradiation to study transportation of cad- mium ion through cell ..... Rowley W S 1993 Yeast bZib proteins mediate pleiotropic drug and metal ...

  1. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ARL

    different stages. Protein and amino acid contents in seeds of mutant are higher than those of the wild .... curled shape, from seedling to maturation phase (Figure. 1D-1F). ..... Arabidopsis seed dermination by stimulating abscisic acid synthesis.

  2. 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.

  3. 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.

  4. ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics.

    Science.gov (United States)

    Gal, Jozsef; Kuang, Lisha; Barnett, Kelly R; Zhu, Brian Z; Shissler, Susannah C; Korotkov, Konstantin V; Hayward, Lawrence J; Kasarskis, Edward J; Zhu, Haining

    2016-10-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.

  5. Molecular analysis of waxy mutants in rice

    International Nuclear Information System (INIS)

    Yatou, O.; Amano, E.

    1990-01-01

    Full text: The 'waxy' gene is a structural gene coding a glycosyl transferase which synthesises amylose in the endosperm tissue. 'Non-waxy' rice cultivars have an active gene and their amylose content is 18-25% depending upon gene performance and modifier genes. In 'waxy' rice, no amylose is found because the enzyme is absent. In mutants induced by gamma rays, neutrons, EI or EMS, amylose content ranged from 0 to 20%, i.e. there are intermediate phenotypes as well. Some of them had the same amount of the enzyme as a 'non-waxy' cultivar, even fully 'waxy' mutants showed a certain amount of the enzyme. This suggests that in mutants there may be no structural change in the enzyme gene but the enzyme produced might be less active. By molecular analysis of the mutants' genes it was found that only two mutants induced by thermal neutrons show structural alterations, the changes in other mutants are either too small to be detected by Southern analysis or are outside the structural gene in question. (author)

  6. Microarray Expression Analyses of Arabidopsis Guard Cells and Isolation of a Recessive Abscisic Acid Hypersensitive Protein Phosphatase 2C MutantW⃞

    Science.gov (United States)

    Leonhardt, Nathalie; Kwak, June M.; Robert, Nadia; Waner, David; Leonhardt, Guillaume; Schroeder, Julian I.

    2004-01-01

    Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell–expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription–PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)–regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type–specific genomic scale analyses of gene function. PMID:14973164

  7. Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

    Science.gov (United States)

    Chakravartty, Vandana

    2012-01-01

    Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues

  8. 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

  9. Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging.

    Directory of Open Access Journals (Sweden)

    Astrid Cornils

    2016-12-01

    Full Text Available The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT. Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.

  10. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  11. Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2013-07-01

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

  12. Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

    Science.gov (United States)

    Ghezelbash, Gholam Reza; Nahvi, Iraj; Emamzadeh, Rahman

    2014-08-01

    The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

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

    Science.gov (United States)

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

    2012-01-01

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

  14. Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

    DEFF Research Database (Denmark)

    Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

    2002-01-01

    as two recessive mutants, designated joe1 and 2, that overexpress the reporter. Genetic analysis indicated that reporter overexpression in the joe mutants requires COI. joe1 responded to MeJA with increased anthocyanin accumulation, while joe2 responded with decreased root growth inhibition. In addition...... activity was also induced by the protein kinase inhibitor staurosporine and antagonized by the protein phosphatase inhibitor okadaic acid. FLUC bio-imaging, RNA gel-blot analysis and progeny analyses identified three recessive mutants that underexpress the FLUC reporter, designated jue1, 2 and 3, as well...

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

    Science.gov (United States)

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

    2014-03-01

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

  16. Characterization of Sendai virus persistently infected L929 cells and Sendai virus pi strain: recombinant Sendai viruses having Mpi protein shows lower cytotoxicity and are incapable of establishing persistent infection

    International Nuclear Information System (INIS)

    Nishio, Machiko; Tsurudome, Masato; Ito, Morihiro; Kawano, Mitsuo; Komada, Hiroshi; Ito, Yasuhiko

    2003-01-01

    It is commonly accepted that the temperature-sensitive phenotype of Sendai virus (SeV) persistently infected cells is caused by the M and/or HN proteins. Expression level of the L, M, HN, and V proteins is extremely low in L929 cells persistently infected with SeVpi (L929/SeVpi cells) incubated at 38 deg. C. The HN protein quickly disappears in L929/SeVpi cells following a temperature shift up to 38 deg. C, and pulse-chase experiments show that the Lpi, HNpi, and Mpi proteins are unstable at 38 deg. C. Following a temperature shift either upward or downward, M protein is translocated into the nucleus and then localizes to the perinuclear region. None of virus-specific polypeptides are detected in the cells primarily infected with SeVpi and incubated at 38 deg. C and virus proteins are not pulse-labeled at 38 deg. C, indicating that temperature-sensitive step is at an early stage of infection. The Mpi protein is transiently located in the nucleus of the SeVpi primarily infected cells. Recombinant SeVs possessing the HNpi or/and Mpi proteins are not temperature-sensitive. The HN protein is expressed at very low levels and the F protein localizes to the perinuclear region in rSeV(Mpi)-infected cells incubated at 38 deg. C for 18 h. rSeVs having the Mpi protein exhibit lower cytotoxicity and are incapable of establishing persistent infection. Amino acid 116 of the Mpi protein is related to the nuclear translocation and lower cytopathogenesis, whereas aa183 is involved in the interaction between M protein and viral glycoproteins

  17. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    Directory of Open Access Journals (Sweden)

    Charalampos Rallis

    2014-01-01

    Target of rapamycin complex 1 (TORC1, which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

  18. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

    Directory of Open Access Journals (Sweden)

    Christelle Folio

    2017-11-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

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

    Science.gov (United States)

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

    2011-09-01

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

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

    Science.gov (United States)

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

    2015-06-30

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

  1. Prion propagation in cells expressing PrP glycosylation mutants.

    Science.gov (United States)

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

    2011-04-01

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

  2. 15N NMR relaxation studies of calcium-loaded parvalbumin show tight dynamics compared to those of other EF-hand proteins

    DEFF Research Database (Denmark)

    Baldellon, C; Alattia, J R; Strub, M P

    1998-01-01

    Dynamics of the rat alpha-parvalbumin calcium-loaded form have been determined by measurement of 15N nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed using spectral density functions and the Lipari-Szabo formalism. The major dynamic features...... for the rat alpha-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix-loop-helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4...... properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity...

  3. Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

    Science.gov (United States)

    Komatsu, Setsuko; Nanjo, Yohei; Nishimura, Minoru

    2013-02-21

    Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Variation in Yield and Physicochemical Quality Traits among Mutants of Japonica Rice Cultivar Wuyujing 3

    OpenAIRE

    Abacar, Jose Daniel; Zhao-miao, Lin; Xin-cheng, Zhang; Cheng-qiang, Ding; She, Tang; Zheng-hui, Liu; Shao-hua, Wang; Yan-feng, Ding

    2016-01-01

    To select elite germplasms, 112 mutants derived from japonica rice cultivar Wuyujing 3 were evaluated. The yield components such as panicle number per square meter, grain number per panicle, and grain weight were measured. The quality traits such as percentage of chalky grains (PCG), brown rice yield (BRY), milled rice yield (MRY), degree of milling (DM), amylose content (AC), protein content (PC), and relationships among traits were inverstigated. Results showed that grain yield ranged from ...

  5. Neurobehavioral performances and brain regional metabolism in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Jacquelin, C; Lalonde, R; Jantzen-Ossola, C; Strazielle, C

    2013-09-01

    As disabled-1 (DAB1) protein acts downstream in the reelin signaling pathway modulating neuronal migration, glutamate neurotransmission, and cytoskeletal function, the disabled-1 gene mutation (scrambler or Dab1(scm) mutation) results in ataxic mice displaying dramatic neuroanatomical defects similar to those observed in the reeler gene (Reln) mutation. By comparison to non-ataxic controls, Dab1(scm) mutants showed severe motor coordination impairments on stationary beam, coat-hanger, and rotorod tests but were more active in the open-field. Dab1(scm) mutants were also less anxious in the elevated plus-maze but with higher latencies in the emergence test. In mutants versus controls, changes in regional brain metabolism as measured by cytochrome oxidase (COX) activity occurred mainly in structures intimately connected with the cerebellum, in basal ganglia, in limbic regions, particularly hippocampus, as well as in visual and parietal sensory cortices. Although behavioral results characterized a major cerebellar disorder in the Dab1(scm) mutants, motor activity impairments in the open-field were associated with COX activity changes in efferent basal ganglia structures such as the substantia nigra, pars reticulata. Metabolic changes in this structure were also associated with the anxiety changes observed in the elevated plus-maze and emergence test. These results indicate a crucial participation of the basal ganglia in the functional phenotype of ataxic Dab1(scm) mutants. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.

    Science.gov (United States)

    Sahly, I; Bar Nachum, S; Suss-Toby, E; Rom, A; Peretz, A; Kleiman, J; Byk, T; Selinger, Z; Minke, B

    1992-01-01

    Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant. Images PMID:1309615

  7. Characterization of a mutant of Escherichia coli B/R defective in mutation frequency decline

    International Nuclear Information System (INIS)

    George, D.L.

    1974-01-01

    A mutant of Escherichia coli B/r designated mfd is very deficient in the ability to exhibit mutation frequency decline (MFD), the characteristic loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with ultraviolet light (uv). This mutant is known to excise pyrimidine dimers very slowly, although it is as uv-resistant as its mfd + B/r parent strain. We have found that the mfd mutant performs the initial incision step of excision repair normally, but repairs the resulting single-strand breaks much more slowly than the mfd + strain. In spite of the slow dimer excision in the mfd mutant, single-strand DNA breaks do not accumulate during postirradiation incubation, implying that incision and excision are well corrdinated. the prolonged postirradiation lag in cell division and DNA synthesis which accompany slow excision in the mfd strain indicates that resumption of these processes of optimal rates is linked to the timing of excision repair. The normal uv-resistance of the mfd mutant also suggests such coordination and shows that the rate of excision repair is independent of its ultimate efficiency in the removal of potentially lethal uv-induced damage. (U.S.)

  8. Thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Nagai, K; Some, H; Tamura, G

    1976-01-01

    Thermonsensitive division mutants were derived from Bacillus subtilis Marburg 168 thy trp/sub 2/ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48/sup 0/C was investigated. In the absence of uracil, the mutant cells grew normally at 37/sup 0/C and stopped dividing after temperature shift to 48/sup 0/C resulting in filaments of two to four times length of normal rods. The total cell number after the temperature shift increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48/sup 0/C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48/sup 0/C or when uracil was introduced to the culture at 48/sup 0/C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48/sup 0/C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature. No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.

  9. Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage*

    Science.gov (United States)

    Comeaux, Evan Q.; Cuya, Selma M.; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C.; Mobley, James A.; Bjornsti, Mary-Ann; van Waardenburg, Robert C. A. M.

    2015-01-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

  10. The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

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    Sven Vilain

    2012-01-01

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

  11. Variation in Yield and Physicochemical Quality Traits among Mutants of Japonica Rice Cultivar Wuyujing 3

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    Jose Daniel Abacar

    2016-01-01

    Full Text Available To select elite germplasms, 112 mutants derived from japonica rice cultivar Wuyujing 3 were evaluated. The yield components such as panicle number per square meter, grain number per panicle, and grain weight were measured. The quality traits such as percentage of chalky grains (PCG, brown rice yield (BRY, milled rice yield (MRY, degree of milling (DM, amylose content (AC, protein content (PC, and relationships among traits were inverstigated. Results showed that grain yield ranged from 2.15 to 12.49 t/hm2 with a mean of 6.4 t/hm2 and number of grains per square meter contributed for 94.64% in grain yield variation. For quality traits, all rice mutants had short size (grain length ≤ 5.5 mm and bold shape (grain length to width ratio = 1.10–2.00. Most of rice mutants (87.5% had PCG values below 20%. All mutants had MRY values above 50%, AC values below 20%, and PC values below 10%. Percentage of chalky grains was significantly negatively correlated with MRY and positively correlated with DM. BRY and MRY were significantly negatively correlated with DM. PC was significantly and positively correlated with MRY and negatively correlated with DM, while AC had no significant correlation with these quality traits. It was concluded that there were 25 rice mutants which fulfilled the major requirements of Jiangsu standard japonica rice such as low percentage of chalky grains, low amylose content, optimal protein content, and which could be used as elite germplasms. Thus the mutants identified may lead to significant progress in improvement of rice quality.

  12. Activation of AMP-activated protein kinase in response to temperature elevation shows seasonal variation in the zebra mussel, Dreissena polymorpha.

    Science.gov (United States)

    Jost, Jennifer A; Keshwani, Sarah S; Abou-Hanna, Jacob J

    2015-04-01

    Global climate change is affecting ectothermic species, and a variety of studies are needed on thermal tolerances, especially from cellular and physiological perspectives. This study utilized AMP-activated protein kinase (AMPK), a key regulator of cellular energy levels, to examine the effects of high water temperatures on zebra mussel (Dreissena polymorpha) physiology. During heating, AMPK activity increased as water temperature increased to a point, and maximum AMPK activity was detected at high, but sublethal, water temperatures. This pattern varied with season, suggesting that cellular mechanisms of seasonal thermal acclimatization affect basic metabolic processes during sublethal heat stress. There was a greater seasonal variation in the water temperature at which maximum AMPK activity was measured than in lethal water temperature. Furthermore, baseline AMPK activity varied significantly across seasons, most likely reflecting altered metabolic states during times of growth and reproduction. In addition, when summer-collected mussels were lab-acclimated to winter and spring water temperatures, patterns of heat stress mirrored those of field-collected animals. These data suggest that water temperature is the main driver of the seasonal variation in physiology. This study concluded that AMPK activity, which reflects changes in energy supply and demand during heat stress, can serve as a sensitive and early indicator of temperature stress in mussels. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. PNRI mutant variety: Cordyline 'Afable'

    International Nuclear Information System (INIS)

    Aurigue, Fernando B.

    2012-01-01

    Cordyline 'Afable', registered by the Philippine Nuclear Research Institute as NSIC 2009 Or-83, is an induced mutant developed from Cordyline 'Kiwi' by treating stem cuttings with acute gamma radiation from a Cobalt-60 source. The new mutant is identical to Cordyline 'Kiwi' in growth habit but differs in foliage color, and exhibits field resistance to Phytophthora sp., a fungus that causes leaf blight and rot in Ti plants. Results of this mutation breeding experiment showed that leaf color was altered by gamma irradiation and resistance to fungal diseases was improved. It also demonstrated how mutations that occur in nature may be generated artificially. Propagation of cordyline 'Afable' is true-to-type by vegetative propagation methods, such as separation of suckers and offshoots, shoot tip cutting, and top cutting. Aside from landscaping material, terrarium or dish-garden plant, it is ideal as containerized plant for indoor and outdoor use. The leaves or shoots may be harvested as cut foliage for flower arrangements. (author)

  14. The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

    Science.gov (United States)

    Lim, Khai Lone; Amir, Amirah; Lau, Yee Ling; Fong, Mun Yik

    2017-08-11

    The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

  15. Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Dusenbery, D B; Sheridan, R E; Russell, R L

    1975-06-01

    The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.

  16. Study on ionizing radiosensitivity of respiratory deficiency yeast mutants

    International Nuclear Information System (INIS)

    Mao Shuhong; Chinese Academy of Sciences, Beijing; Jin Genming; Wei Zengquan; Xie Hongmei

    2006-01-01

    The radiosensitivity of respiratory deficiency yeast mutants has been studied in this work. The mutants which were screened from the yeasts after ionizing irradiation were irradiated with 12 C 6+ at different doses. Because of the great change in its mitochondria and mitochondrial DNA, the respiratory deficiency yeast mutants show radio-sensitivity at dose less than 1 Gy and radioresistance at doses higher than 1 Gy. (authors)

  17. Molecular mechanism of action of pharmacoperone rescue of misrouted GPCR mutants: the GnRH receptor.

    Science.gov (United States)

    Janovick, Jo Ann; Patny, Akshay; Mosley, Ralph; Goulet, Mark T; Altman, Michael D; Rush, Thomas S; Cornea, Anda; Conn, P Michael

    2009-02-01

    The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

  18. Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant.

    Science.gov (United States)

    Johnson, E M; Sears, B B

    1990-01-01

    Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.

  19. Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

    Directory of Open Access Journals (Sweden)

    Md Zahid Kamal

    Full Text Available Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

  20. 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)

  1. Finding Inhibitors of Mutant Superoxide Dismutase-1 for Amyotrophic Lateral Sclerosis Therapy from Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Hung-Jin Huang

    2014-01-01

    Full Text Available Superoxide dismutase type 1 (SOD1 mutations cause protein aggregation and decrease protein stability, which are linked to amyotrophic lateral sclerosis (ALS disease. This research utilizes the world’s largest traditional Chinese medicine (TCM database to search novel inhibitors of mutant SOD1, and molecular dynamics (MD simulations were used to analyze the stability of protein that interacted with docked ligands. Docking results show that hesperidin and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG have high affinity to mutant SOD1 and then dopamine. For MD simulation analysis, hesperidin and THSG displayed similar value of RMSD with dopamine, and the migration analysis reveals stable fluctuation at the end of MD simulation time. Interestingly, distance between the protein and ligand has distinct difference, and hesperidin changes the position from initial binding site to the other place. In flexibility of residues analysis, the secondary structure among all complexes does not change, indicating that the structure are not affect ligand binding. The binding poses of hesperidin and THSG are similar to dopamine after molecular simulation. Our result indicated that hesperidin and THSG might be potential lead compound to design inhibitors of mutant SOD1 for ALS therapy.

  2. RAPD analysis on male sterility mutant of Lilium asiatic hybrids 'pollyanna' induced by irradiation

    International Nuclear Information System (INIS)

    Jia Yuehui; Zhao Xiangyun; Zhang Kezhong; Huang Shangwu; Lu Changxun

    2005-01-01

    RAPD analysis of 80 random 10-mer primers on Lilium Asiatic hybrids 'pollyanna' and its 20 phenotype male sterility mutants induced by irradiation was carried out. Of the tested primers, 31 primers could produced ideal amplification bands on all materials, 4 primers generated stable different polymorphic bands among 9 mutants and 'pollyanna'. Different polymorphic bands of 7-18 were found among 9 mutants and 'pollyanna'. It was showed that 9 mutants were phenotype male sterility mutant of 'pollyanna'. (authors)

  3. 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

  4. 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.

  5. 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''

  6. 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

  7. 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.

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

    Science.gov (United States)

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

    2013-06-27

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

  9. Genetic cytological and biochemical study of a tomato chlorophyll mutant of the xanthic type, obtained by irradiation of the seeds

    International Nuclear Information System (INIS)

    Lefort, M.; Duranton, J.; Galmiche, J.M.; Roux, E.

    1958-01-01

    Irradiation of Lycopersicum aesculantum seeds with increasing doses of X-rays and thermal neutrons leads to the appearance of chlorophyll mutations in the descendants of the irradiated seeds. A genetic study of one of these mutants of the xanthic type showed that it was a recessive mutant with typical mono-genetic separation, while the cytological study demonstrated that the differentiation of the plast stopped at the stage of elementary lamella. Finally it is shown that in the light, the mutation brings about a very large deviation of the carbon metabolism towards the synthesis of amino acids and proteins, at the expense of that of glucosides. (author) [fr

  10. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

    Directory of Open Access Journals (Sweden)

    Yeon Jeong Kim

    Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

  11. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    International Nuclear Information System (INIS)

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E.

    1991-01-01

    The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus

  12. Temperature sensitive riboflavin mutants of Penicillium vermiculatum Dangeard

    International Nuclear Information System (INIS)

    Mitra, J.; Chaudhari, K.L.

    1974-01-01

    Two temperature sensitive UV induced riboflavin mutants rib 1 and rib 6 have been physiologically and genetically characterized. The two mutants behave differently with regard to their temperature sensitivity. The rib 1 mutant exhibits a leaky growth in minimal medium between 15 0 C and 30 0 C but grows well when the medium is supplemented with riboflavin. At 35 0 C the growth response of the mutant is at its max. and at 40 0 C and below 15 0 C it ceases to grow. The rib 6 mutant which is red brown in colour shows wild type character at temp. below 25 0 C in minimal medium but requires riboflavin at 30 0 C and above. Heterokaryotic analysis revealed the nonallelic nature of the two temperature mutants. Genetic tests of allelic relationship between riboflavin markers by crossing were also done. (author)

  13. Macrophage inflammatory protein-1α shows predictive value as a risk marker for subjects and sites vulnerable to bone loss in a longitudinal model of aggressive periodontitis.

    Science.gov (United States)

    Fine, Daniel H; Markowitz, Kenneth; Fairlie, Karen; Tischio-Bereski, Debbie; Ferrandiz, Javier; Godboley, Dipti; Furgang, David; Gunsolley, John; Best, Al

    2014-01-01

    Improved diagnostics remains a fundamental goal of biomedical research. This study was designed to assess cytokine biomarkers that could predict bone loss (BL) in localized aggressive periodontitis. 2,058 adolescents were screened. Two groups of 50 periodontally healthy adolescents were enrolled in the longitudinal study. One group had Aggregatibacter actinomycetemcomitans (Aa), the putative pathogen, while the matched cohort did not. Cytokine levels were assessed in saliva and gingival crevicular fluid (GCF). Participants were sampled, examined, and radiographed every 6 months for 2-3 years. Disease was defined as radiographic evidence of BL. Saliva and GCF was collected at each visit, frozen, and then tested retrospectively after detection of BL. Sixteen subjects with Aa developed BL. Saliva from Aa-positive and Aa-negative healthy subjects was compared to subjects who developed BL. GCF was collected from 16 subjects with BL and from another 38 subjects who remained healthy. GCF from BL sites in the 16 subjects was compared to healthy sites in these same subjects and to healthy sites in subjects who remained healthy. Results showed that cytokines in saliva associated with acute inflammation were elevated in subjects who developed BL (i.e., MIP-1α MIP-1β IL-α, IL-1β and IL-8; p<0.01). MIP-1α was elevated 13-fold, 6 months prior to BL. When MIP-1α levels were set at 40 pg/ml, 98% of healthy sites were below that level (Specificity); whereas, 93% of sites with BL were higher (Sensitivity), with comparable Predictive Values of 98%; p<0.0001; 95% C.I. = 42.5-52.7). MIP-1α consistently showed elevated levels as a biomarker for BL in both saliva and GCF, 6 months prior to BL. MIP-1α continues to demonstrate its strong candidacy as a diagnostic biomarker for both subject and site vulnerability to BL.

  14. Macrophage inflammatory protein-1α shows predictive value as a risk marker for subjects and sites vulnerable to bone loss in a longitudinal model of aggressive periodontitis.

    Directory of Open Access Journals (Sweden)

    Daniel H Fine

    Full Text Available Improved diagnostics remains a fundamental goal of biomedical research. This study was designed to assess cytokine biomarkers that could predict bone loss (BL in localized aggressive periodontitis. 2,058 adolescents were screened. Two groups of 50 periodontally healthy adolescents were enrolled in the longitudinal study. One group had Aggregatibacter actinomycetemcomitans (Aa, the putative pathogen, while the matched cohort did not. Cytokine levels were assessed in saliva and gingival crevicular fluid (GCF. Participants were sampled, examined, and radiographed every 6 months for 2-3 years. Disease was defined as radiographic evidence of BL. Saliva and GCF was collected at each visit, frozen, and then tested retrospectively after detection of BL. Sixteen subjects with Aa developed BL. Saliva from Aa-positive and Aa-negative healthy subjects was compared to subjects who developed BL. GCF was collected from 16 subjects with BL and from another 38 subjects who remained healthy. GCF from BL sites in the 16 subjects was compared to healthy sites in these same subjects and to healthy sites in subjects who remained healthy. Results showed that cytokines in saliva associated with acute inflammation were elevated in subjects who developed BL (i.e., MIP-1α MIP-1β IL-α, IL-1β and IL-8; p<0.01. MIP-1α was elevated 13-fold, 6 months prior to BL. When MIP-1α levels were set at 40 pg/ml, 98% of healthy sites were below that level (Specificity; whereas, 93% of sites with BL were higher (Sensitivity, with comparable Predictive Values of 98%; p<0.0001; 95% C.I. = 42.5-52.7. MIP-1α consistently showed elevated levels as a biomarker for BL in both saliva and GCF, 6 months prior to BL. MIP-1α continues to demonstrate its strong candidacy as a diagnostic biomarker for both subject and site vulnerability to BL.

  15. Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

    Science.gov (United States)

    Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

    1991-09-01

    Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association